Real-time qPCR is a powerful assay to estimate the 171 R/Q alleles at the PrP locus directly in a flock's raw milk: a comparison with the targeted next-generation sequencing.

The hazard to human health represented by transmissible spongiform encephalopathies in sheep is one of the major reasons for implementing the genetic selection plan to break down prion diseases. The problem is particularly important because of the risk of disease transmission from ewe to lamb via milk or colostrum. In order to establish an active and convenient monitoring of the flocks already undergone genetic selection and thus, indirectly increase consumers' security, the challenge of the work was quantifying the classical scrapie risk in bulk milk. A new quantitative real-time PCR assay for the estimation of the 171 R and Q allelic frequencies in a DNA pool representative of all the lactating ewes present in a flock was optimized and validated "in field". The repeatability range was 3.69-5.27 for R and 4.20-5.75 for Q. The ruggedness of the allele frequencies resulted 4.26 for R and 4.77 for Q. Regarding the validation "in field", none of the considered sources of variability (flock, month, number of genotyped animals and somatic cell count) showed a significant effect on flock and milk at the linear model. The targeted next-generation sequencing was also tested to evaluate its applicability in this context. Results show that the real-time PCR assay could represent a valid tool for the determination of 171 R/Q allele frequencies in bulk milk. The implementation of a service for breeder self-control along the production chain would aim to increase the production of high-security dairy products, while monitoring over time of the effects of genetic selection in the flocks.

Updating on the fungal composition in Sardinian sheep's milk by culture-independent methods.

This work applies culture-independent methods for the characterization of fungal populations (yeasts and moulds) naturally occurring in Sardinian ewe's milk sampled in the Italian areas with the largest dairy production (Sardinia and Lazio regions). Sequences of the D1/D2 variable domains at the 5' end of the 26S rRNA gene were obtained by amplification of DNA directly isolated from milk, and this allowed identification of a total of 6 genera and 15 species of fungi. Among the 6 identified genera Geotrichum spp., Candida spp., Phaeosphaeriopsis spp., Pestalotiopsis spp. and Cladosporium spp. belong to the phylum of Ascomycota, while Cryptococcus spp. is part of the phylum of Basidiomycota. In particular, two genera (Pestalotiopsis and Phaeosphaeriopsis) and two species (Plectosphaerella cucumerina and Pryceomyces carsonii) have never been reported in dairy ecosystems before. Results provide evidence that several moulds and yeasts, previously described only in ovine cheeses, are transferred directly from raw milk. The knowledge of fungal consortia inhabiting sheep raw milk is a particularly relevant issue because several species are directly involved in cheese making and ripening, determining the typical aroma. On the other hand, spoilage yeasts and moulds are involved in anomalous fermentation of cheese and may be responsible for considerable economic losses and serious risks for consumers' health.

Detection of Clostridium tyrobutyricum in milk to prevent late blowing in cheese by automated ribosomal intergenic spacer analysis.

Clostridium tyrobutyricum has been identified as the main causal agent of the late blowing defect in cheese, with major effects on quality and commercial value. In this work, for the first time, we applied automated ribosomal intergenic spacer analysis (ARISA) approach to diagnose the presence of C. tyrobutyricum in raw milk before cheesemaking. A species-specific primer set was designed and used for this original application of the ARISA. Sensitivity of detection, reproducibility of the fluorescent PCR assay, and repeatability of the capillary electrophoretic analysis of amplicons were evaluated using DNA extracted from milk added with known amounts of C. tyrobutyricum genome copies, ranging from 3 × 10(6) to 3. Results indicated that the sensitivity of the technique permits to detect the bacterium in all the samples. The reproducibility, evaluated by analyzing 3 sets of serial dilutions, resulted satisfactory, with little deviation within PCR reactions amplifying the same starting amount of template (standard deviations ≤ 0.1, coefficients of variation ≤ 3%). The peaks' fluorescence displayed an evident correspondence with the number of genome copies contained in each dilution. The capillary electrophoretic analysis, tested by running a single PCR product per dilution point in 10 repeats, resulted efficient and highly repeatable, with excellent coefficients of variation ≤ 2% and standard deviations ≤ 0.1 in all the sample sets. This application of ARISA gives good estimates of the total C. tyrobutyricum DNA content allowing a specific, fine-scale resolution of this pollutant species in a complex system as milk. A further advantage linked to the automatization of the process.

