Allele‐specific PCR typing and sequencing of the mitochondrial D‐loop region in four layer breeds

This study aimed to investigate the ability of single nucleotide polymorphism (SNP) haplotypes in chicken mtDNA for presumption of the origins of chicken meat. We typed five SNPs of the D‐loop region in mtDNA by allele‐specific PCR (AS‐PCR) in 556 hens, that is 233 White Leghorn (WL), 50 Dekalb‐TX35 (D‐TX), 140 Barred Plymouth Rock (BPR) and 133 Rhode Island Red (RIR) kept in the National Institute of Livestock and Grassland Science (NILGS, Tsukuba, Japan). Five haplotypes were observed among those chickens by AS‐PCR. WL, D‐TX, BPR and RIR displayed three, two, one and four SNP haplotypes, respectively. By a combination of the haplotypes by AS‐PCR and the breeds, these chickens were classified into 10 groups. After the D‐loop was sequenced in two chickens from every group (20 individuals), 15 SNP sites (including one insertion) and eight sequence haplotypes were observed. In conclusion, haplotype variation was observed in and among the layer breeds of the NILGS. This study demonstrates that SNP haplotypes in mtDNA should be appropriate for the presumption of the origins of chicken meat.     

Genomic evaluation using SNP‐ and haplotype‐based genomic relationship matrices in a closed line of Duroc pigs

A simulation analysis and real phenotype analysis were performed to evaluate the impact of three different relationship matrices on heritability estimation and prediction accuracy in a closed‐line breeding of Duroc pigs. The numerator relationship matrix (NRM), single nucleotide polymorphism (SNP)‐based genomic relationship matrix (GRM) (G_S), and haplotype‐based GRM (G_H) were applied in this study. We used PorcineSNP60 genotype array data (38 114 SNPs) of 831 Duroc pigs with four selection traits. In both heritability estimation and prediction accuracy, the accuracy depended on the number of animals with records. For heritability estimation, a large difference in the results among three relationship matrices was not shown, but the trend of the estimated heritabilities between GRMs, that is G_S < G_H, was shown in this population. For the accuracy of prediction values in test animals, the accuracies of prediction values obtained by two GRMs were higher than that by the NRM in this population. The accuracies obtained by GRMs using animals with no records were lower than that by the NRM using animals with their performance records, but were close to that by the NRM using animals with full‐sib testing records.     

Influences of quorum‐quenching probiotic bacteria on the gut microbial community and immune function in weaning pigs

The aim of this study is to investigate the dynamic gut microbial diversity in weaning swine after administering feed supplemented with probiotic bacteria that specifically inhibit the activity of quorum molecules. Initially, the universal quorum molecule autoinducer‐2 (AI‐2) bioassay results indicated that AI‐2 activity was profoundly inhibited in enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the presence of Lactobacillus acidophilus strain 30SC cell extract, although the growth of EHEC was not affected. Based on plate counting results, bacterial community analysis revealed a specific reduction in coliforms compared to the control, whereas the population of lactobacilli increased in weaning swine in in vivo trials. Supplementation with L. acidophilus strain 30SC did not affect the counts of other communities, such as total aerobes and yeast/mold. In addition, PCR‐denaturing gradient gel electrophoresis analysis showed a significant difference in the 16S rRNA gene products after administering L. acidophilus strain 30SC. Selected bands were sequenced, and most of them were identified as uncultured bacterium clones or a Lactobacillus‐ and Bifidobacterium‐specific community. Therefore, our results indicate that quorum‐quenching probiotic bacteria can significantly modulate the gut microbiota of swine and these beneficial effects can contribute to the improvement of performance and health in the gastrointestinal tract of weaning pigs.     

