Brain microabscesses in a porcine model of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> sepsis

Background

Sepsis caused by Staphylococcus aureus often leads to brain microabscesses in humans. Animal models of haematogenous brain abscesses would be useful to study this condition in detail. Recently, we developed a model of S. aureus sepsis in pigs and here we report that brain microabscesses develop in pigs with such induced S. aureus sepsis.Twelve pigs were divided into three groups. Nine pigs received an intravenous inoculation of S. aureus once at time 0 h (group 1) or twice at time 0 h and 12 h (groups 2 and 3). In each group the fourth pig served as control. The pigs were euthanized at time 12 h (Group 1), 24 h (Group 2) and 48 h (Group 3) after the first inoculation. The brains were collected and examined histopathologically.

Results

All inoculated pigs developed sepsis and seven out of nine pigs developed brain microabscesses. The microabscesses contained S. aureus and were located in the prosencephalon and mesencephalon. Chorioditis and meningitis occurred from 12 h after inoculation.

Conclusions

Pigs with experimental S. aureus sepsis often develop brain microabscesses. The porcine brain pathology mirrors the findings in human sepsis patients. We therefore suggest the pig as a useful animal model of the development of brain microabscesses caused by S. aureus sepsis.

     

<Emphasis Type="Italic">Salmonella enterica</Emphasis> subspecies <Emphasis Type="Italic">enterica</Emphasis> serovar Choleraesuis in a German wild boar population: occurrence and characterisation

Background

The swine-adapted serovar Choleraesuis of Salmonella enterica subspecies enterica is found rarely in domestic pigs in Germany. However, a considerable and increasing number of S. Choleraesuis organisms have been isolated from wild boars in Germany in recent years. To investigate a possible epidemiological context, S. Choleraesuis strains from a regional German wild boar population and other hosts were characterised by genotyping methods.

Results

Macrorestriction analysis, biochemical differentiation and antimicrobial susceptibility typing enabled the identification of several clusters of S. Choleraesuis. Some clusters occurred almost permanently in a certain district, whereas other groups circulated among different wild boar herds in larger regions. Non-porcine hosts were infected with the same cluster as the wild boars.

Conclusions

The emergence of S. Choleraesuis in wild boars might be caused by a higher prevalence in the wild boar population, but also the higher awareness to infections with African swine fever may have resulted in a higher number of examined animals. Separation of wild boar populations and, as a result, also the diverse S. Choleraesuis organisms might be due to natural barriers and artificial barriers like arterial roads. The findings of S. Choleraesuis in domestic pigs emphasize the importance of strict biosecurity measures to prevent transmission from wild boars of this but also other pathogens. To avoid risks for humans by zoonotic pathogens regular inspections of meat from wildlife need to be conducted.

     

Association analysis of the glutelin synthesis genes <Emphasis Type="Italic">GluA</Emphasis> and <Emphasis Type="Italic">GluB1</Emphasis> in a <Emphasis Type="Italic">Japonica</Emphasis> rice collection

Glutelin is the most significant seed storage protein and is regarded as an important nutrient quality trait in rice. Research on the genetic basis of the glutelin content distinction in rice will provide more choices for the diets of people with kidney disease and diabetes. The GluA and GluB1 genes play important roles in the process of glutelin synthesis. In this study, 128 Japonica rice accessions with wide geographic distributions were collected to construct the association panel. Among all the 128 accessions, both sequences of the GluA and GluB1 genes were obtained, and nucleotide polymorphisms were detected. A total of 46 SNPs and eight InDels, six SNPs and four InDels were found in the GluA and GluB1 gene sequences, respectively. Eight haplotypes and two haplotypes were classified based on the SNPs in the coding region of the GluA and GluB1 genes, respectively. Moreover, the association of the polymorphic sites in the two genes with glutelin content in the tested population was estimated. The results revealed that five SNPs in the GluA gene, one SNP and one InDel in the GluB1 gene were associated with glutelin content at a significant level (P < 0.01). Corresponding markers were also designed to check the alleles of GluA and GluB1 genes. These results suggested that polymorphisms in the GluA and GluB1 genes in rice could be utilized in molecular marker-assisted selection to improve the nutrient quality of rice breeding programmes.     

