Evaluation of the causality of the low‐density lipoprotein receptor gene (LDLR) for serum lipids in pigs

A significant quantitative trait locus (QTL) for low‐density lipoprotein cholesterol (LDL‐C) and total cholesterol (TC) was identified around the LDLR gene on chromosome 2 (SSC2) in a White Duroc × Erhualian F₂ resource population and Sutai pigs in our previous study. However, in previous reports, the causality of LDLR with serum lipids is controversial in pigs. To systematically assess the causality of LDLR with serum lipids, association analyses were successively performed in three populations: Sutai pigs, a White Duroc × Erhualian F₂ resource population and a Duroc × (Landrace × Large White) population. We first performed a haplotype‐based association study with 60K SNP genotyping data and evidenced the significant association with LDL‐C and TC around the LDLR gene region. We also found that there is more than one QTL for LDL‐C and TC on SSC2. Then, we evaluated the causalities of two missense mutations, c.1812C>T and c.1520A>G, with LDL‐C and TC. We revealed that the c.1812C>T SNP showed the strongest association with LDL‐C (P = 5.40 × 10^?11) and TC (P = 3.64 × 10^?8) and explained all the QTL effect in Sutai pigs. Haplotype analysis found that two missense SNPs locate within a 1.93‐Mb haplotype block. One major haplotype showed the strongest significant association with LDL‐C (P = 4.62 × 10^?18) and TC (P = 1.06 × 10^?9). However, the c.1812C>T SNP was not identified in the White Duroc × Erhualian intercross, and the association of c.1520A>G with both LDL‐C and TC did not achieve significance in this F₂ population, suggesting population heterogeneity. Both missense mutations were identified in the Duroc × (Landrace × Large White) population and showed significant associations with LDL‐C and TC. Our data give evidence that the LDLR gene should be a candidate causative gene for LDL‐C and TC in pigs, but heterogeneity exists in different populations.     

A genetic comparison of pig, cow and trout isolates of Lactococcus garvieae by PFGE analysis

Aims: Genetic comparison of Lactococcus garvieae isolated from mammals and fish. Methods and Results: One hundred and ninety‐seven L. garvieae isolates obtained from trout (n = 153), cow (n = 7) and pigs (n = 37) were genetically characterized by determining their pulsed‐field gel electrophoresis (PFGE) profiles after macrorestriction with Bsp120I. Overall, L. garvieae isolates from pigs, cow and trout exhibited distinct PFGE patterns, with a low genetic relationship between them. Isolates from trout generated two pulsotypes [Genetic diversity (GD) 0·01] showing that the fish isolates were more genetically homogenous than the others. The L. garvieae isolates from cows displayed five (GD 0·71) different pulsotypes, while the swine isolates displayed 13 different pulsotypes (GD 0·35). Twenty‐one of the 37 swine strains (56·8%) were grouped in a single cluster that included two closely related (93% similarity) pulsotypes. These pulsotypes exhibited a high frequency of isolation from different organs of the animals, and they were also broadly distributed among herds, suggesting a wide distribution across the swine population. This suggests that L. garvieae might be able to colonize different organs of the swine cardio‐respiratory system. Conclusions: Results indicate that most L. garvieae isolates from pigs and trout exhibited a distinct genetic background. Significance and Impact of the Study: The present study describes the isolation of L. garvieae from both diseased and healthy pigs for the first time, and the findings suggest that pigs could be a previously unknown reservoir of this pathogen.     

Effect of climatic conditions on enzootic pneumonia of pigs

Enzootic pneumonia of pigs is a common worldwide problem affecting mainly growing pigs. It is caused byMycoplasma suipneumoniae (M. hyopneumoniae) but the pneumonia is usually complicated byM. hyorhinis and bacteria. The experimental evidence on the effect of temperature, UV light and drying on the survival ofM. suipneumoniae is reviewed and related to the data available onM. pneumoniae M. mycoides subsp.mycoides andM. gallisepticum which cause respiratory disease in man, cattle and chickens respectively. The external and internal climatic conditions which influence the severity of enzootic pneumonia in housed pigs are discussed. Possible further experiments withM. suipneumoniae are discussed in relation to the problem of cultivating one of the most fastidious of all known mycoplasmas.Presented at the Seventh International Biometeorological Congress, 18–25 August 1975, College Park, Maryland, USA.     