Changes of leukocyte counts and expression of pro‐ and anti‐inflammatory cytokines in peripheral leukocytes in periparturient dairy cows with retained fetal membranes

In dairy cows, retained fetal membranes (RFM) affect reproductive performance. The aim of this study was to examine the leukocyte counts and the gene expression of tumour necrosis factor α (TNFα), interleukin 1β (IL‐1β), IL‐8, and IL‐10 in polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) in cows with (n = 5) or without (n = 5) RFM during the peripartum period. The lymphocyte counts in RFM cows were higher than those in control cows throughout the experiment (p < .05). The expression of IL‐8 in PMNs of control cows was higher (p < .05) compared with that of RFM cows postpartum. In cows with RFM, IL‐1β expression was higher (p < .05) in PMNs at 6 weeks postpartum whereas the expression of IL‐1β was lower (p < .05) in PBMCs at 4 weeks postpartum. The expression of IL‐10 in PBMCs of control cows was higher (p < .05) than that of RFM cows at 2 weeks prepartum and 4 weeks postpartum. Taken together, our data indicate that changes of gene expression of pro‐ and anti‐inflammatory cytokines in RFM cows might be associated with the delayed placental separation and development of uterine inflammation in RFM cows.     

Real‐time RT‐PCR for the detection and quantitative analysis of equine rhinitis viruses

Reasons for performing study: Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. Objectives: To develop a sensitive, rapid, real‐time RT‐PCR (rRT‐PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. Methods: TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5′ UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT‐PCR were verified by virus isolation and ERBV positive samples were verified by rRT‐PCR using a different set of primers. Results: The detection limit of the rRT‐PCR for both viruses was 10–100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co‐circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT‐PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp). Conclusions: The rRT‐PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV. Potential relevance: These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.     

Preparation and evaluation of cefquinome‐loaded gelatin microspheres and the pharmacokinetics in pigs

Cefquinome (CEF) is widely used for veterinary clinical applications because of its broad spectrum and high efficiency. However, frequent administrations are required due to its short elimination half‐life. In this study, cefquinome sulfate gelatin microspheres (CEF‐GMS) were prepared as a sustained‐release formulation using emulsion chemical cross‐linking technique. Physical properties, stability, sustained‐release property in vitro, and pharmacokinetics in pigs were assessed. The morphology of CEF‐GMS showed a good sphericity with porous structure on the surface, and the mean diameter was 8.80 ± 0.78 μm, with 90.60 ± 3.98% of the total in the range of 5–20 μm. There were no significant changes of all estimated indexes in the stability tests. In vitro drug release study showed that the release of CEF from CEF‐GMS was much slower than that from crude CEF in a release medium. Pharmacokinetic characteristics were evaluated following intramuscular administration of CEF‐GMS or Cefquinome sulfate injection (CEF‐Inj) in pigs at a dosage of 4 mg CEF/kg body weight. The plasma drug concentration–time data of CEF‐GMS and CEF‐Inj were both best fitted by two‐compartment models with first‐order absorption, and the elimination half‐life of CEF‐GMS was almost 10 times that of CEF‐Inj. Overall, CEF‐GMS might be used as a sustained‐release formulation of CEF for veterinary clinical applications.     

Estimation of the diagnostic accuracy of the invA-gene-based PCR technique and a bacteriological culture for the detection of Salmonella spp. in caecal content from slaughtered pigs using Bayesian analysis

The goal of this study was to estimate the accuracy of the invA-gene-based polymerase chain reaction (PCR) and a culture technique based on pre-enrichment with buffered peptone water, three selective enrichment media (selenite, tetrathionate and Rappaport-Vassiliadis broths) and four selective, solid media (Xylose-Lysine-Tergitol-4, Salmonella/Shigella, Hekton-Enteric and MacConkey), for the detection of Salmonella organisms from caecal samples from slaughter pigs. For this purpose a latent-class (Bayesian) approach was used. Two hundred and three slaughtered pigs were used after grouping them into two groups of 96 and 107 animals. Sensitivity (Se) was estimated to be 56% (95% probability interval 40, 76) for culture and 91% (81, 97) for PCR. The specificity (Sp) of the PCR was 88% (80, 95) while the Sp of the culture had been considered 100% in the statistical analysis as all culture-positive samples were confirmed by serotyping. PCR Se was not affected by the Salmonella serotypes present in the samples analysed. Accordingly, a minimum of 25.5% of the pigs was estimated to harbour Salmonella organisms in their faeces. It was concluded that bacteriology on caecal samples alone was a poor diagnostic method, and that the PCR method could be considered a cost-effective alternative to culture in Salmonella monitoring programmes. However, given the moderate Sp of this molecular technique, PCR-positive samples should be further confirmed through bacteriology.     