Diversity of fungal flora in raw milk from the Italian Alps in relation to pasture altitude.

The present paper explores the diversity of mycobiota inhabiting raw milk sampled at different altitudes (1400 m, 1800 m, 2200 m) from cows grazing Alpine pastures of Valle d'Aosta (North-Western Italian Alps). To this aim, multilocus sequencing was performed at barcodes commonly used for fungal identification (ITS1, D1/D2 domains of the 26S rRNA gene, and part of the ß-tubulin gene). A total of 31 species were detected, most of them yeasts, followed by moulds and by 2 sequences of macroscopic fungi. Several yeasts and moulds were well-characterized inhabitants of the dairy environment, known to positively contribute to cheesemaking. Among these, Candida was the most represented genus with a tendency to cluster at the highest altitudes (6 over 8 observations at ≥ 1800 m), and Kluyveromyces marxianus the most abundant single species, retrieved at all altitudes. The environmental ascomycetous Atrotorquata lineata, never put in relation with food nor described outside North-America, was another species among those most frequently retrieved and was detected in 6 milks at 1400 and 1800 m. The remaining fungi, in general never reported in milk, were mostly environmental. Many of them resulted associated with plants as pathogens or symbionts. Finally, the highest sampled altitude yielded a significant fungal diversity (17 species). This work enlarges the knowledge of fungal consortia inhabiting raw milk and introduces microbial ecology among the altitude-dependent factors, in the composition of Alpine pastures, with the potential of shaping the properties of milks and cheeses, together with the already described physical, chemical and botanical variables.

Differential gene expression in cumulus oocyte complexes collected by ovum pick up from repeat breeder and normally fertile Holstein Friesian heifers.

The aim of this study was to establish whether perturbed gene expression during cumulus oocyte development causes repeat breeding in cattle. In this study, a repeat breeder was defined as a normal estrous cycling animal that did not become pregnant after three inseminations despite the absence of clinically detectable reproductive disorders. Transcripts of genes extracted from cumulus oocyte complexes (COC) that were collected from three repeat breeder and three normally fertile Holstein Friesian heifers were compared. Up to 40 COC were collected from each heifer by means of repeated sessions of ovum pick up in the absence of hormonal stimulation; immediately plunged into liquid nitrogen; and stored at -80°C until analysis. For each heifer, RNA was extracted from the pooled COC and hybridized on GeneChip(®) Bovine Gene Array (Affymetrix). Analysis of gene expression profiles of repeat breeder and control COC showed that 178 genes were differentially expressed (log2 fold change>1.5). Of these genes, 43 (24%) were up-regulated and 135 (76%) were down-regulated in repeat breeder relative to control heifers. This altered pattern of expression occurred in genes involved in several cellular biological processes and cellular components such as metabolism, angiogenesis, substrate/ion transport, regulation/signaling, cell adhesion and cytoskeleton. From these, 13 genes potentially involved in cumulus oocyte growth were subjected to validation by qRT-PCR and nine genes (annexin A1, ANXA1; lactoferrin, LTF; interferon stimulated exonuclease 20kDa, ISG20/HEM45; oxidized low density lipoprotein receptor 1, OLR1; fatty acid desaturase 2, FADS2; glutathione S-transferase A2 and A4, GSTA2 and GSTA4; glutathione peroxidase 1, GPX1; endothelin receptor type A, EDNRA) were confirmed to be differentially expressed. This study identified potential marker genes for fertility in dairy cattle.

Identification of microbiota present on the surface of Taleggio cheese using PCR-DGGE and RAPD-PCR.

