High-throughput DNA sequence analysis elucidates novel insight into the genetic basis of adaptation in local sheep.

Understanding how evolutionary factors related to climate adaptation and human selection have influenced the genetic architecture of domesticated animals is of great interest in biology. In the current study, by using 304 whole genomes from different geographical regions (including Europe, north Africa, Southwest Asia, east Asia, west Africa, south Asia, east Africa, Australia and Turkey), We evaluate global sheep population dynamics in terms of genetic variation and population structure. We further conducted comparative population analysis to study the genetic underpinnings of climate adaption to local environments and also morphological traits. In order to identify genomic signals under selection, we applied fixation index (FST) and also nucleotide diversity (θπ) statistical measurements. Our results revealed several candidate genes on different chromosomes under selection for local climate adaptation (e.g. HOXC12, HOXC13, IRF1, FGD2 and GNAQ), body size (PDGFA, HMGA2, PDE3A) and also morphological related traits (RXFP2). The discovered candidate genes may offer newel insights into genetic underpinning of regional adaptation and commercially significant features in local sheep.

Unmapped short reads from whole-genome sequencing indicate potential infectious pathogens in german black Pied cattle.

When resequencing animal genomes, some short reads cannot be mapped to the reference genome and are usually discarded. In this study, unmapped reads from 302 German Black Pied cattle were analyzed to identify potential pathogenic DNA. These unmapped reads were assembled and blasted against NCBI's database to identify bacterial and viral sequences. The results provided evidence for the presence of pathogens. We found sequences of Bovine parvovirus 3 and Mycoplasma species. These findings emphasize the information content of unmapped reads for gaining insight into bacterial and viral infections, which is important for veterinarians and epidemiologists.

Copy number variants and fixed duplications among 198 rhesus macaques (Macaca mulatta).

The rhesus macaque is an abundant species of Old World monkeys and a valuable model organism for biomedical research due to its close phylogenetic relationship to humans. Copy number variation is one of the main sources of genomic diversity within and between species and a widely recognized cause of inter-individual differences in disease risk. However, copy number differences among rhesus macaques and between the human and macaque genomes, as well as the relevance of this diversity to research involving this nonhuman primate, remain understudied. Here we present a high-resolution map of sequence copy number for the rhesus macaque genome constructed from a dataset of 198 individuals. Our results show that about one-eighth of the rhesus macaque reference genome is composed of recently duplicated regions, either copy number variable regions or fixed duplications. Comparison with human genomic copy number maps based on previously published data shows that, despite overall similarities in the genome-wide distribution of these regions, there are specific differences at the chromosome level. Some of these create differences in the copy number profile between human disease genes and their rhesus macaque orthologs. Our results highlight the importance of addressing the number of copies of target genes in the design of experiments and cautions against human-centered assumptions in research conducted with model organisms. Overall, we present a genome-wide copy number map from a large sample of rhesus macaque individuals representing an important novel contribution concerning the evolution of copy number in primate genomes.

Identification of differentially expressed long non-coding RNAs and messenger RNAs involved with muscle development in Dazu black goats through RNA sequencing.

This study aimed to explore the genetic basis of muscle development in goats. The transcriptome dataset for differentially expressed lncRNAs (DELs) and differentially expressed genes (DEGs) of goat muscle at different developmental stages were obtained using RNA-Seq. A total of 447,806,481 and 587,559,465 clean reads in the longissimus dorsi muscle of Dazu black goats between 75d embryonic stage and 1d after birth were generated through Illumina paired-end sequencing, and their mapping rates were 89.82 and 90.99%, respectively. Moreover, 4517 DEGs and 648 DELs were identified, and 4784 lncRNA-mRNA targeting relationships were predicted. Gene function annotation results showed that 4101 DEGs were significantly enriched to 1098 GO terms, and 2014 DEGs were significantly enriched to 40 KEGG pathways, including many GO terms and pathways related to muscle development, such as cell differentiation and Wnt signaling pathway. Then, 10 DELs and 20 DEGs were randomly selected for RT-qPCR verification, and the agreement rate between the verification and RNA-Seq results was 90%, indicating the high reliability of the RNA-Seq data analysis. In conclusion, this study obtained several mRNAs and lncRNAs related to the muscle development of Dazu black goats and identified several targeted regulatory pairs of lncRNA-mRNA. This study may serve as a reference to understand the genetic basis and molecular mechanism of muscle development in goats.

