DNA from African swine fever (ASF) virus was isolated and was characterized by two restriction enzymes, SmaI and EcoRI. Although both enzymes can distinguish Vero cell-adapted ASF isolates by characteristic restriction endonuclease cleavage patterns, all ASF isolates examined exhibited a high degree of similarity, as measured by co-migration of most of the DNA fragments. The molecular weight of ASF DNA, based on size estimates of DNA fragments from cleavage patterns, ranged from 93 x 10(6) to 100 x 10(6). Virus genome heterogeneity was observed in uncloned, cell culture-adapted ASF isolates as well as in a plaque-purified virus after serial passage in Vero cells. In contrast to the rather minor differences in restriction pattern among the Vero cell-adapted isolates, a major alteration in restriction endonuclease cleavage sites was observed during adaptation of the wild-type virus to cell culture.

The incubation of swine peripheral blood mononuclear cells (PBMC) with African swine fever (ASF) virus preparations strongly inhibited the proliferative response of lymphocytes to PHA and other lectins. The inhibition, which persisted after inactivation of the virus by UV radiation, was dependent upon the dose and the time that virus preparations were present in cultures. When virus preparations were fractionated by ultracentrifugation, the inhibitory activity resulted to be soluble, whereas no activity was found in the sedimented viral fraction. However, the preincubation during 4 days of this sedimented fraction with swine PBMC, before the addition of the mitogen, restored the inhibitory activity. The results obtained suggest that the inhibition is mediated by one or more soluble factors released by swine PBMC after coincubation with ASF virus in a time dependent process. These factors show a molecular weight between 40 and 80 kDa by gel filtration chromatography. The inhibitory activity described in the present paper is an indication of inhibition of lymphocyte function produced by ASF virus which can help to understand how this virus escapes from the host immune system.

We describe the use of an in vivo human bronchial xenograft model of cystic fibrosis (CF) and non-CF airways to investigate pathophysiological alterations in airway surface fluid (ASF) volume (Vs) and Cl content. Vs was calculated based on the dilution of an impermeable marker, [3H]inulin, during harvesting of ASF from xenografts with an isosmotic Cl-free solution. These calculations demonstrated that Vs in CF xenographs (28 +/- 3.0 microliter/cm2; n = 17) was significantly less than that of non-CF xenografts (35 +/- 2. 4 microliter/cm2; n = 30). The Cl concentration of ASF ([Cl]s) was determined using a solid-state AgCl electrode and adjusted for dilution during harvesting using the impermeable [3H]inulin marker. Cumulative results demonstrate small but significant elevations (P < 0.045) in [Cl]s in CF (125 +/- 4 mM; n = 27) compared with non-CF (114 +/- 4 mM; n = 48) xenografts. To investigate potential mechanisms by which CF airways may facilitate a higher level of fluid absorption yet retain slightly elevated levels of Cl, we sought to evaluate the capacity of CF and non-CF airways to absorb both 22Na and 36Cl. Two consistent findings were evident from these studies. First, in both CF and non-CF xenografts, 22Na and 36Cl were always absorbed in an equal molar ratio. Second, CF xenografts hyperabsorbed ( approximately 1.5-fold higher) both 22Na and 36Cl compared with non-CF xenografts. These results substantiate previously documented findings of elevated Na absorption in CF airways and also suggest that the slightly elevated [Cl]s found in this study of CF xenograft epithelia does not occur through a mechanism of decreased apical permeability to Cl.

Adenovirus E4orf4 protein induces the death of human cancer cells and Saccharomyces cerevisiae. Binding of E4orf4 to the B/B55/Cdc55 regulatory subunit of protein phosphatase 2A (PP2A) is required, and such binding inhibits PP2A(B55) activity leading to dose-dependent cell death. We found that E4orf4 binds across the putative substrate binding groove predicted from the crystal structure of B55α such that the substrate p107 can no longer interact with PP2A(B55α). We propose that E4orf4 inhibits PP2A(B55) activity by preventing access of substrates and that at high E4orf4 levels this inhibition results in cell death through the failure to dephosphorylate substrates required for cell cycle progression. However, E4orf4 is expressed at much lower and less toxic levels during a normal adenovirus infection. We suggest that in this context E4orf4 largely serves to recruit novel substrates such as ASF/SF2/SRSF1 to PP2A(B55) to enhance adenovirus replication. Thus E4orf4 toxicity probably represents an artifact of overexpression and does not reflect the evolutionary function of this viral product.