UNLABELLED: Microbial DNA from 9 batches of Taleggio PDO cheese sampled at various times during ripening, brines, swabs of wooden shelves used for cheese dry-salting, and 13 commercial cheeses were analyzed by denaturing gradient gel electrophoresis (PCR-DGGE) and/or random amplification of polymorphic DNA (RAPD-PCR). Sequencing allowed the detection of 12 genera, 27 species, and 2 unclassified bacteria. Molecular analysis allowed for the detection of microorganisms not previously associated with Taleggio such as Lactobacillus paracasei, Carnobacterium maltaromaticum, Bacillus licheniformis, Corynebacterium variabile, Psychrobacter cibarius, and Staphylococcus carnosus. For the first time Massilia spp. was detected in a dairy ecosystem. PRACTICAL APPLICATION: Indigenous species and strains of bacteria identified by this study could be used for the selection of dairy cultures to be employed routinely by manufacturers to control the Taleggio cheese production. The new cultures may give the bases for driving dairy processes and, consequently, control the typical flavor resulting from metabolic actions of environmental microorganisms.

An association analysis between OXT genotype and milk yield and flow in Italian Mediterranean river buffalo.

The aim of this study was to evaluate possible associations between three SNPs at the oxytocin locus (AM234538: g.28C>T; g.204A>G and g.1627G>T) and two productive traits, milk yield and milkability, in Italian Mediterranean river buffaloes. Effects of parity, calving season and month of production were also evaluated. A total of 41 980 test-day records belonging to 219 lactations of 163 buffalo cows were investigated. The allele call rate was 98·8% and the major allele frequency for all the investigated loci was 0·76. The OXT genotype was significantly associated with milk yield (P=0·029). The TT genotype showed an average daily milk yield approximately 1·7 kg higher than GT buffaloes. Such a difference represents about 23% more milk/d. A large dominance effect (-1·17±0·43 kg) was estimated, whereas the contribution of OXT genotype (r(2)(OXT)) to the total phenotypic variance in milk yield was equal to 0·06. The TT genotype showed higher values also for the milk flow, even though the estimated difference did not reach a level of statistical significance (P=0·07). Such an association, among the first reported for the oxytocin locus in ruminants, should be tested on a population scale and possible effects on milk composition traits should be evaluated in order to supply useful indications for the application of marker-assisted selection programmes in river buffaloes.

Internal transcribed spacer as a target to assess yeast biodiversity in Italian Taleggio PDO cheese.

UNLABELLED: Three batches of soft smear-ripened Taleggio PDO cheese were made in Northern Italy during the summertime 2010. A total of 129 isolates cultured from cheese surface were examined by using PCR-based methods and sequencing of both the ITS1 region and D1 and D2 domains of the 26S rRNA gene. Sequence analysis of isolates brought to the identification of 6 species: Debaryomyces hansenii, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Pichia guilliermondii, and Torulaspora delbrueckii. Analysis of DNA directly extracted from 45 cheese surfaces permitted to detect 2 additional species Candida sake and Candida etchellsii. D. hansenii was predominant and widespread whereas the other yeast species were detected less frequently. To determine the relationships between yeast community and the environment, 39 isolates from wooden boxes used for dry salting of cheese were analyzed as well. Sequencing of ITS1 region allowed to identify D. hansenii, T. delbrueckii, and K. lactis. ITS1 multiple sequence alignments of D. hansenii detected in wooden boxes showed an in-del polymorphism at position 169. ITS1 secondary structures of yeasts were modeled to explore new applications of this region for molecular identification purposes. PRACTICAL APPLICATION: This study used molecular analysis to identify adventitious yeast population present in the surface of Taleggio smear-ripened cheese. D. hansenii was found predominant in pasteurized milk, in dry salting equipment, and in all cheese samples until the end of ripening.

A DNA array based assay for the characterization of microbial community in raw milk.

An oligonucleotide array based on PCR method containing a combination of probes for taxonomic markers and species-specific virulence genes was developed for the simultaneous identification of 14 Lactic Acid Bacteria (LAB) and food-borne pathogenic bacteria in raw milk. The hypervariable regions V3 and V6 of 16S, together with a variable region of 23S rRNA genes and several genes specific for virulence factors were selected. Universal primers and multiplex PCR were used for the rapid differentiation of bacterial species and low concentration of specific pathogenic and spoilage bacteria were detected in milk. The dominant species such as Streptococcus thermophilus, Enterococcus faecalis, Lactococcus lactis, andLeuconostoc lactis were identified by indirect-labelling reactions based upon incorporation of amino-allyl dUTPs. The results regarding food-borne pathogens detection showed highly specific hybridisation patterns with the genomic DNA from Campylobacter jejuni, Escherichia coli, Salmonella thyphimurium, Listeria monocytogenes and Yersinia enterocolitica. A clear differentiation of dominant species present in a complex microbial community such as raw milk was achieved by the application of short oligonucleotide probes which discriminate sequences differing by few nucleotides.