Temperature-related activity of <Emphasis Type="Italic">Gomphiocephalus hodgsoni</Emphasis> (Collembola) mitochondrial DNA (COI) haplotypes in Taylor Valley,Antarctica

Gomphiocephalus hodgsoni (Collembola) is the most common and widely distributed arthropod in the Dry Valleys of southern Victoria Land, and is genetically diverse with over 70 mitochondrial cytochrome c oxidase subunit I (COI) haplotypes. There is also considerable physiological variation among G. hodgsoni individuals in their cold tolerance and metabolic activity. Here, we assessed genetic haplotypes of G. hodgsoni relative to the environmental conditions during which individuals were active. We sequenced the COI region of 151 individuals collected in pitfall traps from three sites within Taylor Valley and found 19 unique haplotypes that separated into two distinct lineages (1.6 % divergence), with one lineage comprising 80 % of the sequenced population. During two-hourly sampling, air temperature was the strongest predictor of activity between the two lineages (R ² = 0.56), and when combined with subsurface soil temperature, relative humidity and photosynthetically active radiation, explanatory power increased to R ² = 0.71. With steadily increasing air temperatures predicted for much of Antarctica, it is likely that some haplotypes will have a selective advantage and potentially result in decreased genetic variability within populations. We suggest that temporal monitoring of the relative proportions of COI haplotypes or other appropriate genetic markers may provide a subtle measure of biological responses to environmental changes within Antarctic terrestrial ecosystems.     

Association analysis of the SNP (rs345476947) in the <Emphasis Type="Italic">FUT2</Emphasis> gene with the production and reproductive traits in pigs

The FUT2 gene was considered as an important candidate for pathogenic infections, while the potential associations between this gene and the production and reproductive traits of pigs have not been explored. In this study, we detected the genetic variants of porcine FUT2 gene and analyzed the associations of the polymorphisms with FUT2 mRNA expression and production and reproductive traits (age at 100 kg, backfat thickness at 100 kg, eye muscle thickness, the number of newborn piglets, the number of weaned piglets, and birth weight) in 100 Large White sows. One single nucleotide polymorphism (SNP) (rs345476947, C→T) in the intron of FUT2 and three genotypes (TT, CT and CC) were determined. Association analysis revealed significant associations between this SNP with the number of newborn piglets and weaned piglets. Furthermore, individuals with the TT genotype had significantly higher numbers of newborn piglets and weaned piglets than those with the CC genotype (P?<?0.05). Quantitative PCR analysis showed that FUT2 expression in individuals with CC genotype was significantly higher than those with TT and CT genotypes in the liver and lymph gland (P?<?0.05) and higher than that of CT in the spleen, kidney, and duodenum (P?<?0.05). These findings indicated that the TT genotype may be a favorable genotype for the reproductive traits of pigs. Our study revealed the genetic variants of the FUT2 gene and identified a promising candidate SNP (rs345476947) associated with the reproductive traits, which has the potential to be applied in selective breeding of pigs.     

Novel SNPs of the bovine <Emphasis Type="Italic">NUCB2</Emphasis> gene and their association with growth traits in three native Chinese cattle breeds

In this study, polymorphism in the exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 of bovine NUCB2 gene was detected by PCR-SSCP and DNA sequencing methods in 686 individuals from three Chinese cattle breeds. Two haplotypes (M and N), three observed genotypes (MM, MN and NN) and two SNPs (NC_007313: g. 27451G>A, NC_007313: g. 27472T>C) were detected. The frequencies of haplotypes M and N in inland Chinese three breeds were 0.531–0.721 and 0.279–0.469 respectively. The studied showed that Nanyang, Jiaxian Red and Qinchuan cattle populations were in Hardy–Weinberg equilibrium at SNPs locus of NUCB2 gene (P > 0.05). Polymorphism of the NUCB2 gene was shown to be associated with growth traits in Qingchuan and Nanyang cattle breed. The linkage of two mutant sites in the bovine NUCB2 gene had significant effects on body length, body weight, heart girth, and average daily gain at 24 months (P < 0.05). Results of this study suggested that the NUCB2-gene-specific SNP may be a useful marker for growth traits in future marker-assisted selection programmes in inland Chinese cattle.     