High-throughput analysis of lncRNA in cows with naturally infected Staphylococcus aureus mammary gland.

LncRNA (long non-coding RNA) is an RNA molecule with a length between 200 and 100,000 nt. It does not encode proteins and is involved in a variety of intracellular processes, becoming a research hotspot of genetics. To identify key lncRNAs associated with dairy mastitis, we collected mammary epithelial tissue samples of Normal disease-free Holstein cows (HCN) and unhealthy Holstein cows with Staphylococcus aureus (HCU) and performed RNA sequencing (RNA-seq) on the samples. A total of 270 differentially expressed lncRNAs and 500 differentially expressed mRNAs were identified by high-throughput sequencing and bioinformatics analysis. Furthermore, Hydrolase activity is the most enriched in GO, and ErbB signaling pathway is significantly enriched in KEGG. In addition, through qPCR validation of 5 candidate lncRNAs in HCN and HCU, four differentially expressed lncRNAs MSTRG.498, MSTRG57.1, MSTRG.41.1 and MSTRG 124.1 were confirmed to have significant differentially expressed in cow mastitis. Also, lncRNA MSTRG.498 and its target gene, SMC4, might directly or indirectly play a role in cow mastitis. The regulatory network of lncRNA-miRNA-mRNA has been inferred from a bioinformatics perspective, which may assist understand the underlying molecular mechanism of lncRNAs involved in regulating mastitis in cows. Our findings will provide meaningful resources for further research on the regulatory function of lncRNAs in cow mastitis.

Management and analysis of high-throughput sequence data for infectious animal diseases.

Advances in technology and decreasing costs have accelerated the use of high-throughput sequencing (HTS) for both diagnosis and characterisation of infectious animal diseases. High-throughput sequencing offers several advantages over previous techniques, including rapid turnaround times and the ability to resolve single nucleotide changes among samples, both of which are important for epidemiological investigations of outbreaks. However, due to the plethora of genetic data being routinely generated, the storage and analysis of these data are proving challenging in their own right. In this article, the authors provide insight into the aspects of data management and analysis that should be considered before adopting HTS for routine animal health diagnostics. These elements fall largely into three interrelated categories: data storage, data analysis and quality assurance. Each has numerous complexities and may need to be adapted as HTS evolves. Making appropriate strategic decisions about bioinformatic sequence analysis early on in project development will help to avert major issues in the long term.

Les avancées technologiques dans le domaine du séquençage à haut débit (SHD) et la diminution des coûts liés à cette technique en ont accéléré l'utilisation à des fins de diagnostic et de caractérisation des maladies animales infectieuses. Le séquençage à haut débit offre plusieurs avantages par rapport aux techniques antérieures, en particulier la rapidité de son exécution et une résolution de l'ordre d'un seul changement de nucléotide parmi plusieurs échantillons, ce qui présente un grand intérêt lors des enquêtes épidémiologiques sur les foyers. Néanmoins, la pléthore de données génétiques générées en routine par le SHD devient un véritable problème en termes de stockage et d'analyse de ces données. Les auteurs apportent un éclairage sur les aspects de la gestion et de l'analyse des données qu'il convient de prendre en compte avant d'adopter le SHD pour le diagnostic de routine en santé animale. Ces éléments relèvent de trois catégories étroitement reliées : le stockage de données, l'analyse de données et l'assurance qualité. Chacun de ces aspects présente de nombreuses complexités et nécessitera sans doute d'être adapté à mesure que le SHD évolue. Lorsqu'elles sont prises dès la phase initiale d'un projet, des décisions stratégiques appropriées en matière d'analyse bio-informatique de séquences peuvent contribuer à éviter des problèmes majeurs sur le long terme.