Identification and quantification of alphaS1, alphaS2, beta, and kappa-caseins in water buffalo milk by reverse phase-high performance liquid chromatography and mass spectrometry.

A method for the simultaneous quantitation of alpha(S1), alpha(S2), beta, and kappa-caseins in water buffalo (Bubalus bubalis) milk using reverse phase high-performance liquid chromatography was developed. The molecular masses of the peaks separated by the described chromatographic protocol were determined by ESI-MS. alpha(S1)- and kappa-caseins were found to be heteromorphic in several individual milk samples. In particular, alpha(S1)-casein showed two peaks with a molecular mass of 23,490 Da and 23,516 Da, and kappa-casein showed three peaks with molecular masses of 19,165 Da, 19,177 Da, and 19,247 Da. Only one form for beta-casein (24,033 Da) and alpha(S2)-casein (22,741 Da) were detected. The mean values of casein fraction concentration observed throughout the individual samples were 8.89 gL(-1) with a relative standard deviation (RSD) of 20% for alpha(S1)-casein, 5.08 gL(-1) with a RSD of 25% for alpha(S2)-casein, 20.91 gL(-1) with a RSD of 16% for beta-casein, and 4.13 gL(-1) with a RSD of 24% for kappa-casein. Linear and second-order polynomial correlations with total nitrogen were calculated for all casein fractions.

Study of microbial diversity in raw milk and fresh curd used for Fontina cheese production by culture-independent methods.

The bacterial populations of raw milk employed for the production of Fontina cheese in alpine farms located in different valleys and altitudes (from 700 to 2246 m above sea level) were investigated by culture independent techniques. Total microbial DNA was isolated from milk and curd samples and used as template in Polymerase Chain Reaction (PCR) to study the hypervariable V3 region of the bacterial 16S rRNA gene and analyzed by Denaturing Gradient Gel Electrophoresis (DGGE). Representative bands of DGGE patterns were sequenced for identification purposes. The use of universal primer for PCR-DGGE allowed the description of the bacterial community, not only for the presence of lactic acid bacteria, but also for other adventitious species. DGGE profiles obtained from milk and fresh curd samples were generally different and typical for each farm, although some recurrent bands were observed. Cluster analysis of DGGE profiles did not show high similarity among samples and it was probably dependent on the different geographical areas of pastures. Some Lactic Acid Bacteria (LAB) recurred in many samples (Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis, Lactococcus lactis, Leuconostoc lactis) indicating that alpine milk is a preferential niche for their colonization. The microbiota included not only mesophilic and thermoresistant LAB but also adventitious bacteria (Macrococcus caseolyticus, Rothia spp.) and psychrotrophic bacteria (Chryseobacterium spp., Pseudomonas spp.), that were found in almost all samples, but disappeared after the warming up at 47-48 degrees C of coagulated milk. Pantoea spp. was primarily found in curds and only with a low incidence in milk samples, indicating the environmental origin. Finally the sequencing data confirmed the presence of E. faecium, E. faecalis and S. thermophilus as major species present in the curd. These species were found also in raw milk, proving its importance as source of the typical fermenting microflora.

Quantification of bovine casein fractions by direct chromatographic analysis of milk. Approaching the application to a real production context.

The ability to quantify the casein content by an exact and cost-effective approach represents an issue of crucial importance in the dairy industry as the natural variations in milk protein concentration can markedly affect the yield of the cheesemaking processes, thus causing a direct and significant economic impact on the producers. In this work, the separation and quantification of alpha(s1)-, alpha(s2)-, kappa- and beta-casein was carried out by direct RP-HPLC analysis of milk. The identification of each casein was established by electrospray ionization mass spectrometry. The data show that this method is able to effectively separate the bovine casein fractions, it provides simplified analytical conditions (with special regard to mobile phase composition and gradient profile) and faster separation while ensuring adequate precision to achieve reliable quantifications in milk samples from dairy production.