Molecular phylogeography and paleodistribution modeling of the boreal tree species <Emphasis Type="Italic">Ulmus lamellosa</Emphasis> (T.Wang et S. L. Chang) (Ulmaceae) in China

The uplift of mountains and climatic oscillations are important for understanding of the demographic history and genetic structure of species. We investigated the biogeographic history of the boreal tree species Ulmus lamellosa (Ulmaceae) in China, by using a combined phylogeographic and paleodistribution modeling approach. In this study, 14 populations of endangered U. lamellosa were analyzed by using chloroplast DNA (cpDNA) sequences. A high level of genetic differentiation (Φ _ST = 86.22%) among populations with a significant phylogeographic pattern (N _ST > G _ST, P < 0.05) was found in U. lamellosa. Ten haplotypes were detected by combining chloroplast DNA data, and haplotype 3 (H3) was found to be common and widespread. The intraspecific divergence of all U. lamellosa cpDNA haplotypes (9.27 Ma; 95% HPD 5.17–13.33 Ma) most probably began in the late Miocene. The pairwise difference among haplotypes and neutrality tests (Tajima’s D and Fu’s Fs statistic) indicated that populations of U. lamellosa, except group I, have not experienced recent sudden expansions. Multiple refuge areas were identified across the entire distribution ranges of U. lamellosa. The low level of gene flow (Nm = 0.14) among populations may have resulted from isolation resulting from distance and complex topography during climatic oscillations; this isolation was probably the major process that shaped the present distribution of haplotypes. These results support the hypothesis that U. lamellosa persisted in situ during glaciations and occupied multiple localized glacial refugia, contrary to the hypotheses of large-scale range contraction and long-distance southward migration.     

Secreted expression of truncated capsid protein from porcine circovirus type 2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To achieve secreted expression of the truncated capsid protein from porcine circovirus type 2 (PCV2) in Pichia pastoris.

Results

A truncated cap gene (tcap) with a deleted N-terminal nuclear localization signal was optimized and synthesized. Effective secreted expression was achieved in P. pastoris GS115. The high-productive recombinant strain for tCap was grown in a 5 l bioreactor and the productivity of tCap in supernatant reached 250 μg/ml. Furthermore, serum antibody test demonstrated that adjuvant-assisting tCap induced a significant increase of specific PCV2-Cap antibody over time in mice and a similar antibody level in pigs compared with a commercial Cap-based subunit vaccine.

Conclusion

This work establishes a secreted expression strategy in P. pastoris for the production of PCV2 Cap with superior bioactivity, and this strategy might provide potential uses in developing Cap-based subunit vaccine in the future.

     

Plasma matrix metalloproteinase-9 levels, <Emphasis Type="Italic">MMP</Emphasis>-<Emphasis Type="Italic">9</Emphasis> gene haplotypes,and cardiovascular risk in obese subjects

Plasma matrix metalloproteinase (MMP)-9 is a predictor of cardiovascular mortality, and MMP-9 polymorphisms affect plasma MMP-9 levels. However, no study examined whether MMP-9 haplotypes affect MMP-9 levels in obese adults. We examined whether MMP-9 polymorphisms and haplotypes are associated with obesity, and whether they affect MMP-9 levels in obese subjects. We examined the plasma levels of MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in 105 subjects with normal weight (controls), 100 obese subjects, and 156 obese subjects with ≥3 metabolic risk factors (MRFs). We determined genotypes for three polymorphisms: C-1562T (rs3918242), Q279R (A>G, rs17576), and R668Q (G>A, rs17577). MMP-9 levels and activity (MMP-9/TIMP-1 ratio) were higher in obese subjects than in controls (P < 0.05). However, MMP-9 levels were higher in obese subjects with ≥3 MRFs than in obese subjects (P < 0.05). Obese subjects with ≥3 MRFs carrying the GA+AA genotypes for R668Q (G>A) polymorphism had higher MMP-9 levels than subjects carrying the AA genotype (P < 0.05). The “T, G, A” haplotype was more common in both groups of obese subjects than in controls (OR 3.95 and 4.39, respectively; P < 0.01). Notably, obese subjects with ≥3 MRFs carrying the “T, G, A” haplotype had higher MMP-9 levels than subjects carrying the “C, A, G” reference haplotype (P < 0.05). The “T, G, A” haplotype was associated with an increased risk of obesity and affected MMP-9 levels in obese subjects with ≥3 MRFs. Our findings suggest that plasma MMP-9 levels and MMP-9 haplotypes may help to discriminate obese subjects at an increased cardiovascular risk.     