Los avances tecnológicos y la reducción de los costos han acelerado el uso de la secuenciación de alto rendimiento (SAR) con fines de diagnóstico y caracterización de enfermedades animales infecciosas. La secuenciación de alto rendimiento presenta varias ventajas en comparación con otras técnicas anteriores, en particular ciclos más rápidos y una resolución que permite detectar diferencias de un solo nucleótido entre las muestras, aspectos ambos de gran importancia para el estudio epidemiológico de brotes infecciosos. Sin embargo, debido al sinnúmero de datos genéticos que constantemente se generan, no es de extrañar que esté resultando problemático almacenar y analizar los datos obtenidos. Los autores arrojan luz sobre los aspectos de la gestión y el análisis de datos que conviene tener en cuenta antes de aplicar la SAR a las labores sistemáticas de diagnóstico en sanidad animal. Estos elementos corresponden a grandes líneas a tres categorías relacionadas entre sí: el almacenamiento de datos; el análisis de datos; y la garantía de calidad. Cada una de ellas presenta multitud de complicaciones y exige un proceso permanente de adaptación a medida que la técnica de secuenciación va evolucionando. El hecho de adoptar las buenas decisiones estratégicas sobre el análisis bioinformático de secuencias en los primeros momentos de la concepción de un proyecto ayudará a evitar importantes problemas a largo plazo.

Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis.

OBJECTIVES: To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK). PROCEDURE: Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n = 11 and n = 4, respectively) and IUK patients (n = 22 and n = 8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined. RESULTS: The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples. CONCLUSION: A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.

Extracting turkey coronaviruses from the intestinal lumen of infected turkey embryos yields full genome data with good coverage by NGS.

Currently, turkey coronaviruses (TCoV) are isolated from homogenized intestines of experimentally infected embryos to ensure a maximum recovery of viral particles from all components of the intestines. However, the process of homogenization also ensures release of a significant amount of cellular RNAs into the sample that hinders downstream viral genome sequencing. This is especially the case for next generation sequencing (NGS) which sequences molecules at random. This characteristic means that the heavily abundant cellular RNA in the sample drowns out the minority viral RNA during the sequencing process and, consequently, very little to no viral genome data are obtained. To address this problem, a method was developed, in which 10 descendent isolates of the European strain of TCoV were recovered uniquely from the intestinal lumen without homogenization of the tissue. For nine out of 10 samples, NGS produced viral RNA reads with good coverage depth over the entire TCoV genomes. This is a much-needed new, simple and cost effective method of isolating TCoV that facilitates downstream NGS of viral RNA and should be considered as an alternative method for isolating other avian enteric coronaviruses in the interest of obtaining full-length genome sequences.

Assessment of the performance of different imputation methods for low-coverage sequencing in Holstein cattle.

Low-coverage sequencing (LCS) followed by imputation has been proposed as a cost-effective genotyping approach for obtaining genotypes of whole-genome variants. Imputation performance is essential for the effectiveness of this approach. Several imputation methods have been proposed and successfully applied in genomic studies in human and other species. However, there are few reports on the performance of these methods in livestock. Here, we evaluated a variety of imputation methods, including Beagle v4.1, GeneImp v1.3, GLIMPSE v1.1.0, QUILT v1.0.0, Reveel, and STITCH v1.6.5, with varying sequencing depth, sample size, and reference panel size using LCS data of Holstein cattle. We found that all of these methods, except Reveel, performed well in most cases with an imputation accuracy over 0.9; on the whole, GLIMPSE, QUILT, and STITCH performed better than the other methods. For species with no reference panel available, STITCH followed by Beagle would be an optimal strategy, whereas for species with reference panel available, QUILT would be the method of choice. Overall, this study illustrated the promising potential of LCS for genomic analysis in livestock.