We have developed an automated method to assess bone age of children using a digital hand atlas. The hand atlas consists of two components. The first component is a database which is comprised of a collection of 1400 digitized left hand radiographs from evenly distributed normally developed children of Caucasian (CA), Asian (AS), African-American (AA) and Hispanic (HI) origin, male (M) and female (F), ranged from 1- to 18-year-old; and relevant patient demographic data along with pediatric radiologists' readings of each radiograph. This data is separate into eight categories: CAM, CAF, AAM, AAF, HIM, HIF, ASM, and ASF. In addition, CAM, AAM, HIM, and ASM are combined as one male category; and CAF, AAF, HIF, and ASF are combined as one female category. The male and female are further combined as the F & M category. The second component is a computer-assisted diagnosis (CAD) module to assess a child bone age based on the collected data. The CAD method is derived from features extracted from seven regions of interest (ROIs): the carpal bone ROI, and six phanlangeal PROIs. The PROIs are six areas including the distal and middle regions of three middle fingers. These features were used to train the 11 category fuzzy classifiers: one for each race and gender, one for the female, one male, and one F & M, to assess the bone age of a child. The digital hand atlas is being integrated with a PACS for validation of clinical use.

The present study was performed to evaluate the potential for clinical application of digital linear tomosynthesis in imaging hip prostheses. Volumetric x-ray digital linear tomosysnthesis was used to image hip prostheses. The tomosynthesis was compared to metal artifact reduction (MAR) computed tomography (CT), and non-MAR CT scans of a prosthesis case. The effectiveness of this method in enhancing visibility of a prosthesis case was quantified in terms of the signal-to-noise ratio (SNR), and removal of ghosting artifacts in a prosthesis case was quantified in terms of the artifact spread function (ASF). In the near in-focus plane, the contrast is greater in the MAR CT or tomosynthesis relative to the non-MAR CT. The order of ASF performance of the algorithm was as follows: (1) tomosynthesis; (2) MAR-CT; (3) non-MAR CT. The potential usefulness of digital linear tomosynthesis for evaluation of hip prostheses was demonstrated. Further studies are required to determine the ability of digital linear tomosynthesis to quantify the spatial relationships between the metallic components of these devices as well as to identify bony changes with diagnostic consequences.

METHODS

A retrospective study of the effectiveness of Amicar (epsilon aminocaproic acid).

OBJECTIVE

Evaluate the effectiveness of Amicar in decreasing perioperative blood loss and transfusion requirements in same-day anterior (ASF) and posterior spinal fusion (PSF) with segmental spinal instrumentation (SSI) for idiopathic scoliosis.

BACKGROUND

Preliminary prospective, prospective randomized double-blind, and fibrinogen studies have demonstrated Amicar to be effective in decreasing perioperative blood loss in patients with idiopathic scoliosis undergoing PSF with SSI. Increased fibrinogen secretion is a possible explanation.

METHODS

There were 73 consecutive patients divided into 3 study groups based on the administration of Amicar: Group 1 (n = 16), no Amicar; Group 2 (n = 18), Amicar for the PSF with SSI only; and Group 3 (n = 39), Amicar for both ASF and PSF with SSI. All patients were managed using the same general anesthesia technique, intraoperative procedure, postoperative care path, and indications for transfusion (hemoglobin <7 g/dL). Total perioperative blood loss (estimated intraoperative blood loss for both procedures and measured postoperative chest tube and PSF wound suction drainage) and total transfusion requirements between groups were compared using one-way ANOVA.

RESULTS

There were statistically significant decreases in mean estimated intraoperative PSF with SSI, total perioperative blood loss, and transfusion requirements in the 2 Amicar groups. However, Amicar had no significant effect on estimated intraoperative ASF blood loss, chest tube drainage, or PSF wound suction drainage. Total perioperative blood loss and transfusion requirements (cell saver, autologous, directed, and allogeneic blood) were: 3442.8 +/- 1344.0 mL and 1537.1 +/- 905.1 mL in Group 1; 2089.8 +/- 684.0 mL and 485.2 +/- 349.8 mL in Group 2; and 2184.1 +/- 1163.7 mL and 531.5 +/- 510.5 mL in Group 3. There were no Amicar related complications.