A genome‐wide scan for quantitative trait loci affecting respiratory disease and immune capacity in Landrace pigs

Respiratory disease is the most important health concern for the swine industry. Genetic improvement for disease resistance is challenging because of the difficulty in obtaining good phenotypes related with disease resistance; however, identification of genes or markers associated with disease resistance can help in the genetic improvement of pig health. The purpose of our study was to investigate whether quantitative trait loci (QTL) associated with disease resistance were segregated in a purebred population of Landrace pigs that had been selected for meat production traits and mycoplasmal pneumonia of swine (MPS) scores over five generations. We analysed 1395 pigs from the base to the fifth generation of this population. Two respiratory disease traits [MPS scores and atrophic rhinitis (AR) scores] and 11 immune‐capacity traits were measured in 630–1332 animals at 7 weeks of age and when the animal's body weight reached 105 kg. Each of the pigs, except sires in the base population, was genotyped using 109 microsatellite markers, and then, QTL analysis of the full‐sib family population with a multi‐generational pedigree structure was performed. Variance component analysis was used to detect QTL associated with MPS or AR scores, and the logarithm of odds (LOD) score and genotypic heritability of the QTL were estimated. Five significant (LOD > 2.51) and 18 suggestive (LOD > 1.35) QTL for respiratory disease traits and immune‐capacity traits were detected. The significant QTL for Log‐MPS score, located on S. scrofa chromosome 2, could explain 87% of the genetic variance of this score in this analysis. This is the first report of QTL associated with respiratory disease lesions.     

Hydrogen peroxide production and myo‐inositol metabolism as important traits for virulence of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia. In our previous work, we reconstructed the metabolic models of this species along with two other mycoplasmas from the respiratory tract of swine: Mycoplasma hyorhinis, considered less pathogenic but which nonetheless causes disease and Mycoplasma flocculare, a commensal bacterium. We identified metabolic differences that partially explained their different levels of pathogenicity. One important trait was the production of hydrogen peroxide from the glycerol metabolism only in the pathogenic species. Another important feature was a pathway for the metabolism of myo‐inositol in M. hyopneumoniae. Here, we tested these traits to understand their relation to the different levels of pathogenicity, comparing not only the species but also pathogenic and attenuated strains of M. hyopneumoniae. Regarding the myo‐inositol metabolism, we show that only M. hyopneumoniae assimilated this carbohydrate and remained viable when myo‐inositol was the primary energy source. Strikingly, only the two pathogenic strains of M. hyopneumoniae produced hydrogen peroxide in complex medium. We also show that this production was dependent on the presence of glycerol. Although further functional tests are needed, we present in this work two interesting metabolic traits of M. hyopneumoniae that might be directly related to its enhanced virulence.     

Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae

Background  

Mycoplasma hyopneumoniae is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP). Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422.     

In vivo effect of a TLR5 SNP (C1205T) on Salmonella enterica serovar Typhimurium infection in weaned,specific pathogen‐free Landrace piglets

