Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2

Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.     

An exploratory study of behavioural differences between young pigs susceptible and non-susceptible to stress syndrome

The behaviour of eight stress-susceptible and eight normal juvenile pigs was compared by visual observation to test the possibility for pre-determining, to some degree, the stress susceptibility on behavioural grounds. Data were analysed according to observation time and the physiological status of each animal. During the feeding period (08.00–10.00 h), the general level of activity of all the pigs was higher, as expected; 13 of 20 behaviours were more frequent.From 08.00 to 10.00 h, stress-susceptible animals drank more often and investigated the environment and their peers by naso-ear and naso-anal contacts more frequently than normal animals, while they were seen lying less often. However, frequency of all behaviours was similar from 10.00 to 12.00 h. It is suggested that behavioural recognition of stress-susceptible swine could be a simple, reliable and non-stressful method, although it still remains complementary to physiological detection.     

Mapping of the porcine oestrogen receptor 2 gene and association study with litter size in Iberian pigs

Genes encoding the oestrogen receptors (ESR) are considered candidate genes for prolificacy traits due to the key role these molecules play in the regulation of reproductive physiology. In this paper, we report the assignment of the pig ESR2 gene to porcine chromosome 1 by radiation hybrid mapping. Most of the ESR2 cDNA was sequenced from Iberian pig ovarian RNA samples and one A/G single nucleotide polymorphism (SNP) was found at exon 5, being associated with a Met/Val substitution at position 949. This SNP was genotyped using a PCR-RFLP (Hsp92II) protocol and its potential effect on litter size was evaluated in two Iberian pig populations. However, no statistically significant association between the ESR2 polymorphism and litter size was found.     

Prediction of susceptibility to the porcine stress syndrome

An experiment designed to compare different predictors of porcine stress syndrome (PSS) was conducted. Animals were exposed to the anesthetic gas, halothane, and their reactions monitored to determine susceptibility or resistance to PSS. Two blood creatine phosphokinase (CPK) traits plus their logs10 were examined as predictors of PSS susceptibility. These were Sigma CPK, Antonik CPK, log Sigma CPK, and log Antonik CPK. The accuracy of these predictors varied from 87 percent to 91 percent in agreement with halothane-determined stress susceptibility. In addition, the relationship of PSS and blood types systems (AO,H) were studied. Two blood types, (+,-/-) and (-,+/+), were consistently stress susceptible while three blood types, (+,a/a), (+,a/c), and (+,c/-), were consistently stress resistant. However, one blood type (+,a/-) contained both stress-susceptible and stress-resistant individuals.     

猪esr基因反转录转座子插入多态性及其与大白猪生产性能的关联性分析

雌激素受体(Estrogen receptor,esr)介导雌激素影响相关基因表达,从而调控哺乳动物的生长和繁殖机能.为了探讨esr基因的反转录转座子多态性对猪生长性能的影响,文中应用比较基因组学和生物信息学方法,预测猪esr基因的反转录转座子插入位点,采用PCR方法验证不同品种猪中插入多态性,并将该基因型与大白猪性能...     

Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs

Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions. Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype. Three independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60-day intervals. A total of 315 plaque-cloned viruses were recovered from 21 pigs over the 367-day observation period and compared to the original plaque-cloned virus by virus neutralization assay, monoclonal antibody analysis, and sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. Variants were detected by day 7 postinoculation, and multiple variants were present concurrently in every pig sampled over the observation period. Sequence analysis showed ORFs 1b and 7 to be highly conserved. In contrast, sequencing of ORF 5 disclosed 48 nucleotide variants which corresponded to 22 amino acid variants. Although no epitopic changes were detected under the conditions of this experiment, PRRS virus was shown to evolve continuously in infected pigs, with different genes of the viral genome undergoing various degrees of change.     

Liposome/DNA systems: correlation between association, hydrophobicity and cell viability

Small unilamellar vesicles associated with plasmid DNA showed maximum association efficiency for a cationic mixture of egg phosphatidylcholine (EPC):1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):di-1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP) (16:8:1 molar ratio) [65%], followed by neutral lipids EPC:1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):cholesterol (Chol) (2:2:1 molar ratio) [30%], and a polymerized formulation 1,2-bis(10,12-tricosadiynoyl)sn-glycero-3-phosphocholine (DC8,9PC):DMPE:Chol (2:2:1 molar ratio) [11%]. The hydrophobicity factor (HF) for these formulations followed the trend DC8,9PC:DMPE:CHOL < EPC:DMPE:Chol < EPC:DOPE DOTAP, and DNA association did not alter this trend. Results suggest that the higher the HF value, the more fluid the membrane and the higher the efficiency of DNA association. On the other hand, no differences were observed in cell toxicity with lipids up to 1 mg/ml in VERO cells.     

Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs

Background

Age-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach.

Results

We observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age.

Conclusions

This study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-653) contains supplementary material, which is available to authorized users.     

HFE genotype frequencies in consecutive reference laboratory specimens: comparisons among referral sources and association with initial diagnosis

We quantified HFE genotype frequencies in specimens submitted by physicians grouped by specialty and determined associations of genotypes with initial diagnosis based on phenotyping in patients evaluated at an iron disorders center. Of 526 specimens (519 from Alabama), these "typical" hemochromatosis-associated genotypes were detected: 85 C282Y/C282Y, 50 C282Y/H63D, and 27 H63D/H63D. Respective frequencies of C282Y/C282Y in specimens from an iron disorders center (n = 156), gastroenterologists (n = 147), hematologists/medical oncologists (n = 85), liver transplant surgeons (n = 11), endocrinologists and rheumatologists (n = 9), and "other sources" (n = 7) were greater (p < 0.05) than in population controls. In 44 patients from an iron disorders center initially diagnosed as "presumed hemochromatosis," 27 (61.4%) had C282Y/C282Y, 10 (22.7%) had C282Y/H63D, and 3 (6.8%) had H63D/H63D. C282Y/C282Y was not detected in 48 patients with "abnormality probably not an iron overload disorder." A total of 20.5% of 44 family members of patients had "typical" hemochromatosis-associated HFE genotypes (7.0% controls; p = 0.02). We conclude that most physicians who submitted specimens identify patients by phenotyping who have greater frequencies of "typical" hemochromatosis-associated HFE genotypes than controls, and that HFE mutation testing is useful in detecting hemochromatosis in family members of persons with hemochromatosis or iron overload.     

Oxidative stress in Rett syndrome: Natural history,genotype, and variants

Abstract

Objectives

Rett syndrome (RTT) is an X-linked autism spectrum disorder caused by mutations in the MeCP2 gene in the great majority of cases. Evidence suggests a potential role of oxidative stress (OS) in its pathogenesis. Here, we investigated the potential value of OS markers (non-protein-bound iron (NPBI) and F₂-isoprostanes (F₂-IsoPs)) in explaining natural history, genotype-phenotype correlation, and clinical heterogeneity of RTT, and gauging the response to omega-3 polyunsaturated fatty acids (ω-3 PUFAs).

Methods

RTT patients (n = 113) and healthy controls were assayed for plasma NPBI and F₂-IsoPs, and intraerythrocyte NPBI. Forty-two patients with typical RTT were randomly assigned to ω-3 PUFAs supplementation for 12 months. NPBI was measured by HPLC and F₂-IsoPs using a gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) technique.

Results

F₂-IsoPs were significantly higher in the early stages as compared with the late natural progression of classic RTT. MeCP2 mutations related to more severe phenotypes exhibited higher OS marker levels than those of milder phenotypes. Higher OS markers were observed in typical RTT and early seizure variant as compared with the preserved speech and congenital variants. Significant reduction in OS markers levels and improvement of severity scores were observed after ω-3 PUFAs supplementation.

Discussion

OS is a key modulator of disease expression in RTT.     

Simple and efficient analysis of disease association with missing genotype data

Missing genotype data arise in association studies when the single-nucleotide polymorphisms (SNPs) on the genotyping platform are not assayed successfully, when the SNPs of interest are not on the platform, or when total sequence variation is determined only on a small fraction of individuals. We present a simple and flexible likelihood framework to study SNP-disease associations with such missing genotype data. Our likelihood makes full use of all available data in case-control studies and reference panels (e.g., the HapMap), and it properly accounts for the biased nature of the case-control sampling as well as the uncertainty in inferring unknown variants. The corresponding maximum-likelihood estimators for genetic effects and gene-environment interactions are unbiased and statistically efficient. We developed fast and stable numerical algorithms to calculate the maximum-likelihood estimators and their variances, and we implemented these algorithms in a freely available computer program. Simulation studies demonstrated that the new approach is more powerful than existing methods while providing accurate control of the type I error. An application to a case-control study on rheumatoid arthritis revealed several loci that deserve further investigations.     

