Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells

An insight into the Chinese traditional seafood market: Species characterization of cephalopod products by DNA barcoding and phylogenetic analysis using COI and 16SrRNA genes

Squids, cuttlefish and octopus are used for the preparation of traditional products sold on the Chinese market without a specific denomination. In this study DNA barcoding and phylogenetic distance analysis of COI and 16S rRNA genes' fragments were used to characterize the most commonly processed species in dried whole, grilled shredded and salted cephalopod preparations. Ninety-five products (23 sold as cuttlefish, 4 as octopus and 68 as squid) purchased in Chinese local markets were analyzed. Overall, the study identified 10 different species: Sepia pharaonis, S. esculenta, S. recurvirostra, S. lycidas in cuttlefish; Amphioctopus marginatus in octopus; Uroteuthis chinensis, U. edulis, Ommastrephes bartramii, Illex argentinus and Dosidicus gigas in squids. This latter species, characterized by a low commercial value, was found in the majority of the samples (50.5%) and in all the shredded products. By comparing the molecular results with the declared macrocategory (cuttlefish, octopus and squid), two cases of misdescription were pointed out, involving shredded cuttlefish and octopus which were identified as D. gigas. Our results are of particular interest in the light of the scarcity of data regarding the identification of cephalopods on international markets and considering that China is one of the leading cephalopod-producing countries

Low frequency of the scrapile resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed

Frequencies of polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene were studied in 1207 pure-bred and cross-bred Italian Biellese rams, a small ovine breed of about 65 000 head in Italy. Aside from the five most common alleles (VRQ, ARQ, ARR, AHQ and ARH), the rare ARK allele was also found, with the highest frequency reported so far in an ovine breed (2·5 %). ARK/--- genotypes had a total frequency of 4·9 %. The resistance-associated ARR allele was seen at a low frequency (8·3 %). Only 1·4 % of animals examined had a resistant ARR/ARR PrP genotype. Semi-resistant (ARR/ARQ, ARR/ARH and ARR/AHQ) PrP genotypes had a total frequency of 12·6 % and PrP genotypes that are associated with high scrapie susceptibility (e.g. VRQ/VRQ and ARQ/ARQ) had a total frequency of 81·1 %. Statistical analysis comparing PrP allele frequencies between pure-bred and cross-bred animals showed that the ARR allele occurred at a significantly lower frequency in pure-bred rams. Furthermore, comparison of PrP allele frequencies between pure-bred rams over 18 months of age and those below 18 months of age showed a significant decrease in the ARR allele in breeding rams over 18 months of age. Based on these results, breeding for scrapie resistance in the Biellese breed will have to take into account the low frequency of the ARR allele, which also seems to be subject to negative selection by farmers. Further investigation is required to understand whether the ARK allele is also associated with resistance to transmissible spongiform encephalopathie

Extended genetic diversity of bovine viral diarrhea virus and frequency of genotypes and subtypes in cattle in Italy between 1995 and 2013

Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5 \u2032 UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. A unique genetic variant was detected in the majority of herds, but cocirculation of genetic variants was also observed. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction

Bovine viral diarrhea virus 1 (BVDV-1) mutation detection by NGS in virus pairs

BVDV-1 is a RNA virus (Pestivirus). BVDV in persistently infected animals may exist in two biotypes: non-cytopathic (ncp) and cytopathic (cp), with the latter causing fatal mucosal disease. Mutations leading from ncp to cp biotype include nucleotide substitutions or insertions of viral or cellular sequences. Since the viral population is mixed, detection of such mutation from NGS data is a bioinformatic challenge. Here we present a bioinformatic approach to detect such mutations. Supernatants of five isolates were processed as follows: RNA extraction, cDNA and ds-cDNA synthesis, and finally Nextera XT library preparation protocol. Part of the samples were also processed according to NEBNext Ultra RNA protocol. Libraries were sequenced on MiSeq. After adapter trimming, de novo assembly was performed with Trinity, generating contigs, that were characterized by a blast search. BVDV contigs were further blasted against Bos genus sequences, and bovine contigs were blasted against the drafted strain-specific viral genome. Read mapping against the generated contigs (bwa) was used to detect nucleotide substitutions. A full genome sequence has been retrieved for all the 5 samples, plus additional shorter contigs. In one sample, 3 contigs showed a 77-251bp insertion of the bovine ubiquitin C gene. In all the samples, a variable number of SNPs were detected in different gene regions. Most of the SNPs were detected in the NS2/NS3 region. In some cases, BVDV short contigs were composed by reassorted viral sequences. Blast analysis of short contigs led to the identification of probable insertions of both bovine and viral sequences. Furthermore, since BVDV mutation rate is high, using a strain-specific draft genome as a reference in NGS data analysis allows better results, such as SNP identification, since using a different reference increases variability, and thus tangle a correct identification of ncp/cp substitution