Toll‐like receptor 5 is a pattern‐recognition receptor for bacterial flagellin. We previously reported that a single nucleotide polymorphism (SNP) of swine TLR5, C1205T, impairs recognition of Salmonella typhimurium (ST) flagellin and ethanol‐killed Salmonella Choleraesuis (SC). In the present study, weaned, specific pathogen‐free (SPF) Landrace piglets with CC, CT or TT genotypes were orally infected with ST (L‐3569 strain) to determine the effect of this specific SNP on ST infection in vivo. Eighteen ST‐infected piglets (six each with CC, CT, or TT) exhibited fever and diarrhea for 1 week after infection. TT piglets had the longest duration of fever. TT piglets had the greatest mean diarrhea score during the experimental period, followed by CT and CC piglets. Fecal ST shedding was greater in CT and TT pigs than CC pigs from 2 days after infection. Serum haptoglobin concentration increased in ST‐infected piglets and to greater extents in CT and TT pigs than CC pigs. Daily weight gain was lower in infected pigs, particularly TT piglets, than control pigs. To the best of our knowledge, this study is the first to demonstrate that impairment of TLR recognition affects pig susceptibility to disease in vivo. Thus, piglets with the T allele of swine TLR5 (C1205T) exhibit impaired resistance to ST infection. Furthermore, elimination of the T allele of this SNP from Landrace pigs would lead to enhancement of their resistance to ST infection.

     

Developmental patterns of serum leptin levels, <Emphasis Type="Italic">leptin</Emphasis> gene expression in adipose tissue and <Emphasis Type="Italic">Ob-Rb</Emphasis> gene expression in hypothalamus of Erhualian and Large White pigs

The present study was aimed to investigate the developmental patterns of leptin mRNA expression in dorsal subcutaneous adipose tissue and Ob-Rb mRNA expression in hypothalamus in pigs of different breeds and sexes. Erhualian gilts and boars and Large White boars were sampled at birth, 3, 20, 30, 45, 90, 120 and 180 days of age, respectively. Serum concentration of leptin was measured with RIA and single tube semi-quantitative RT-PCR was applied to determine the relative abundances of mRNA expression using 18S rRNAas an internal standard. The results showed that leptin mRNA expression in adipose tissue increased with age and displayed both sex and breed differences. In Erhualian pigs, females expressed higher leptin mRNA compared with males, and Erhualian boars showed higher abundance of leptin mRNA than Large White boars (P < 0.01). Serum leptin levels were in good agreement with adipose leptin mRNA, displaying similar sex and line differences. In contrast, expression of Ob-Rb mRNA in hypothalamus exhibited a distinctive pattern, decreased gradually after birth, and then increased till weaning. After weaning, Ob-Rb gene expression decreased gradually with age but rose gradually again from 120 to 180 days of age in Erhualian pigs. The expression of Ob-Rb mRNA was higher in Large White pigs than that in Erhualian pigs (P< 0.01). The results suggest that the serum leptin level and leptin gene expression in adipose tissue highly correlate with adiposity.     

Development of a quantitative Real‐Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma hyopneumoniae strain 116 required to induce pneumonia in SPF pigs

Aims: A triplex real‐time PCR assay to quantify Mycoplasma hyopneumoniae in specimens from live and dead pigs was developed and validated. The minimal dose of Myc. hyopneumoniae required to induce pneumonia in specific pathogen‐free pigs was determined. Methods and Results: This TaqMan test simultaneously detected three genes encoding the proteins P46, P97 and P102. All Myc. hyopneumoniae strains analysed were detected, including strains isolated in three countries (France, England and Switzerland) and from several pig farms (n = 33), and the test was specific. The estimated detection thresholds were 1·3 genome equivalents (μl^?1) for the targets defined in p97 and p102 genes and 13 genome equivalents (μl^?1) for the segment defined in the p46 gene. This test was used to quantify Myc. hyopneumoniae in specimens sampled from experimentally infected pigs. In live pigs, c. 10⁷, 10⁸ and 10¹⁰ genome equivalents (ml^?1) of Myc. hyopneumoniae were detected in the nasal cavities, tonsils and trachea samples, respectively. In dead pigs, 10⁸–10¹⁰ genome equivalents (ml^?1) of Myc. hyopneumoniae were detected in the lung tissue with pneumonia. The estimated minimal dose of Myc. hyopneumoniae required to induce pneumonia was 10⁵ colour‐changing units (CCU) per pig (corresponding to 10⁸ mycoplasmas). Conclusion: The triplex RT‐PCR test was validated and can be used for testing samples taken on the pig farms. Significance and Impact of the Study: This test should be a very useful tool in pig herds to control enzootic pneumonia or healthy carrier pigs and to study the dynamics of Myc. hyopneumoniae infections.     