Occurrence and distribution of multiple antibiotic-resistant <Emphasis Type="Italic">Enterococcus</Emphasis> and <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. from Indian poultry: in vivo transferability of their erythromycin,tetracycline and vancomycin resistance

The objective of this study was to determine the occurrence and distribution of antibiotic resistant (AR) lactic acid bacteria (LAB) in Indian poultry. LAB from poultry farm feces (n = 21) and samples from slaughter houses comprising chicken intestine (n = 46), raw meat (n = 23), and sanitary water (n = 4) were evaluated and compared with those from organic chicken (OC) collected from nearby villages. Screening studies showed 5–7 log units higher erythromycin (ER), tetracycline (TC) and vancomycin (VAN) resistant LAB from conventional poultry chicken (CC) compared to OC. Molecular characterization of isolated cultures (n = 32) with repetitive-PCR profiling and 16S rRNA gene sequencing revealed their taxonomical status as Enterococcus faecium (n = 16), Enterococcus durans (n = 2), Lactobacillus plantarum (n = 10), Lactobacillus pentosus (n = 1) and Lactobacillus salivarius (n = 3). The isolates were found to harbor erm(B), msr(C), msr(A/B), tet(M), tet(L) and tet(K) genes associated with Tn916 and Tn917 family transposons. Expression studies through real-time PCR revealed antibiotic-induced expression of the identified AR genes. In vitro and in vivo conjugational studies revealed transfer of ER and TC resistant (ER^R and TC^R) genes with transfer frequencies of 10^?7 and 10^?4 transconjugants recipient^?1, respectively. Although no known VAN resistance (VAN^R) genes were detected, high phenotypic resistance was observed and was transferable to the recipient. From a public health point of view, this study reports Indian poultry as a major source of high levels of AR bacteria contaminating the food chain and the environment. Thus, urgent and determined strategies are needed to control the spread of multiple AR bacteria.     

Involvement of Zn Depletion in Cd-Induced Toxicity on Prenatal Bone Formation in Rat

This experiment was conducted to evaluate the effects of chromium methionine with/without zinc sulfate or zinc amino acid complex on the growth performance, carcass traits, meat quality, serum parameters, endocrine parameters, and antioxidant status of growing-finishing pigs. A total of 180 (32.0 ± 1.7 kg body weight, BW) crossbred pigs (Duroc × Landrace × Yorkshire) were used in a completely randomized design with three dietary treatments and 10 replicates per treatment (five pens of barrows and five pens of gilts with six pigs per replicate). Three treatments were corn-soybean meal-based diets supplemented with 100 mg Zn/kg from zinc sulfate (ZnSO₄), 100 mg Zn/kg from ZnSO₄ + 0.2 mg Cr/kg from chromium methionine complex (CrMet), or 50 mg Zn/kg from ZnSO₄ + 50 mg Zn/kg from zinc amino acid complex (ZnAA) + 0.2 mg Cr/kg from CrMet, respectively. The experiment lasted 105 days, of which was divided into three stages including phase 1 (30 to 50 kg BW), phase 2 (50 to 80 kg BW), and phase 3 (80 to 110 kg BW). Results showed that supplementation with CrMet and ZnAA improved (P < 0.05) the feed conversion of the pigs in phase 2, phase 3, and the overall experiment. Hot carcass weight, dressing percentage, and a longissimus dorsi muscle area were increased (P < 0.05) in pigs fed with diets supplemented with both CrMet and ZnAA compared with pigs fed with diets containing only ZnSO₄ (P < 0.05). There was also an increase (P < 0.01) pH_24 h in the longissimus dorsi muscle in pigs fed with diets supplemented with CrMet and ZnAA. The concentration of serum glucose in pigs fed with diets containing CrMet and ZnAA was decreased (P < 0.05) compared with that in pigs fed with the diet containing ZnSO₄. Supplementation with CrMet and ZnAA increased (P < 0.05) the circulating levels of insulin and decreased (P < 0.05) cortisol. There was an increase (P < 0.05) in total serum antioxidant capacity and Cu/Zn superoxide dismutase activity as well as a decrease (P < 0.05) in serum malondialdehyde concentrations in pigs fed with diets supplemented with CrMet and ZnAA compared with pigs fed with the diet supplemented only with ZnSO₄. In conclusion, supplementation of CrMet only or CrMet together with ZnAA improved feed conversion, carcass traits, and meat quality in the growing-finishing pigs.     

