Different intestinal tropism of the G2b Taiwan porcine epidemic diarrhea virus-Pintung 52 strain in conventional 7-day-old piglets.

The group 2b (G2b) porcine epidemic diarrhea virus (PEDV) that emerged in 2013 has since caused devastating diseases and economic loss. The full-length genome of the G2b Taiwan PEDV-Pintung 52 (PEDV-PT) strain and its intestinal tropism by evaluating the pathological changes in the original PEDV-PT infected field piglet and orally inoculation of either 10, 103, or 105 50% tissue culture infective dose/mL (TCID50/mL) of the plaque-purified PEDV-PT-Passage 5 (P5) in 7-day-old conventional piglets were analyzed. Phylogenetic analysis of the full-length genome indicated that the G2b Taiwan PEDV-PT strain was closely related to the North American G2b PEDV strains. Some pathological features of the G2b Taiwan PEDV-PT infection, including the absence of lesions and antigen signal in the crypt epithelial cells of the jejunum and ileum and in the villus enterocytes of the duodenum and colon, were different from those of infections by the North American G2b PEDV strains. This difference in the intestinal tropism of the G2b Taiwan PEDV-PT strain highlights the importance of studying the pathogenicities of different PEDV variants. Moreover, similar distributions of PEDV antigens and lesions in the G2b Taiwan PEDV-PT infected field piglet and its plaque-purified isolate, PEDV-PT-P5, inoculated piglets indicating that the plaque-purified PEDV-PT-P5 viral stock could facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing the G2b PEDV infection.

Note: A method for minimizing oxide formation during elevated temperature nanoindentation.

A standardized method to protect metallic samples and minimize oxide formation during elevated-temperature nanoindentation was adapted to a commercial instrument. Nanoindentation was performed on Al (100), Cu (100), and W (100) single crystals submerged in vacuum oil at 200 °C, while the surface morphology and oxidation was carefully monitored using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The results were compared to room temperature and 200 °C nanoindentation tests performed without oil, in order to evaluate the feasibility of using the oil as a protective medium. Extensive surface characterization demonstrated that this methodology is effective for nanoscale testing.

Magnetically enhanced high-specificity virus detection using bio-activated magnetic nanoparticles with antibodies as labeling markers.

This study describes magnetically driven suppression of cross-reactions among molecules. First, the magnetic nanoparticles are coated with bio-probes and dispersed in liquid. The bio-probes can then bind with homologous or heterologous bio-targets. When alternating-current (ac) magnetic fields are applied, magnetic nanoparticles rotate driven by ac magnetic fields. Thus, the bio-targets bound on the surface of magnetic nanoparticles experience a centrifugal force. The centrifugal force can be manipulated by adjusting the angular frequency of the rotating magnetic nanoparticles. The angular frequency is determined by the applied ac magnetic field frequency. Since the binding force for good binding is much higher than that of poor binding, frequency manipulation is needed for the centrifugal force to be higher than the poor-binding force but lower than the good-binding force. Therefore, poor binding which contributes to cross reactions between molecules can be suppressed efficiently by control of the ac magnetic field frequency.

Immune responses to foot-and-mouth disease virus in pig farms after the 1997 outbreak in Taiwan.

This paper reports on a retrospective study of the antibody responses to structural and non-structural proteins of FMD virus O Taiwan 97 in six pig herds in Taiwan in the year after the 1997 Taiwanese FMD outbreak. All herds were vaccinated against FMD after the outbreak as part of the countrywide control program. Three of the herds had confirmed FMD infections (herds N, O and P) and three herds remained non-infected (herds K, L and M). The serum neutralizing antibody titers and the non-structural protein ELISA (NSP) antibody responses in sows and 1-month-old pigs in the infected herds were higher than in the non-infected herds, but over time a number of positive NSP reactors were detected. From the serological studies and the herd monitoring and investigations it was considered that the FMD NSP positive reactors may not have constituted a true reservoir of FMD virus infection especially in herds where susceptible pigs were no longer present post-exposure or post-vaccination. Pigs vaccinated with an unpurified FMD type O vaccines being used at that time also showed false positive responses for NSP antibodies.

Immune responses of pigs to commercialized emulsion FMD vaccines and live virus challenge.