High-throughput sequencing reveals differential expression of miRNAs in yak and cattleyak epididymis.

Cattleyaks (CY) are interspecific hybrids between cattle (Bos taurus) and yak (Bos gruniens, YK) exhibiting the same prominent adaptability and higher performances than YK. The main problem of this crossbreeding is that the males are sterile. Different series of events of spermatogenesis coordinate to regulate gene expressing, involving microRNAs (miRNAs). As non-coding ribonucleic acids (ncRNAs), miRNAs predominantly facilitate the regulation of gene expression at post-transcriptional stages and play important roles in the acquisition and maintenance of male fertility in reproduction. The function of miRNA in the male reproductive system extends from the testis into the epididymis, regulating gene expression and contributing to regional gene expression variations. RNA sequencing on biological replicates, we described differentially expressed miRNAs profiles for tissue from epididymis of YK and CY. In the present study, High-throughput sequencing analysis showed that 55 differentially expressed (DE) miRNAs were identified in the epididymis of YK and CY. Among these, 43 DE miRNAs were upregulated while the remaining 12 DE miRNAs were downregulated between epididymis of YK and CY. In addition, we identified that the top most important DE miRNAs, bta-miR-449c, bta-miR-539, bta- miR-136, bta-miR-504, bta-miR-31 and bta-miR-222 were involved in the process of sperm maturation in epididymis CY. It was identified that the bta-miR-449c and bta-miR-222 may play major roles in the process of sperm maturation, sperm quality, sperm count, sperm production and male infertility of CY. Furthermore, GO and KEGG analyses were performed to classify the functions of target genes for DE miRNAs. In addition, RT-qPCR validation of the DE miRNAs and its targeted genes revealed that putative miRNAs are involved in the male CY infertility by altering the gene expression. Present findings may not only increase our understanding of the molecular mechanisms regulated by the miRNAs in epididymis, but also provide a valuable information to understand the male infertility mechanism of CY.

Taxonomy, not locality, influences the cloacal microbiota of two nearctic colubrids: a preliminary analysis.

BACKGROUND: The gut microbiota is an emerging frontier in wildlife research and its importance to vertebrate health and physiology is becoming ever more apparent. Reptiles, in particular snakes, have not received the same attention given to other vertebrates and the composition of their wild gut microbiome remains understudied. The primary goal of this work was to describe the cloacal microbiota of two Colubrids, the Eastern Gartersnake (Thamnophis sirtalis sirtalis) and the Northern Watersnake (Nerodia sipedon sipedon), and if their cloacal microbiota differed as well as if it did between a wetland and upland population of the former species. METHODS AND RESULTS: We utilized next-generation sequencing of cloacal swabs-a non-destructive proxy for the gut microbiota. The cloacal microbiome of Eastern Gartersnakes (N = 9) was like those of other snakes being comprised of Proteobacteria, Bacteroidetes, and Firmicutes, while that of Northern Watersnakes (N = 6) was dominated by Tenericutes. Seven microbial operational taxonomic units (OTUs), all members of Proteobacteria, were shared among all individuals and were indicative of a core microbiome in Eastern Gartersnakes, but these OTUs were not particularly relevant to Northern Watersnakes. The latter had greater OTU richness than did Eastern Gartersnakes, and habitat did not have any apparent effect on the microbial community composition in Eastern Gartersnakes. CONCLUSIONS: Our findings suggest host taxonomy to be a determining factor in the cloacal microbiota of snakes and that Tenericutes are associated with aquatic habitats. This is the first report to examine the cloacal microbiome of these species and provides a useful foundation for future work to build upon.

Signature selection analysis reveals candidate genes associated with production traits in Iranian sheep breeds.