Mapping and fine mapping of quantitative trait loci for the number of vertebrae in a White Duroc × Chinese Erhualian intercross resource population

The number of vertebrae is associated with body size and meat production in pigs. To identify quantitative trait loci (QTL) for the number of vertebrae, phenotypic values were measured in 1029 individuals from a White Duroc × Chinese Erhualian intercross F₂ population. A whole genome scan was performed with 194 microsatellite markers in the F₂ population. Four genome‐wide significant QTL and eight chromosome‐wide significant QTL for the number of vertebrae were identified on pig chromosomes (SSC) 1, 2, 6, 7, 10 and 12. The most significant QTL was detected on SSC7 with a confidence interval of 1 cM, explaining 42.32% of the phenotypic variance in the thoracic vertebral number. The significant QTL on SSC1, 2 and 7 confirmed previous reports. A panel of 276 animals representing seven Western and Chinese breeds was genotyped with 34 microsatellite markers in the SSC7 QTL region. No obvious selective sweep effect was observed in the tested breeds, indicating that intensive selection for enlarged body size in Western commercial breeds did not wipe out the genetic variability in the QTL region. The Q alleles for increased vertebral number originated from both Chinese Erhualian and White Duroc founder animals. A haplotype block of approximately 900 kb was found to be shared by all Q‐bearing chromosomes of F₁ sires except for one distinct Q chromosome. The critical region harbours the newly reported VRTN gene associated with vertebral number. Further investigations are required to confirm whether VRTN or two other positional candidate genes, PROX2 and FOS, cause the QTL effect.     

Trajectories to extinction: spatial dynamics of the contraction of geographical ranges

Aim We examined the range contraction of 309 declining species of animals and plants to determine if the contraction dynamics better matched predictions based on the demographic characteristics of historical populations (demographic hypothesis) or based on the contagion‐like spread of extinction forces (contagion hypothesis). Location Species included in the analysis came from all biogeographic regions. Methods We obtained range maps for 309 species from literature or through personal correspondence with authorities. Hypotheses were contrasted by examining the sequence of changes in the proportion (C) of the remnant range that fell within the central region of the historical range. Monte Carlo simulations and polynomial regressions were employed to examine changes in C during the process of range contraction. Results The results of the Monte Carlo simulations indicated that more species had observed range contractions consistent with the contagion hypothesis than expected by chance (z‐score = 2.922, P = 0.002). The Monte Carlo analysis also indicated that the number of species whose observed range contractions were consistent with the demographic hypothesis was no greater than expected by chance (z‐score = 0.337, P = 0.367). The results of the polynomial regression analysis for the two most common taxonomic groups (mammals and birds) and for all geographical regions (Australia, Africa, Eurasia, and North America) we examined also supported the contagion hypothesis. Main conclusions Most of the examined range contractions are consistent with the contagion hypothesis and that the most likely contagion is human related disturbance. These results have important implications for the conservation of endangered species.     

Three novel quantitative trait loci for skin thickness in swine identified by linkage and genome‐wide association studies

Skin is the largest organ in the pig body and plays a key role in protecting the body against pathogens and excessive water loss. Deciphering the genetic basis of swine skin thickness would enrich our knowledge about the skin. To identify the loci for porcine skin thickness, we first performed a genome scan with 194 microsatellite markers in a White Duroc × Erhualian F₂ intercross. We identified three genome‐wide significant QTL on pig chromosomes (SSC) 4, 7 and 15 using linkage analysis. The most significant QTL was found on SSC7 with a small confidence interval of ~5 cM, explaining 23.9 percent of phenotypic variance. Further, we conducted a genome‐wide association study (GWAS) using Illumina PorcineSNP60 Beadchips for the F₂ pedigree and a population of Chinese Sutai pigs. We confirmed significant QTL in the F₂ pedigree and replicated QTL on SSC15 in Chinese Sutai pigs. A meta‐analysis of GWASs on both populations detected a genomic region associated with skin thickness on SSC4. GWAS results were generally consistent with QTL mapping. Identical‐by‐descent analysis defined QTL on SSC7 in a 683‐kb region harboring an interesting candidate gene: HMGA1. On SSC15, the linkage disequilibrium analysis showed a haplotype block of 2.20 Mb that likely harbors the gene responsible for skin thickness. Our findings provide novel insights into the genetic basis of swine skin thickness, which would benefit further understanding of porcine skin function.     