The immune response to structural and non-structural proteins (NSPs) was studied on sequential serum samples in swine from O/Taiwan/97 FMDV challenge studies, outbreaks and after vaccination. The results showed that pigs vaccinated with a commercial vaccine prior to or after infection maintained high neutralizing antibody titers with gradual decline from peak titers over the duration of this study. However, neutralizing antibody titers in non-vaccinated pigs only reached moderate levels 2-4 weeks post infection and remained low thereafter. For the 3B and 3ABC NSP antibody ELISA responses, there were gradually decreasing levels of NSP antibody over time. In multiple vaccinations, all pigs showed significant increases in neutralizing antibodies after booster vaccination. For the 3B NSP antibody ELISA after vaccination, the mean S/P ratios for pigs vaccinated with all three FMD vaccines were all below the 0.23 cut-off value set by the manufacture, but some sera from individual vaccinated pigs gave results above this cut-off after primary or secondary vaccination. However, with the 3ABC NSP antibody ELISA, all sera from vaccinated pigs had negative results for NSP antibody for all time points.

Comparison of sensitivity and specificity in three commercial foot-and-mouth disease virus non-structural protein ELISA kits with swine sera in Taiwan.

Three commercialized ELISA kits for the detection of antibodies to the non-structural proteins (NSPs) of FMD virus were compared, using sera from uninfected, vaccinated, challenged and naturally infected pigs. The kinetics of the antibody response to NSPs was compared on sequential serum samples in swine from challenge studies and outbreaks. The results showed that ELISA A (UBI) and ELISA B (CEDI) had better sensitivity than that of the 3ABC recombinant protein-based ELISA C (Chekit). The peak for detection of antibodies to NSPs in ELISA C was significantly delayed in sera from natural infection and challenged swine as compared to the ELISA A and B. The sensitivity of the three ELISAs gradually declined during the 6-month post-infection as antibodies to NSP decline. ELISA kits A and B detected NSP antibody in 50% of challenged pigs by the 9-10th-day and 7-8th-day post-challenge, respectively. ELISA B and C had better specificity than ELISA A on sequential serum samples obtained from swine immunized with a type O FMD vaccine commercially available in Taiwan. Antibody to NSPs before vaccination was not detected in swine not exposed to FMD virus, however, antibody to NSPs was found in sera of some pigs after vaccination. All assays had significantly lower specificity when testing sera from repeatedly vaccinated sows and finishers in 1997 that were tested after the 1997 FMD outbreak. However, when testing sera from repeatedly vaccinated sows or finishers in 2003-2004, the specificity for ELISAs A, B and C were significantly better than those in 1997. This effect was less marked for ELISA A. The ELISA B was the best test in terms of the highest sensitivity and specificity and the lowest reactivity with residual NSP in vaccinates.

Release characteristics of microspheres prepared by co-spray drying Actinobacillus pleuropneumoniae antigens and aqueous ethyl-cellulose dispersion.

Using formalin inactivated Actinobacillus pleuropneumoniae antigens and aqueous ethylcellulose dispersions, microspheres of oral vaccines were developed by a co-spray drying process. The present study attempted to determine whether the dosage formulations of microspheres could form enteric matrices. To assess the enteric characteristics, an in vitro dissolution test was performed with the AQ6-AP microspheres; 95% of the A. pleuropneumoniae protein was released within 3 h at pH 7, but there was no release at pH 1.5. The scanning microscopy revealed that the surface structure of AQ6-AP microspheres became porous at neutral pH. The SDS-PAGE analysis showed that the release rate of proteins from the microspheres was pH dependent not only for the AQ6-AP formulation but also when antigens of A. pleuropneumoniae were replaced with porcine serum. The results suggest that the A. pleuropneumoniae antigens were entrapped in the AQ6 microspheres under the acidic conditions. In a mouse model, oral immunization with AQ6-AP microspheres containing A. pleuropneumoniae evoked systemic IgG and mucosal IgA responses against A. pleuropneumoniae antigens. Thus, the present method may further provide an opportunity to develop oral vaccines and mucosal immunity.

Molecular epidemiological studies on foot-and-mouth disease type O Taiwan viruses from the 1997 epidemic.

Sequence diversity was assessed of the complete VP1 gene directly amplified from 49 clinical specimens during an explosive foot-and-mouth disease (FMD) outbreak in Taiwan. Type O Taiwan FMD viruses are genetically highly homogenous, as seen by the minute divergence of 0.2-0.9% revealed in 20 variants. The O/HCP-0314/TW/97 and O/TCP-022/TW/97 viral variants dominated FMD outbreaks and were prevalent in most affected pig-raising areas. Comparison of deduced amino acid sequences around the main neutralizable antigenic sites on the VP1 polypeptide showed no significant antigenic variation. However, the O/CHP-158/TW/97 variant had an alternative critical residue at position 43 in antigenic site 3, which may be due to selective pressure in the field. Two vaccine production strains (O1/Manisa/Turkey/69 and O1/Campos/Brazil/71) probably provide partial heterologous protection of swine against O Taiwan viruses. The type O Taiwan variants clustered in sublineage A1 of four main lineages in the phylogenetic tree. The O/Hong Kong/9/94 and O/1685/Moscow/Russia/95 viruses in sublineage A2 are closely related to the O Taiwan variants. The causative agent for the 1997 epidemic presumably originated from a single common source of type O FMD viruses prevalent in neighboring areas.