BACKGROUND: Sheep were among the first animals to be domesticated. They are raised all over the world and produce a major scale of animal-based protein for human consumption and play an important role in agricultural economy. Iran is one of the important locations for sheep genetic resources in the world. Here, we compared the Illumina Ovine SNP50 BeadChip data of three Iranian local breeds (Moghani, Afshari and Gezel), as a population that does not undergone artificial breeding programs as yet, and five other sheep breeds namely East Friesian white, East Friesian brown, Lacaune, DorsetHorn and Texel to detect genetic mechanisms underlying economical traits and daptation to harsh environments in sheep. RESULTS: To identify genomic regions that have been targeted by positive selection, we used fixation index (Fst) and nucleotide diversity (Pi) statistics. Further analysis indicated candidate genes involved in different important traits such as; wool production included crimp of wool (PTPN3, NBEA and KRTAP20-2 genes), fiber diameter (PIK3R4 gene), hair follicle development (LHX2 gene), the growth and development of fiber (COL17A1 gene)), adaptation to hot arid environments (CORIN gene), adaptive in deficit water status (CPQ gene), heat stress (PLCB4, FAM107B, NBEA, PIK3C2B and USP43 genes) in sheep. CONCLUSIONS: We detected several candidate genes related to wool production traits and adaptation to hot arid environments in sheep that can be applicable for inbreeding goals. Our findings not only include the results of previous researches, but also identify a number of novel candidate genes related to studied traits. However, more works will be essential to acknowledge phenotype- genotype relationships of the identified genes in our study.

Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing.

BACKGROUND: Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP . RESULTS: We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. CONCLUSIONS: Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

Blood cultures and blood microbiota analysis as surrogates for bronchoalveolar lavage fluid analysis in dogs with bacterial pneumonia.

BACKGROUND: Diagnosis of canine bacterial pneumonia relies on airway lavage to confirm septic, suppurative inflammation, and a positive bacterial culture. Considering risks of bronchoalveolar lavage fluid (BALF) collection, minimally invasive methods like culture or next generation sequencing of blood would be appealing. In dogs with bacterial pneumonia, our study aims included (1): determining proportion of agreement between cultivable bacteria in BALF and blood (2); characterizing BALF, blood, and oropharyngeal (OP) microbiota and determining if bacteria cultured from BALF were present in these communities; and (3) comparing relatedness of microbial community composition at all three sites. Bacterial cultures were performed on BALF and blood. After DNA extraction of BALF, blood and OP, 16S rRNA amplicon libraries were generated, sequenced, and compared to a bacterial gene sequence database. RESULTS: Disregarding one false positive, blood cultures were positive in 2/9 dogs (5 total isolates), all 5 isolates were present in BALF cultures (16 total isolates). Based on sequencing data, all sites had rich and diverse microbial communities. Comparing cultured BALF bacterial genera with sequenced taxa, all dogs had ≥1 cultured isolate present in their microbiota: cultured BALF isolates were found in microbiota of BALF (12/16), blood (7/16), and OP (6/11; only 7 dogs had OP swabs). Of 394 distinct taxa detected in BALF, these were present in 75% OP and 45% blood samples. BALF community composition was significantly different than OP (p = 0.0059) and blood (p = 0.0009). CONCLUSIONS: Blood cultures are insensitive but specific for cultured BALF bacteria in canine bacterial pneumonia. Cultivable BALF bacteria were present in BALF, blood and OP microbiota to differing degrees.

Concordance rate in cattle and sheep between genotypes differing in Illumina GenCall quality score.