Evaluation of the heat‐killed and dried cell preparation of Enterococcus faecalis against villous atrophy in early‐weaned mice and pigs

Early weaning induces villous atrophy in the small intestine (SI) of piglets. Oral administration of live lactic acid bacteria (LAB) can improve villous shortening. In this study, we evaluated the oral administration of a heat‐killed and dried cell preparation of Enterococcus faecalis (a LAB) strain EC‐12 against villous atrophy in early‐weaned mice (Experiment 1) and pigs (Experiments 2 and 3). Twelve 16‐days‐old mice were divided into two groups in Experiment 1: gavage of EC‐12 (10 mg/kg body weight (BW)/day), or control. On day 21, SI was collected. Eighteen 21‐day‐old pigs were divided into two groups in Experiment 2: gavage of EC‐12 (10 mg/kg BW/day), or control. After 10 days, the villous height of jejunum was measured. Six 21‐day‐old pigs were divided into two groups in Experiment 3: the basal diet supplemented with EC‐12 at 0.05%‐fed group, or the basal diet‐fed group. After 10 days, the villous height of jejunum was measured. The villous heights in SI were significantly higher by EC‐12 administration in all experiments. EC‐12 successfully improved the villous atrophy in the early‐weaned mice and pigs when EC‐12 was administered orally.     

IBV荧光定量PCR检测方法的建立及对感染鸡体内病毒的检测

IBV（infectious bronchitis virus）属于冠状病毒γ群,可以引起鸡的呼吸道疾病,造成严重的损失,现在对IBV诊断方法的研究进行得十分广泛,建立了多种方法。本研究建立了IBV的Real-Time PCR检测方法,最低检测灵敏度为10 copies/μL,可以有效地区别其他禽源病毒,板内差异为0.1%～0.7%,板间差异为0.7%～1.6%,证明该方法具有较高的灵敏性、特异性和稳定性,并对分离到的野毒株ck/HHLJ/HH13进行了病毒感染试验的检测,在鸡体内的气管肺脏肾脏中都具有较高的浓度,其中气管和肾脏中病毒的滴度一直维持很高的水平,至感染后21 d才逐渐消失,病理组织学可见肾脏和气管病变最严重,证实了所分离到的野毒株ck/HHLJ/HH13是具有很强毒力的肾型IBV毒株。     

Sensitive detection of the c‐KIT c.1430G>T mutation by mutant‐specific polymerase chain reaction in feline mast cell tumours

Here, we describe the establishment of mutant‐specific polymerase chain reaction (PCR) for detection of a c‐KIT c.1430G>T mutation in feline mast cell tumours. Several mutations in feline c‐KIT have been identified, with the c.1430G>T mutation accounting for a significant portion of feline mast cell tumour mutations. The c.1430G>T mutation in c‐KIT exon 9 was detected in 15.7% (11 of 70) of samples by mutant‐specific PCR but in only 7.1% (5 of 70) by PCR–restriction fragment length polymorphism (RFLP) in the genomic DNA isolated from 70 formalin‐fixed paraffin‐embedded sections or cells collected by fine needle aspiration. Mutant‐specific PCR showed remarkably higher detection rate than did PCR–RFLP. DNA sequence analysis did not always yield identical results to those of mutant‐specific PCR, suggesting heterogeneity of tumour cells. Mutant‐specific PCR is a valid and efficient screening tool for detection of the c‐KIT c.1430G>T point mutation in feline mast cell tumours compared with PCR–RFLP and sequencing analysis.     

Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)

Adhesion tests are complex, time‐consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.     