Identification of the copper-zinc superoxide dismutase activity in Mycoplasma hyopneumoniae.

Copper-zinc superoxide dismutase (Cu/ZnSOD), a key enzyme in defense against toxic oxygen-free radicals, is widespread in eukaryotes and several species of gram-negative bacteria. The presence of this enzyme in Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary pathogen of mycoplasmal pneumonia in pigs, was examined since the polyclonal antibody against bovine Cu/ZnSOD was dominantly cross-reactive with the M. hyopneumoniae Cu/ZnSOD from whole cellular proteins. In situ activity staining on SDS-PAGE showed that the molecular mass of M. hyopneumoniae Cu/ZnSOD in reducing form was approximately 17kDa. The presence of Cu and Zn ions at the active site of the enzyme was confirmed on the basis of inhibition by KCN and by H(2)O(2). The activity of M. hyopneumoniae Cu/ZnSOD on both SDS- and native-polyacrylamide gels was completely inhibited by 2mM KCN and the gels showed no iron-containing SOD (FeSOD) or manganese-containing SOD (MnSOD) in the crude extracts. The activity of M. hyopneumoniae Cu/ZnSOD in crude extract was 70units/mg protein and was 55% inhibited by 5mM KCN and 56% inactivated by 40mM H(2)O(2). This enzyme was growth-stage dependent and evidenced markedly higher production during the early log phase. Different expression levels of Cu/ZnSOD activity in field isolates were also detected. Taken together, the presence of Cu/ZnSOD in M. hyopneumoniae was identified for the first time.

Complete nucleotide sequence of pig (Sus scrofa) mitochondrial genome and dating evolutionary divergence within Artiodactyla.

The complete nucleotide sequence of the pig (Sus scrofa) mitochondrial genome, containing 16613bp, is presented in this report. The genome is not a specific length because of the presence of the variable numbers of tandem repeats, 5'-CGTGCGTACA in the displacement loop (D-loop). Genes responsible for 12S and 16S rRNAs, 22 tRNAs, and 13 protein-coding regions are found. The genome carries very few intergenic nucleotides with several instances of overlap between protein-coding or tRNA genes, except in the D-loop region. For evaluating the possible evolutionary relationships between Artiodactyla and Cetacea, the nucleotide substitutions and amino acid sequences of 13 protein-coding genes were aligned by pairwise comparisons of the pig, cow, and fin whale. By comparing these sequences, we suggest that there is a closer relationship between the pig and cow than that between either of these species and fin whale. In addition, the accumulation of transversions and gaps in pig 12S and 16S rRNA genes was compared with that in other eutherian species, including cow, fin whale, human, horse, and harbor seal. The results also reveal a close phylogenetic relationship between pig and cow, as compared to fin whale and others. Thus, according to the sequence differences of mitochondrial rRNA genes in eutherian species, the evolutionary separation of pig and cow occurred about 53-60 million years ago.

Characterization and functional analysis of the porcine lactoferrin gene promoter.

Lactoferrin, a ferric binding glycoprotein found in milk, can possibly prevent microbial infection of the mammary gland and gastrointestinal tract. To define the regulation of the porcine lactoferrin gene (pLTF), we cloned its 5'-flanking region from a porcine liver genomic library and analyzed the 5' upstream region of approx. 4kb, two exons, and an intron. The transcription start site was localized by primer extension to residue G, which is 41 nucleotides upstream from the ATG start codon. The pLTF 5'-flanking region possesses several putative cis-acting regulatory elements found in both housekeeping and inducible genes; to define their function, they were inserted into a chloramphenicol acetyltransferase reporter construct. The region up to -156 sufficed for basic promoter activity, whereas the region up to -780 was required for maximal promoter activity in porcine testis cells (STcells), kidney cells (PK15 cells) and human mammary epithelial cells (HBL-100 cells). Detailed analysis of this proximal region by DNase I footprinting and electrophoretic mobility shift assays reveals that the ubiquitous factors SP1, AP2 and the mammary gland-specific factor (MGF) might play significant roles in regulating the transcription of the pLTF gene.