Proper quality control of data prior to downstream analyses is fundamental to ensure integrity of results; quality control of genomic data is no exception. While many metrics of quality control of genomic data exist, the objective of the present study was to quantify the genotype and allele concordance rate between called single nucleotide polymorphism (SNP) genotypes differing in GenCall (GC) score; the GC score is a confidence measure assigned to each Illumina genotype call. This objective was achieved using Illumina beadchip genotype data from 771 cattle (12 428 767 genotypes in total post-editing) and 80 sheep (1 557 360 SNPs genotypes in total post-editing) each genotyped in duplicate. The called genotype with the lowest associated GC score was compared to the genotype called for the same SNP in the same duplicated animal sample but with a GC score of >0.90 (assumed to represent the true genotype). The mean genotype concordance rate for a GC score of <0.300, 0.300-0.549, and ≥0.550 in the cattle (sheep in parenthesis) was 0.9467 (0.9864), 0.9707 (0.9953), and 0.9994 (0.99997) respectively; the respective allele concordance rate was 0.9730 (0.9930), 0.9849 (0.9976), and 0.9997 (0.99998). Hence, concordance eroded as the GC score of the called genotype reduced, albeit the impact was not dramatic and was not very noticeable until a GC score of <0.55. Moreover, the impact was greater and more consistent in the cattle population than in the sheep population. Furthermore, an impact of GC score on genotype concordance rate existed even for the same SNP GenTrain value; the GenTrain value is a statistical score that depicts the shape of the genotype clusters and the relative distance between the called genotype clusters.

Correlation Between KIT Expression and c-Kit Mutations in 2 Subtypes of Canine Oral Melanocytic Neoplasms.

c-Kit mutations have been reported in 15% to 40% of certain human melanoma subtypes, including those histologically similar to canine oral malignant melanomas. Therapeutic response to tyrosine kinase inhibitors has been demonstrated in those human patients. As canine oral malignant melanomas tend to have a poor prognosis despite aggressive surgical removal, evaluation of KIT expression and identification of c-Kit mutations in canine oral melanocytic neoplasms was performed to determine if there is any indication that tyrosine kinase inhibitor drugs might effectively treat any of these cases. This study evaluated 27 canine oral malignant melanomas and 12 canine histologically well-differentiated oral melanocytic neoplasms for activating c-Kit mutations, determined differences in immunohistochemical expression of KIT and c-Kit mutation status, and determined if KIT expression could predict c-Kit mutation status. Among samples that contained intraepithelial nests of neoplastic melanocytes in the KIT-labeled sections, KIT was expressed within cells in these nests in 22/23 (96%) malignant melanomas and 5/7 histologically well-differentiated neoplasms. KIT was expressed in 10% to 30% of neoplastic melanocytes in the lamina propria in 3/24 (13%) malignant melanomas, but 0/9 (0%) histologically well-differentiated neoplasms. Next-generation sequencing identified 85 variants in c-Kit, including 9 nonsynonymous mutations that resulted in amino acid changes predicted to affect protein function. c-Kit mutations with predicted deleterious protein effects were more common in malignant melanomas (8/27 [30%] vs 1/12 [8%]). There was no apparent relationship between detected c-Kit mutations and KIT expression. These results do not support the use of therapies that target c-Kit.

Investigation of donkey milk bacterial diversity by 16S rDNA high-throughput sequencing on a Cyprus donkey farm.

The interest in milk originating from donkeys is growing worldwide due to its claimed functional and nutritional properties, especially for sensitive population groups, such as infants with cow milk protein allergy. The current study aimed to assess the microbiological quality of donkey milk produced in a donkey farm in Cyprus using culture-based and high-throughput sequencing techniques. The culture-based microbiological analysis showed very low microbial counts, whereas important food-borne pathogens were not detected in any sample. In addition, high-throughput sequencing was applied to characterize the bacterial communities of donkey milk samples. Donkey milk mostly composed of gram-negative Proteobacteria, including Sphingomonas, Pseudomonas, Mesorhizobium, and Acinetobacter; lactic acid bacteria, including Lactobacillus and Streptococcus; the endospores forming Clostridium; and the environmental genera Flavobacterium and Ralstonia, detected in lower relative abundances. The results of the study support existing findings that donkey milk contains mostly gram-negative bacteria. Moreover, it raises questions regarding the contribution of (1) antimicrobial agents (i.e., lysozyme, peptides) in shaping the microbial communities and (2) bacterial microbiota to the functional value of donkey milk.

Perspectives and challenges in validating new diagnostic technologies.

This paper focuses on several new diagnostic technologies, which are set to dominate the testing landscape in the near future and have applications in animal health diagnostics, namely: next-generation sequencing, assays to detect biomarkers, and point-of-care tests. An example of real-time loop-mediated isothermal amplification validation is also provided. Validating these new technologies presents several challenges, which are addressed in this paper.