High-performance liquid chromatography of governing liquid to detect illegal bovine milk's addition in water buffalo Mozzarella: comparison with results from raw milk and cheese matrix.

A method to detect fraudulent addition of bovine milk in water buffalo Mozzarella cheese by gradient high-performance liquid chromatography (RP-HPLC), relying on the measurement of quantity ratios within beta-lactoglobulin protein family, is described. Analyses were performed on raw milk, cheese matrix and cheese governing liquid using a C4 column and UV detection. This work demonstrated that bovine milk addition during cheesemaking can be detected in governing liquid of Mozzarella down to the EU law limit of 1% as well as in raw milk and cheese matrix. A significant lowering of peaks' areas and heights was observed in cheese matrix and governing liquid samples in comparison with the corresponding milk ones, possibly due to proteins' degradation during the cheesemaking process. The results show that, unlike previous works reported, the use of a matrix-specific calibration curve is essential in order to achieve a proper quantitation of beta-lactoglobulin proteins, thus allowing a reliable estimation of bovine milk addition.

A single nucleotide polymorphism in the sheep kappa-casein coding region.

Genetic polymorphisms in CSN3 gene in Pag (Croatia), Sarda (Italy) and Pramenka (Serbia) sheep breeds were investigated. A single nucleotide polymorphism (SNP) was localized by sequence analysis (sequence submitted to GenBank under accession AY237637) relying on an original primer pair. Primers for sequencing (kappa-casF and kappa-casR) were designed on the available CSN3 sequences to amplify the genomic region encoding the major part of the mature protein (exon 4). An SNP was detected at position 237 of the sheep kappa-casein mRNA (reference sequence: GenBank X51822), where a thymine was substituted for a cytosine. The SNP was typed by conventional PCR and SYBR Green I-based real-time PCR. C and T alleles were discriminated using a dedicated set of primers that consisted of one common forward primer (SNP-TC) and two reverse primers (SNP-T and SNP-C), the latter two differing in the 3' end base and in the presence of a 12 bp poly-G tail in SNP-C. The SNP was found in both the heterozygous and the homozygous state in Sarda and Pramenka breeds, and in the heterozygous state only in the Pag breed. The observed allelic frequencies of the SNP were 0.12 in Pag, 0.27 in Sarda and 0.45 in Pramenka.

Water buffalo (Bubalus bubalis): complete nucleotide mitochondrial genome sequence.

In this work, we report the whole sequence of the water buffalo (Bubalus bubalis) mitochondrial genome. The water buffalo mt molecule is 16.355 base pair length and shows a genome organization similar to those reported for other mitochondrial genome. These new data provide an useful tool for many research area, i.e. evolutionary study and identification of food origin.

The complete coding region sequence of river buffalo (Bubalus bubalis) SRY gene.

The Y-linked SRY gene is responsible for testis determination in mammals. Mutations in this gene can lead to XY Gonadal Dysgenesis, an abnormal sexual phenotype described in humans, cattle, horses and river buffalo. We report here the complete river buffalo SRY sequence in order to enable the genetic diagnosis of this disease. The SRY sequence was also used to confirm the evolutionary divergence time between cattle and river buffalo 10 million years ago.

The complete nucleotide sequence of goat (Capra hircus) mitochondrial genome. Goat mitochondrial genome.

The goat mtDNA sequences reported to date are fragmentary. By using both in silico cloning procedure and conventional molecular biology techniques we have determined the complete nucleotide sequence of the goat (Capra hircus) mitochondrial genome. The length of the sequence was 16.640 bp. Genes responsible for 12S and 16S rRNAs, 22 tRNAs and 13 protein-coding regions are found. The genome organization is conformed to those of other mitochondrial genomes. Comparison between the 13 protein coding genes of goat, cow and sheep reveals that the difference range from 1.2 to 12.2% with a mean of 7.3% between goat and cow and from 0 to 15.6% (mean 4.7%) between goat and sheep.