Pneumocephalus and respiratory depression after accidental dural puncture during epidural analgesia--a case report.

A case of pneumocephalus and respiratory depression after dural puncture during lumbar epidural analgesia is reported. The loss of resistance to air technique was employed to identify the epidural space. Severe respiratory depression and stuporous consciousness developed one hour after a bolus of 2 mg morphine was given epidurally at the end of operation. With computerized tomographic brain scanning and continuous observation of clinical course, the neurologic symptoms were thought to be a mixed complication of pneumocephalus and possible intrathecal morphine overdose. We suggest that in order to avoid iatrogenic pneumocephalus by inadvertent dural puncture in the attempt to identify the epidural space the use of the loss of resistance to normal saline technique or the hanging-drop technique is more reliable than the loss of resistance to air technique. A small test dose prior to a full dose is given and should not be omitted to further confirm the proper placement of the epidural catheter during epidural analgesia.

Altered adenosine triphosphatase activities in pigs with naturally occurring hypertrophic cardiomyopathy.

The purpose of this study was to determine whether myocardial adenosine triphosphatase (ATPase) activities were reduced in pigs with naturally occurring hypertrophic cardiomyopathy (HCM). The selection of hearts for the HCM and the normal control groups depended on histological examination. Specific ATPase activity and 5'-nucleotidase activity were measured in left ventricular myocardium obtained from HCM (n = 7) and normal control (n = 7) animals. The histological features of HCM included marked disorientation of muscle cells, thickening of the intramural coronary arterial wall with a narrowed lumen, endocardial fibrosis and myocardial fibrosis. The HCM group showed significant increases in both heart weight (32%) and heart weight to body weight ratio (46%). The total ATPase activity in crude homogenates from the HCM group was significantly decreased by 16%. Azide-sensitive ATPase (mitochondrial ATPase) activity, ouabain-sensitive ATPase (Na+, K+-ATPase) activity, basal Mg(2+)-ATPase activity and Ca(2+)-ATPase activity were all significantly decreased by 18%, 30%, 20% and 50%, respectively. In contrast, no significant decrease was found in the mean values for 5'-nucleotidase activity. These results suggest that myocardial ATPase activities are suppressed in pigs with naturally occurring HCM.

Mouse lipocortin I gene structure and chromosomal assignment: gene duplication and the origins of a gene family.

Using cDNA probes obtained from library screening and anchored polymerase chain reaction, we have isolated and characterized three overlapping mouse genomic clones that contain the mouse lipocortin I (Lipo I) structural gene. Restriction enzyme mapping, Southern blotting, and DNA sequencing were carried out on the cloned genomic DNA. Lipo I spans about 17 kb and is divided into 13 exons encoding a protein of 346 amino acid residues. The promoter region of the gene has a TATA box and a CCAAT box located upstream of the transcription initiation site at -31 and -76 bp, respectively. Analysis of the strain distribution pattern of Lipo 1 in BXD and AKXD collections of recombinant inbred strains establishes that Lipo I is located on chromosome 19 in close proximity to Ea-4. While no striking relationship exists between the exon/intron structure of the gene and the four repeated 70-amino-acid domains of the protein, three of the four 17-amino-acid repeats believed to be responsible for Ca2+/phospholipid binding are encoded by the last codon of one exon and the first 16 codons of the following exon. This pattern supports the gene duplication theory, and the similarity in gene structure between mouse Lipo I and Lipo II suggests they have a recent evolutionary ancestor.

The mouse lymph node homing receptor is identical with the lymphocyte cell surface marker Ly-22: role of the EGF domain in endothelial binding.

The lymph node homing receptor core polypeptide (mLHRc) is composed of a tandem collection of domains: a lectin domain, an epidermal growth factor (EGF) domain, and two repeats common in complement regulatory proteins. Here we demonstrate localization of mLHRc to chromosome 1, the portion syntenic with chromosome 1 in man. This locus is inseparable in mouse strains from the murine lymphocyte cell surface marker Ly-22. The data indicate that Ly-22 is an allelic determinant on the LHR resulting from a single amino acid interchange within the EGF domain. Cross-blocking experiments demonstrate that anti-Ly-22 and MEL-14 recognize independent epitopes and that Ly-22 is distinct from the carbohydrate binding region. Application of anti-Ly-22 in the in vitro binding assay shows inhibition of binding of lymphocytes to high endothelial venules (HEVs). The localization of the Ly-22 epitope in this novel chimeric protein suggests direct participation of the EGF domain in the adhesion of lymphocytes to HEV.