Selection of Potential Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> with Inhibitory Activity Against <Emphasis Type="Italic">Salmonella</Emphasis> and Fecal Coliform Bacteria

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.     

Impact of elevated CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> rooted stem cuttings inoculated with <Emphasis Type="Italic">Frankia</Emphasis>

Impact of different levels of elevated CO ₂ on the activity of Frankia (Nitrogen-fixing actinomycete) in Casuarina equisetifolia rooted stem cuttings has been studied to understand the relationship between C. equisetifolia, Frankia and CO₂. The stem cuttings of C. equietifolia were collected and treated with 2000 ppm of Indole Butyric Acid (IBA) for rooting. Thus vegetative propagated rooted stem cuttings of C. equisetifolia were inoculated with Frankia and placed in the Open top chambers (OTC) with elevated CO₂ facilities. These planting stocks were maintained in the OTC for 12 months under different levels of elevated CO₂ (ambient control, 600 ppm, 900 ppm). After 12 months, the nodule numbers, bio mass, growth, and photosynthesis of C. equisetifolia rooted stem cuttings inoculated with Frankia were improved under 600 ppm of CO₂. The rooted stem cuttings of C. equisetifolia inoculated with Frankia showed a higher number of nodules under 900 ppm of CO₂ and cuttings without Frankia inoculation exhibited poor growth. Tissue Nitrogen (N) content was also higher under 900 ppm of CO₂ than ambient control and 600 ppm levels. The photosynthetic rate was higher (17.8 μ mol CO₂ m^?2 s^?1) in 900 ppm of CO₂ than in 600 ppm (13.2 μ mol CO₂ m^?2 s^?1) and ambient control (8.3 μ mol CO₂ m^?2 s^?1). This study showed that Frankia can improve growth, N fixation and photosynthesis of C. equietifolia rooted stem cuttings under extreme elevated CO₂ level conditions (900 ppm).     

Microsatellite-based genetic diversity analyses of <Emphasis Type="Italic">sugary1</Emphasis>-, <Emphasis Type="Italic">shrunken2</Emphasis>- and double mutant- sweet corn inbreds for their utilization in breeding programme

Sweet corn has recently experienced sharp rise in demand worldwide. Recessive sugary1 (su1) and shrunken2 (sh2) that enhances kernel sweetness have been abundantly used in sweet corn breeding. Analyses of genetic diversity among sweet corn inbreds assume great significance for their effective utilization in hybrid breeding. A set of 48 diverse sweet corn genotypes encompassing su1su1, sh2sh2 and su1su1/sh2sh2 types were analyzed using 56 microsatellite markers. A total of 213 alleles with mean of 3.8 alleles per locus were generated. Two unique- and 12 rare- alleles were identified. The average PIC and genetic dissimilarity was 0.50 and 0.73, respectively. Cluster analysis grouped the inbreds into three major clusters, with each of the su1su1-, sh2sh2- and su1su1/sh2sh2-types were broadly clustered together. Principal coordinate analyses also depicted the diverse origin of the genotypes. The study identified inbreds for synthesis of pools and pedigree populations to develop novel inbreds. The study led to the identification of prospective heterotic combinations in various genetic backgrounds (sh2sh2 × sh2sh2, su1su1 × su1su1, su1su1/sh2sh2 × su1su1/sh2sh2, sh2sh2 × su1su1/sh2sh2 and su1su1 × su1su1/sh2sh2).     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.