Carine Nezer

A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig

Most traits and disorders have a multifactorial background indicating that they are controlled by environmental factors as well as an unknown number of quantitative trait loci (QTLs). The identification of mutations underlying QTLs is a challenge because each locus explains only a fraction of the phenotypic variation. A paternally expressed QTL affecting muscle growth, fat deposition and size of the heart in pigs maps to the IGF2 (insulin-like growth factor 2) region. Here we show that this QTL is caused by a nucleotide substitution in intron 3 of IGF2. The mutation occurs in an evolutionarily conserved CpG island that is hypomethylated in skeletal muscle. The mutation abrogates in vitro interaction with a nuclear factor, probably a repressor, and pigs inheriting the mutation from their sire have a threefold increase in IGF2 messenger RNA expression in postnatal muscle. Our study establishes a causal relationship between a single-base-pair substitution in a non-coding region and a QTL effect. The result supports the long-held view that regulatory mutations are important for controlling phenotypic variation.

An imprinted QTL with major effect on muscle mass and fat deposition maps to the IGF2 locus in pigs.

An imprinted QTL with major effect on muscle mass and fat deposition maps to the IGF2 locus in pigs

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

Abstract. A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand2-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect.

Results of a whole genome scan targeting QTL for growth and carcass traits in a Piétrain × Large White intercross

We herein report the results of a whole genome scan performed in a Piétrain × Large White intercross counting 525 offspring to map QTL influencing economically important growth and carcass traits. We report experiment-wide significant lod scores (> 4.6 for meatiness and fat deposition on chromosome SSC2, and for average daily gain and carcass length on chromosome SSC7. Additional suggestive lod scores (> 3.3) for fat deposition are reported on chromosomes SSC1, SSC7 and SSC13. A significant dominance deviation was found for the QTL on SSC1, while the hypothesis of an additive QTL could not be rejected for the QTL on SSC7 and SSC13. No evidence for imprinted QTL could be found for QTL other than the one previously reported on SSC2.

Comparative sequence analysis of the INS-IGF2-H19 gene cluster in pigs.

IGF2 is the major candidate gene for a paternally expressed Quantitative Trait Locus (QTL) in the pig primarily affecting muscle development. Here we report two sequence contigs together comprising almost 90 kb containing the INS-IGF2 and H19 genes. A comparative sequence analysis of the pig, human, and mouse genomic sequences was conducted to identify the exon/intron organization, all promoters, and other evolutionarily conserved elements. RT-PCR analysis showed that IGF2 transcripts originated from four different promoters and included various combinations of seven untranslated exons together with three coding exons, in agreement with previous findings in other mammals. The observed sequence similarity in intronic and intragenic regions among the three species is remarkable and is most likely explained by the complicated regulation of imprinting and expression of these genes. The general trend was, as expected, a higher sequence similarity between human and pig than between these species and the mouse, but a few exceptions to this rule were noted. This genomic region exhibits several striking features, including a very high GC content, many CpG islands, and a low amount of interspersed repeats. The high GC and CpG content were more pronounced in the pig than in the two other species. The results will facilitate the further characterization of this important QTL in the pig.

Microbiota characterization of a Belgian protected designation of origin cheese, Herve cheese, using metagenomic analysis.

Herve cheese is a Belgian soft cheese with a washed rind, and is made from raw or pasteurized milk. The specific microbiota of this cheese has never previously been fully explored and the use of raw or pasteurized milk in addition to starters is assumed to affect the microbiota of the rind and the heart. The aim of the study was to analyze the bacterial microbiota of Herve cheese using classical microbiology and a metagenomic approach based on 16S ribosomal DNA pyrosequencing. Using classical microbiology, the total counts of bacteria were comparable for the 11 samples of tested raw and pasteurized milk cheeses, reaching almost 8 log cfu/g. Using the metagenomic approach, 207 different phylotypes were identified. The rind of both the raw and pasteurized milk cheeses was found to be highly diversified. However, 96.3 and 97.9% of the total microbiota of the raw milk and pasteurized cheese rind, respectively, were composed of species present in both types of cheese, such as Corynebacterium casei, Psychrobacter spp., Lactococcus lactis ssp. cremoris, Staphylococcus equorum, Vagococcus salmoninarum, and other species present at levels below 5%. Brevibacterium linens were present at low levels (0.5 and 1.6%, respectively) on the rind of both the raw and the pasteurized milk cheeses, even though this bacterium had been inoculated during the manufacturing process. Interestingly, Psychroflexus casei, also described as giving a red smear to Raclette-type cheese, was identified in small proportions in the composition of the rind of both the raw and pasteurized milk cheeses (0.17 and 0.5%, respectively). In the heart of the cheeses, the common species of bacteria reached more than 99%. The main species identified were Lactococcus lactis ssp. cremoris, Psychrobacter spp., and Staphylococcus equorum ssp. equorum. Interestingly, 93 phylotypes were present only in the raw milk cheeses and 29 only in the pasteurized milk cheeses, showing the high diversity of the microbiota. Corynebacterium casei and Enterococcus faecalis were more prevalent in the raw milk cheeses, whereas Psychrobacter celer was present in the pasteurized milk cheeses. However, this specific microbiota represented a low proportion of the cheese microbiota. This study demonstrated that Herve cheese microbiota is rich and that pasteurized milk cheeses are microbiologically very close to raw milk cheeses, probably due to the similar manufacturing process. The characterization of the microbiota of this particular protected designation of origin cheese was useful in enabling us to gain a better knowledge of the bacteria responsible for the character of this cheese.

Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota.

ABSTRACT In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function.

Psychrotrophic lactic acid bacteria associated with production batch recalls and sporadic cases of early spoilage in Belgium between 2010 and 2014

Between 2010 and 2014 several spoilage cases in Belgium occurring in retail foodstuffs prior to the end of shelf-life have been reported to our laboratory. Overall, seven cases involved strictly psychrotrophic lactic acid bacteria (LAB) contamination in packaged and chilled-stored food products. The products derived either from recalls of entire production batches or as specimens of sporadic spoilage manifestations. Some of these samples were returned to the manufacturing companies by consumers who observed the alterations after purchasing the products. The products covered a wide range of foodstuffs (i.e. meat, dairy, vegetable, egg products and composite food) and denoted different spoilage defects. However, the microbiota determined by means of 16S rRNA gene high-throughput sequencing analysis underpin few LAB genera (i.e. Leuconostoc, Lactobacillus, Weissella and Lactococcus), which are frequently encountered nowadays as specific spoilage organisms (SSO) albeit overlooked by mesophilic enumeration methods due to their strictly psychrotrophic character. The present study confirms the spreading of psychrotrophic LAB in Belgian food processing environments leading to unexpected spoilage, corroborating their spoilage dynamics and prevalence in all kinds of packaged and refrigerated foodstuffs in Northern Europe.

Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments

Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques.

High-throughput sequencing analysis reveals the genetic diversity of different regions of the murine norovirus genome during in vitro replication

In this study, we report the genetic diversity and nucleotide mutation rates of five representative regions of the murine norovirus genome during in vitro passages. The mutation rates were similar in genomic regions encompassing partial coding sequences for non-structural (NS) 1-2, NS5, NS6, NS7 proteins within open reading frame (ORF) 1. In a region encoding a portion of the major capsid protein (VP1) within ORF2 (also including the ORF4 region) and a portion of the minor structural protein (VP2), the mutation rates were estimated to be at least one order of magnitude higher. The VP2 coding region was found to have the highest mutation rate.