Low fraction of the 222K PrP variant in the protease-resistant moiety of PrPres in heterozygous scrapie positive goats

The presence of lysine (K) at codon 222 has been associated with resistance to classical scrapie in goats, but few scrapie cases have been identified in 222Q/K animals. To investigate the contribution of the 222K variant to PrPres formation in natural and experimental Q/K scrapie cases, we applied an immunoblotting method based on the use of two different monoclonal antibodies, F99/97.6.1 and SAF84, chosen for their different affinities to 222K and 222Q PrP variants. Our finding that PrPres seems to be formed nearly totally by the 222Q variant provides evidence that the 222K PrP variant confers resistance to conversion to PrPres formation and reinforces the view that this mutation has a protective role against classical scrapie in goats

Phylogeography, phylodynamics and transmission chains of bovine viral diarrhea virus subtype 1f in Northern Italy

Bovine viral diarrhea virus (BVDV) type 1 in Italy is characterized by high genetic diversity, with at least 20 subtypes. Subtype 1f is endemic in a restricted geographic area, meaning that it has local distribution. We investigated the population dynamics of BVDV-1f in Northern Italy and characterized the transmission chains of a subset of samples from Piedmont and Aosta Valley regions. A total of 51 samples from 1966 to 2013 were considered and 5\u2032 UTR sequences were used for phylogeography. A subset of 12 samples was selected for Npro gene sequencing and further characterization of the transmission chains using both molecular and epidemiological data. Phylogeography estimated the root of BVDV-1f tree in Veneto in 1965. Four significant subclades included sequences clustering by region: Lombardy (n\ua0=\ua03), Lombardy and Emilia-Romagna (n\ua0=\ua07), Piedmont (n\ua0=\ua017), Piedmont and Aosta Valley (n\ua0=\ua021). The Piedmont-only subclade has a ladder-like branching structure, while the Piedmont and Aosta Valley subclade has a nearly complete binary structure. In the subset, the outbreak reconstruction identified one sample from Piedmont as the most probable source of infection for the Aosta Valley cases. An ad hoc questionnaire submitted to public veterinarians revealed connections between sampled and non-sampled farms by means of trades, exhibitions and markets. According to the phylogeography, BVDV-1f moved westward, entering from Veneto, and spreading to Lombardy and Emilia-Romagna in the early 1990s, and finally to Piedmont and Aosta Valley in the first decade of 2000s. Both phylogeographic analyses on the whole dataset and on the selection of Npro dataset pointed out that subtype 1f entered Aosta Valley from Piedmont. The integration of molecular and epidemiological data revealed connections between farms, and such approach should be considered in any control plan. In Aosta Valley, the study showed that BVDV1f can be controlled only monitoring the introduction of cattle from Piedmont region

Detection of a phylogenetically divergent eel virus European X (EVEX) isolate in European eels (Anguilla anguilla) farmed in experimental tanks in Italy

There is very little scientific literature about the impact of Eel virus European X (EVEX) on eels held under artificial reproduction condition, and its effective role in eel decline is uncertain and still unclear. Following a disease outbreak of European eels, farmed in an experimental hatchery in Italy, a sample of the affected animals was analyzed to determine the cause of illness. Virological isolation, PCR assay and molecular characterization revealed the presence of EVEX in the diseased animals. The complete N, P, M and G gene regions were amplified by using novel primers specific for EVEX. The EVEX strain identified in this study was phylogenetically close to the known EVEX isolates; however, this Italian isolate diverges and localizes in a separated branch of this cluster. In addition, some unique amino acid changes in three out of four analyzed viral proteins differentiated the Italian virus from reported EVEX sequences which, although few in number, do cover different geographical areas and a 30-year time span. Molecular data are needed to gain further insight into the genetic variability of EVEX, to better define its phylogenetic and evolutionary relationships and to unravel the existence of different circulating strains. Examination of samples caught in the same area failed to detect the presence of EVEX infection. These results strongly indicate that the presence and risk factors associated with EVEX infection are not well established and further study should be performed in this field, in particular on stress and temperature dependence for disease development

Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks

Susceptibility to scrapie in sheep is influenced by polymorphisms of the prion protein (PrP) gene, whereas no strong association between genetics and scrapie has yet been determined in goats due to the limited number of studies on these animals. In this case¿control study on 177 goats from six Italian scrapie outbreaks, the association between PrP alleles and the occurrence of scrapie was studied. Three silent mutations and 11 PrP polymorphisms were identified, of which two polymorphisms (L133Q and M137I) and one silent mutation (T202T) have not been reported previously. Twelve alleles were determined by cloning. Statistical analysis suggested a possible protective role against scrapie for the glutamine to lysine mutation at codon 22