Ultrasonographic and radiographic diagnosis of breast nodules in young women]

Mammography as a priority method of diagnosis of breast nodules in young women is of low efficacy as a result of a dense background of the breast as distinct from echography permitting the detection of abnormal lesion against this background. Altogether 126 patients with clinical manifestations of breast nodules were investigated. Apparatus methods used for investigation of 38 of them, revealed but manifestations of fibrocystic mastopathy. Assessment of the efficacy of x-ray and ultrasound diagnostic methods was performed in 88 patients with breast nodules (the patients varied in age from 14 to 40). Radiodiagnostic accuracy in benign tumors was 54.1%, that in malignant tumors--78.1%. Ultrasound investigation proved to be the most informative method for diagnosis of breast nodules in young women, and the use of a proposed algorithm would permit optimization of the diagnostic process.     

MMP-1 promoter genotype and haplotype association with posterior tibial tendinopathy

Purpose

Posterior tibial tendon (PTT) is particularly vulnerable and its insufficiency is recognized as the main cause of adult acquired flatfoot. Some patients have a predisposition without clinically recognized cause, suggesting that individual characteristics play an important role in tendinopathy. The objective of the present study is to investigate the association of − 519 (rs1144393) matrix metalloproteinase-1 (MMP-1) polymorphism and the − 1607 (rs1799750) and − 519 MMP-1 haplotypes and risk of PTT dysfunction.

Methods

The test group included 50 females who presented PTT dysfunction Grade 2 or 3, and who were submitted to surgical treatment, with histopathological examination of the tendon and magnetic resonance image (MRI) confirming tendinopathy, while the control group was 100 asymptomatic women who present intact PTT at MRI. We analyzed functional polymorphisms MMP-1 and their haplotypes using polymerase chain reaction and restriction fragment length analysis.

Results

There was a significant difference in the presence of the different alleles and genotypes between the control group and test group for the MMP-1 gene (p ≤ 0.01). The G allele of the − 519 MMP-1 polymorphism increased susceptibility to degeneration in the PTT tendon and seems to be a genetic risk factor. Global haplotype analysis indicated a significant difference between both groups (p < 0.0001). Haplotypes G–2G and A–2G had statistically significant risk effect on PTT insufficiency. G–2G, p < 0.001; OR = 5.72 (CI, 2.84–11.52) and A–2G p = 0.002, OR = 3.95 (CI, 1.65–9.44).

Conclusion

According to our results, − 519 MMP-1 isolated and − 1607/− 519 MMP-1 haplotypes are associated to tendinopathy in posterior tibial tendon.     

Cytokine responses in porcine respiratory coronavirus-infected pigs treated with corticosteroids as a model for severe acute respiratory syndrome

The effectiveness and potential immunosuppressive effects of anti-inflammatory glucocorticoids in the lungs of severe acute respiratory syndrome (SARS) patients are undefined. We treated porcine respiratory coronavirus (PRCV)-infected conventional pigs with the corticosteroid dexamethasone (DEX) as a model for SARS. Innate and Th1 cytokines in bronchoalveolar lavage (BAL) and serum were elevated in PRCV-infected pigs compared to controls, but were decreased after DEX treatment in the PRCV-infected, DEX-treated (PRCV/DEX) pigs. Although decreased in BAL, Th2 cytokine levels were higher in serum after DEX treatment. Levels of the proinflammatory cytokine interleukin-6 in BAL and serum were decreased in PRCV/DEX pigs early but increased later compared to those in phosphate-buffered saline-treated, PRCV-infected pigs, corresponding to a similar trend for lung lesions. PRCV infection increased T-cell frequencies in BAL, but DEX treatment of PRCV-infected pigs reduced frequencies of T cells; interestingly B and SWC3a(+) (monocytes/macrophages/granulocytes) cell frequencies were increased. DEX reduced numbers of PRCV-stimulated Th1 gamma interferon-secreting cells in spleen, tracheobroncheolar lymph nodes, and blood. Our findings suggest that future glucocorticoid treatment of SARS patients should be reconsidered in the context of potential local immunosuppression of immune responses in lung and systemic Th1 cytokine-biased suppression.