A novel susceptibility locus in MST1 and gene‐gene interaction network for Crohn's disease in the Chinese population

The incidence of Crohn's disease is increasing in many Asian countries, but considerable differences in genetic susceptibility have been reported between Western and Asian populations. This study aimed to fine‐map 23 previously reported Crohn's disease genes and identify their interactions in the Chinese population by Illumina‐based targeted capture sequencing. Our results showed that the genetic polymorphism A>G at rs144982232 in MST1 showed the most significant association (P = 1.78 × 10^?5; odds ratio = 4.87). JAK2 rs1159782 (T>C) was also strongly associated with Crohn's disease (P = 2.34 × 10^?4; odds ratio = 3.72). Gene‐gene interaction analysis revealed significant interactions between MST1 and other susceptibility genes, including NOD2, MUC19 and ATG16L1 in contributing to Crohn's disease risk. Main genetic associations and gene‐gene interactions were verified using ImmunoChip data set. In conclusion, a novel susceptibility locus in MST1 was identified. Our analysis suggests that MST1 might interact with key susceptibility genes involved in autophagy and bacterial recognition. These findings provide insight into the genetic architecture of Crohn's disease in Chinese and may partially explain the disparity of genetic signals in Crohn's disease susceptibility across different ethnic populations by highlighting the contribution of gene‐gene interactions.     

Efficacy of Influenza Vaccination and Tamiflu? Treatment – Comparative Studies with Eurasian Swine Influenza Viruses in Pigs

Recent epidemiological developments demonstrated that gene segments of swine influenza A viruses can account for antigenic changes as well as reduced drug susceptibility of pandemic influenza A viruses. This raises questions about the efficacy of preventive measures against swine influenza A viruses. Here, the protective effect of vaccination was compared with that of prophylactic Tamiflu® treatment against two Eurasian swine influenza A viruses. 11-week-old pigs were infected by aerosol nebulisation with high doses of influenza virus A/swine/Potsdam/15/1981 (H1N1/1981, heterologous challenge to H1N1 vaccine strain) and A/swine/Bakum/1832/2000 (H1N2/2000, homologous challenge to H1N2 vaccine strain) in two independent trials. In each trial (i) 10 pigs were vaccinated twice with a trivalent vaccine (RESPIPORC® FLU3; 28 and 7 days before infection), (ii) another 10 pigs received 150 mg/day of Tamiflu® for 5 days starting 12 h before infection, and (iii) 12 virus-infected pigs were left unvaccinated and untreated and served as controls. Both viruses replicated efficiently in porcine respiratory organs causing influenza with fever, dyspnoea, and pneumonia. Tamiflu® treatment as well as vaccination prevented clinical signs and significantly reduced virus shedding. Whereas after homologous challenge with H1N2/2000 no infectious virus in lung and hardly any lung inflammation were detected, the virus titre was not and the lung pathology was only partially reduced in H1N1/1981, heterologous challenged pigs. Tamiflu® application did not affect these study parameters.In conclusion, all tested preventive measures provided protection against disease. Vaccination additionally prevented virus replication and histopathological changes in the lung of homologous challenged pigs.     