CONCLUSIONS

Amicar was highly effective in decreasing total perioperative blood loss and transfusion requirements in same-day ASF and PSF with SSI for idiopathic scoliosis. It results in less preoperative autologous blood donation, perioperative blood transfusion, costs, and potential transfusion-related complications. It was most effective in decreasing intraoperative estimated PSF with SSI blood loss. It had no significant effect during the ASF, postoperative chest tube, or PSF wound suction drainage. We now recommend that it be used for the PSF with SSI procedure only.

BACKGROUND

Human monocytes express two naturally occurring forms of circulating tissue factor (TF) - full-length TF, a membrane-spanning protein, and alternatively spliced TF, a soluble molecule. Presence of the variable exon 5 in TF mRNA determines whether the encoded TF protein is transmembrane, or soluble. Recently, an essential SR protein ASF/SF2 was implicated in TF pre-mRNA processing in human platelets.

OBJECTIVE

To examine molecular mechanisms governing regulated processing of TF pre-mRNA in human monocytic cells.

RESULTS

In silico analysis of the human TF exon 5, present only in full-length TF mRNA, revealed putative binding motifs termed exonic splicing enhancers (ESE) for the SR proteins ASF/SF2 and SRp55, which were found to be abundantly expressed in monocytic cell lines THP-1 and SC, as well as monocyte-enriched peripheral blood mononuclear cells (PBMC). Using a splice competent mini-gene reporter system transiently expressed in monocytic cells, it was determined that weakening of either five closely positioned ASF/SF2 ESE (bases 87-117) or a single conserved SRp55 ESE (base 39) results in severe skipping of exon 5. ASF/SF2 and SRp55 were found to physically associate with the identified ESE.

CONCLUSIONS

SR proteins ASF/SF2 and SRp55 appear to interact with the variable TF exon 5 through ESE at bases 39 and 87-117. Weakening of the above ESE modulates splicing of TF exon 5. This study is the first to identify and experimentally characterize cis-acting splicing elements involved in regulated biosynthesis of human TF.

BACKGROUND

Dandruff is a common scalp condition characterized by flakes, pruritus and sometimes mild erythema. These symptoms reflect underlying histopathologic and biochemical events that must be reversed if treatment is to be effective.

OBJECTIVE

This study aimed to better characterize the state of the epidermis in dandruff and to determine how a defined set of skin surface biomarkers of this state change during a successful course of treatment with a potentiated zinc pyrithione (ZPT) shampoo.

METHODS

A population of dandruff sufferers was treated for 3 weeks with a commercial ZPT shampoo or a non-medicated product, and the effect of treatment on adherent scalp flake (ASF) scores was evaluated. Biopsies were taken from lesional sites at baseline and at the end of the study for histomorphometric and histopathologic analysis. Stratum corneum (SC) samples were likewise obtained for evaluation of biochemical markers of inflammation (IL-1α, IL-1RA, IL-8) and barrier integrity (keratin 1, 10, 11; involucrin; SC lipids; human serum albumin). The biomarker profile was evaluated first by comparison with that in non-dandruff subjects at baseline, and then to determine whether any treatment-induced changes were correlated with reductions in flaking in dandruff sufferers.

RESULTS

Taken together, our studies showed that treatment with the ZPT shampoo led to an improvement in the overall scalp condition as assessed by the resolution of flaking, reduction in epidermal thickness and inflammatory biomarkers, and a dramatic improvement in biomarkers of epidermal barrier integrity.

CONCLUSIONS

The combination of biomarkers examined appears to be a good overall descriptor of the health of the scalp in dandruff, and changes in these biomarkers track with tissue-level events that underlie clinical efficacy in the treatment of dandruff by ZPT shampoo. For the first time, we demonstrate a set of tools that extend beyond flaking scores to provide insight into specific biological changes occurring on the scalp to enable an objective assessment of scalp health.