Les auteurs s'intéressent à plusieurs nouvelles technologies de diagnostic appelées à occuper, dans un futur proche, une place de choix dans le paysage du dépistage et dont il existe déjà des applications en santé animale, à savoir : le séquençage de nouvelle génération, la détection de biomarqueurs et les tests utilisables sur le lieu des soins. Ils décrivent par ailleurs l'exemple de la validation d'une amplification isotherme à médiation par boucle en temps réel. La validation de ces nouvelles technologies présente un certain nombre de difficultés, que les auteurs examinent en détail.

Los autores se centran en varias tecnologías de nuevo cuño que están llamadas a dominar el panorama de las pruebas de diagnóstico en un futuro próximo y que tienen aplicaciones de diagnóstico en sanidad animal, a saber: la secuenciación de próxima generación, los ensayos de detección de marcadores biológicos y las pruebas practicadas en el lugar de consulta. También ofrecen un ejemplo de validación de una técnica de amplificación isotérmica mediada por bucles en tiempo real. La validación de estas nuevas tecnologías presenta varias dificultades, que los autores examinan en estas líneas.

Current ocular microbiome investigations limit reproducibility and reliability: Critical review and opportunities.

Enthusiasm for research describing microbial communities using next-generation sequencing (NGS) has outpaced efforts to standardize methodology. Without consistency in the way research is carried out in this field, the comparison of data between studies is near impossible and the utility of results remains limited. This holds true for bacterial microbiome research of the ocular surface, and other sites, in both humans and animals. In addition, the ocular surface remains under-explored when compared to other mucosal sites. Low bacterial biomass samples from the ocular surface lead to further technical challenges. Taken together, two major problems were identified: (1) Normalization of the workflow in studies utilizing NGS to investigate the ocular surface bacteriome is necessary in order to propel the field forward and improve research impact through cross-study comparisons. (2) Current microbiome profiling technology was developed for high bacterial biomass samples (such as feces or soil), posing a challenge for analyses of samples with low bacterial load such as the ocular surface. This article reviews the challenges and limitations currently facing ocular microbiome research and provides recommendations for minimum reporting standards for veterinary ophthalmologists and clinician scientists to limit inter-study variation, improve reproducibility, and ultimately render results from these studies more impactful. The move toward normalization of methodology will expedite and maximize the potential for microbiome research to translate into meaningful discovery and tangible clinical applications.

High-throughput sequencing reveals crucial miRNAs in skeletal muscle development of Bian chicken.

1. Growth performance is significant for chickens. MicroRNAs (miRNAs) have been found to play important roles in the post-transcriptional regulation of skeletal muscle growth. However, the mechanism of miRNAs in this process has not been elucidated.2. This study involved collecting leg muscle from slow- and fast-growing groups of Bian chicken at 16 weeks of age for high-throughput sequencing. A total of 42 differentially expressed miRNAs (DEMs) were identified. Among them, 22 DEMs were up-regulated and 20 DEMs were down-regulated.3. Biological process terms, relating to growth, were found by GO enrichment for target genes of DEMs and KEGG pathway analysis of target genes. This revealed some significantly enriched pathways closely related to skeletal muscle development, such as the calcium signalling pathway, ECM-receptor interaction, lysine degradation, apoptosis and tight junctions. Network interaction analysis of DEMs and target genes showed that the top fifty hub genes were targeted by thirteen DEMs.4. Four important miRNAs (novel_miR_158, novel_miR_144, novel_miR_291, and miR-205a) as well as some other valuable miRNAs, such as gga-miR-214 and gga-miR-3525 were identified. The qPCR results of five DEMs were highly consistent with that of sequencing between the two groups, which proved the reliability of miRNA-seq.5. The study will help to improve the molecular mechanism of miRNAs in chickens and guide future experiments concerning miRNA function in chicken growth.