The use of COLD‐PCR,DHPLC and GeneScanning for the highly sensitive detection of c‐KIT somatic mutations in canine mast cell tumours

The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different ‘driver’ somatic mutations of c‐KIT, together with the wild‐type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild‐type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50–20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5–1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.     

Identical genotypes of community‐associated MRSA (ST59) and livestock‐associated MRSA (ST9) in humans and pigs in rural China

This study investigated the prevalence of MRSA in samples taken in households, with and without backyard pigs in villages in a rural area of Shandong Province, China. Community‐associated MRSA and livestock‐associated MRSA, belonging to ST59 and ST9, respectively, were identified in both humans and pigs. The genotypic and phenotypic comparison of isolates indicates that bidirectional transmission of MRSA has occurred between humans and pigs in the villages.     

Immunohistochemical study of matrix metalloproteinase‐3 (MMP‐3) at the articular cartilage in osteochondrotic pigs

In order to understand the mechanism of osteochondrosis in the pig, articular cartilage was taken from the distal femoral condyles of Duroc pigs exhibiting leg weakness and then examined immunohistochemically for the localization of matrix metalloproteinases‐3 (MMP‐3), one of the enzymes involved in the resolution of cartilage matrix. The articular cartilage had the typical characteristics of osteochondrotic lesions, such as abnormal calcification, clefts of cartilage, disappearance of proteoglycan, and necrotic chondrocytes. The immunoreaction of MMP‐3 was observed in chondrocytes at the boundary between normal cartilage and proteoglycan‐deficient area. Moreover, chondrocytes expressing MMP‐3 showed normal morphology, but the surrounding cartilage matrix did not stain with toluidine blue, which indicated a lack of glycosaminoglycans. These results suggest that MMP‐3 is highly involved in the appearance and expansion of osteochondrotic lesions.     

Effects of mycoplasmal pneumonia of swine (MPS) lung lesion‐selected Landrace pigs on MPS resistance and immune competence in three‐way crossbred pigs

To clarify the genetic influence of mycoplasmal pneumonia of swine (MPS) lesion‐selected Landrace (La) on MPS resistance and immune characteristics in three‐way crossbred pigs (LaWaDa), the LaWaDa pigs were compared with the non‐selected crossbred (LbWbDb) and purebred (La) pigs. The MPS lesion score in the three lines was as follows: La line < LaWaDa line < LbWbDb line, with significant differences among the lines. The proportions of myeloid cells and T cells were lower and higher, respectively, in the LaWaDa pigs compared with those in the other two lines. Messenger RNA (mRNA) expression of interleukin (IL)‐6, IL‐10, transforming growth factor‐β, and interferon‐γ in peripheral blood was significantly increased after vaccination in the La and LaWaDa lines. IL‐4 mRNA expression in the LaWaDa line was intermediate to the La and LbWbDb lines. Furthermore, principal component analysis for immune traits and MPS lesions was executed to clarify the characteristics of each pig line. These findings suggest that the immune responses in the three pig lines are genetically distinct and that MPS resistance and some immunity characteristics from the La line were transmitted to the three‐way crossbred pigs.     

Differential expression of genes encoding neurotrophic factors and their receptors along the septal‐temporal axis of the rat hippocampus

The hippocampus plays a key role in learning and emotional regulation. The hippocampus’ function varies along its septotemporal axis, with the septal pole being more frequently involved in spatial learning and memory, and the temporal pole playing a greater role in emotional behaviors. In this study, we present findings aimed at checking the expression level of the genes encoding neurotrophins and their receptors, including nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and their receptors (TrkA, TrkB and TrkC) in the hippocampus along the septotemporal axis. Using real‐time PCR, several different expression patterns were observed. Remarkably, the expression of both NT‐3 and TrkA genes in the septal hippocampus was higher than in the middle and temporal hippocampus. Higher expression of NT‐3 and TrkA may implicate active neurogenesis in the dentate gyrus (DG) of the septal hippocampus because more neurogenesis occurs in the septal than the temporal DG of rats. Finally, the results obtained in this study emphasize the importance of choosing the hippocampal portion along its septotemporal axis for any hippocampal molecular and biochemical experimental studies.     