Genome‐wide association study identifies quantitative trait loci affecting hematological traits in an F2 intercross between Landrace and Korean native pigs

Changes affecting the status of health and robustness can bring about physiological alterations including hematological parameters in swine. To identify quantitative trait loci (QTL) associated with eight hematological traits (one leukocyte trait, six erythrocyte traits and one platelet trait), we conducted a genome‐wide association study using the PorcineSNP60K BeadChip in a resource population derived from an intercross between Landrace and Korean native pigs. A total of 36 740 SNPs from 816 F₂ progeny were analyzed for each blood‐related trait after filtering for quality control. Data were analyzed by the genome‐wide rapid association using mixed model and regression (GRAMMAR) approach. A total of 257 significant SNPs (P < 1.36 × 10^?6) on SSC3, 6, 8, 13 and 17 were identified for blood‐related traits in this study. Interestingly, the genomic region between 17.9 and 130 Mb on SSC8 was found to be significantly associated with red blood cell, mean corpuscular volume and mean corpuscular hemoglobin. Our results include the identification of five significant SNPs within five candidate genes (KIT, IL15, TXK, ARAP2 and ERG) for hematopoiesis. Further validation of these identified SNPs could give valuable information for understanding the variation of hematological traits in pigs.     

Transcriptome analysis of the endometrium from Chinese Erhualian sows that differ in calcium ion concentration and litter size

Embryonic survival rate, an important factor in the fecundity of sows, is affected by endometrium‐secreting histotroph. A higher concentration of calcium ion has been observed in the uterus of highly prolific Erhualian sows (EH) compared with those of less prolific (EL) sows. This suggests that EH sows have better establishment and maintenance of pregnancies, thus increasing embryonic survival rate during the peri‐implantation period. To understand the mechanisms of how the endometrium‐secreting histotroph affects embryonic survival rate during the Erhualian peri‐implantation period, the expression patterns of endometrial mRNA in the EH and EL sows on day 12 of gestation were analyzed using RNA sequencing technology. A total of 164 differentially expressed genes (DEGs) were identified (P_adj < 0.05, |log₂(FC)| ≥ 1), including 46 upregulated and 118 downregulated genes in EH compared to EL. Gene Ontology enrichment indicated that a subset of DEGs was involved in calcium ion binding and cell adhesion. Solute carrier family 8 member A3 and solute carrier family 24 member 4, identified as upregulated genes (P_adj < 0.05) in EH, were considered key candidate genes expressed in the endometrium affecting embryonic survival rate during the peri‐implantation period. The results improve understanding of the genetic mechanism underlying the variation in litter size of Erhualian pigs during the peri‐implantation period.     

Effect of challenge of pigs previously immunised with inactivated vaccines containing homologous and heterologous <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis> strains

Background  

Mycoplasma hyopneumoniae is the primary cause of enzootic pneumonia in pigs. Although vaccination is an important control tool, the results observed under field conditions are variable. This may be due to antigenic differences between the strains circulating in pig herds and the vaccine strain. This study compared the protective efficacy of four bacterins against challenge infection with a highly virulent field strain of M. hyopneumoniae.     

Proteomic comparative analysis of pathogenic strain 232 and avirulent strain J of <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis>

Mycoplasma hyopneumoniae is an important pathogen of pigs causing enzootic pneumonia of swine. The pathogen remains largely enigmatic as far as the host-pathogen interactions are concerned. In the present study, the protein profiles of two strains of M. hyopneumoniae were compared by two-dimensional gel electrophoresis and mass spectrometry. The results indicate that the major adhesin P97, the 50-kDa protein derived from P159 adhesin, and the 43-kDa cleavage product of P102 are expressed at much higher levels in the pathogenic strain 232. In contrast, the avirulent strain J switches its focus to metabolism and expresses more glyceraldehyde 3-phosphate dehydrogenase in gluconeogenesis and lactate dehydrogenase, pyruvate dehydrogenase, and phosphate acetyltransferase in the pyruvate metabolism pathway. We speculate that the avirulent strain may have developed better capabilities to cope with the rich environment during repeated inoculations. Simultaneously, the capability to infect host cells may become less important so that the adhesion-related protein genes are down-regulated. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 2, pp. 264–271.