Differential gene expression of Eph‐ephrin A1 and LEPR‐LEP with high or low number of embryos in pigs during implantation

The objective of this study was to ascertain whether mRNA and protein expressions of implantation‐related genes (erythropoietin‐producing hepatocellular receptor–ligand A1, Eph‐ephrin A1 and leptin receptor–leptin, LEPR‐LEP) differed between pigs with high and low number of embryos, and whether these differences in gene expression might affect embryo implantation. Experimental pig groups (n = 24) for high and low number of embryos were prepared by altering the number of eggs ovulated in pre‐pubertal gilts treated with 1.5 × (High) or 1.0 × (Low) PG600 ([400 IU PMSG + 200 IU hCG]/dose, AKZO‐NOBEL). Gilts expressing oestrus were artificially inseminated twice and maintained in breeding and gestation until the reproductive tract was collected on day 22 of pregnancy. At slaughter, the reproductive tracts from each pregnant gilt from each treatment were immediately processed to collect samples for RNA and protein analysis. Within each gilt, three conceptus points were sampled, one from each horn and then a random conceptus within the tract. At each conceptus point, endometrial attachment site, chorion–allantois and embryo were collected and immediately frozen in liquid nitrogen. Number of corpus luteum (CL) (35.4 vs. 12.6) and total embryo number (18.8 vs. 10.2) were greater in the high‐embryo compared to the low‐embryo group, respectively (p < .05). Real‐time qPCR results showed that Eph‐ephrin A1 mRNA expression was less in the high‐embryo (p < .05) compared to the low‐embryo group. In addition, Western blotting analysis indicated that Eph‐ephrin A1 and LEP protein expression at endometrial attachment site in high‐embryo was less (p < .05) compared to low‐embryo group. It was also noted that mRNA expression of Eph‐ephrin A1 and LEPR‐LEP was greater in pregnant than non‐pregnant gilts (p < .05). Moreover, mRNA expression of Eph‐ephrin A1 (p < .05) and LEPR‐LEP was greatest at endometrial attachment site among all three tissues. There was a positive correlation between expressions of Eph‐ephrin A1, LEPR‐LEP and embryo length with the correlation coefficient 0.31–0.59. For Eph‐ephrin A1, the highest correlation coefficient appeared between Eph A1 expression and normal embryo number, between ephrin A1 expression and embryo length. For LEPR‐LEP, the highest correlation coefficient appeared between LEPR‐LEP expression and ovary weight (0.79 for both, p < .05), followed by embryo length and weight. The results of this study suggest that low expression of Eph‐ephrin A1 and LEPR‐LEP is somehow related to increased embryo number during implantation and that endometrial attachment site might be the main target tissue of these gene products. Yet, the increased expression of Eph‐ephrin A1 and LEPR‐LEP appeared associated with increased embryo growth (length and weight) and ovary weight, Eph‐ephrin A1 and LEPR‐LEP might play roles in the regulation of embryo implantation in pigs.     

Effect of fructo‐oligosaccharides on nutrient digestibility and digesta retention time in adult guinea pigs

A previous study suggested that addition of fructo‐oligosaccharides (FOS) to the diet improved nitrogen (N) utilization and decreased acid detergent fiber (ADF) digestibility in guinea pigs. The present study was conducted to clarify the relationship between ADF digestibility and gastrointestinal mean retention time (MRT) in guinea pigs under FOS supplementation. Adult male guinea pigs were fed a commercial diet (50 g/day) with either 5% glucose (glucose group) or 5% FOS (FOS group) for 12 days in individual metabolism cages. Unlike the glucose group, N utilization improved, but ADF digestibility significantly (P < 0.05) decreased in the FOS group. MRT of solid digesta also significantly (P < 0.05) decreased in the FOS group compared with that in the glucose group. We concluded that reduction of MRT of solid digesta containing FOS decreased ADF digestibility in guinea pigs.