BACKGROUND

Asthma treatment has broadened from managing clinical markers to incorporate factors that are most meaningful to patients, collectively called health-related quality of life (HQL).

OBJECTIVE

To develop an asthma-specific HQL tool, meeting demands for brevity, usefulness and measurement precision.

METHODS

The 20-item Sydney Asthma Quality of Life Questionnaire (AQLQ) and six additional items were studied using factor analysis, reliability and validity tests among asthma patients 14 and older.

RESULTS

The 15-item Integrated Therapeutics Group Asthma Short Form (ITG-ASF) retains the validity of the AQLQ with improved scaling properties and interpretability. The ITG-ASF yields 6 scores: Symptom-Free Index, Functioning with Asthma, Psychosocial Impact of Asthma, Asthma Energy and Asthma-Confidence in Health and a Total. All items correlated 0.40 or higher with their hypothesized scales and passed discriminant validity tests, with scaling success rates from 75 to 100%. Reliability exceeded the minimum of 0.70 for group comparisons. Ceiling and floor effects were acceptable. Scales were valid in relation to changes in asthma severity and lung function. The best predictor of asthma severity (National Asthma Education and Prevention Program (NAEPP) staging) was the Symptom-Free Index. A Spanish translation is available, Chinese-American is forthcoming. The reading grade level is 4.8.

CONCLUSIONS

Development of the ITG-ASF was a data-driven process maximizing measurement precision and breadth while minimizing burden. The ITG-ASF is a brief, comprehensive and empirically valid tool that complements traditional markers of the outcomes of asthma care.

The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay is presented for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR products. Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis with BsmA I or Ban II, respectively, are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical signs. A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than five hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease.

Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.

BACKGROUND

The pathogenesis of inflammatory bowel disease (IBD) is complex and multifaceted including genetic predisposition, environmental components, microbial dysbiosis, and inappropriate immune activation to microbial components. Pathogenic bacterial provocateurs like adherent and invasive E. coli have been reported to increase susceptibility to Crohn's disease. Serum-derived bovine immunoglobulin/protein isolate (SBI) is comprised primarily of immunoglobulins (Igs) that bind to conserved microbial components and neutralize exotoxins.

OBJECTIVE

To demonstrate that oral administration of SBI may modulate mucosal inflammation following colonization with E. coli, LF82, and exposure to dextran sodium sulfate (DSS).

METHODS

Defined microbiota mice harboring the altered Schaedler flora (ASF) were administered SBI or hydrolyzed collagen twice daily starting 7 days prior to challenge with E. coli LF82 and continuing for the remainder of the experiment. Mice were treated with DSS for 7 days and then evaluated for evidence of local and peripheral inflammation.

RESULTS

Igs within SBI bound multiple antigens from all eight members of the ASF and E. coli LF82 by western blot analysis. Multiple parameters of LF82/DSS-induced colitis were reduced following administration of SBI, including histological lesion scores, secretion of cytokines and chemokines from cecal biopsies, intestinal fatty acid binding protein (I-FABP) and serum amyloid A from plasma.

CONCLUSIONS

Oral administration of SBI attenuated clinical signs of LF82/DSS-induced colitis in mice. The data are consistent with the hypothesis that SBI immunoglobulin binding of bacterial antigens in the intestinal lumen may inhibit the inflammatory cascades that contribute to IBD, thus attenuating DSS-induced colitis.

Several lines of evidence suggest that the auditory evoked potential (AEP) augmenting/reducing slope may serve as a biological marker of central serotonergic activity. According to Hegerl and Juckel (Biol. Psychiatry, 33, 1993, 173), reduced serotonergic activity is hypothesized to increase the slope of the AEP amplitude stimulus intensity function (ASF-slope). Hints for this hypothesis were investigated by employing the acute tryptophan depletion paradigm in 18 healthy females. A within-subject, placebo controlled double-blind cross over design was used for that purpose. Subjects ingested both a 50 g amino-acid drink with (placebo condition) and without tryptophan (depletion condition). With respect to the N1/P2-slope, test-retest reliability of a 1 week interval ranged between r=0.56 and 0.58 for the pre-ingestion baseline recording sessions. Affect was not altered by tryptophan depletion and not related to the ASF-slope. The comparison between placebo and depletion conditions did not reveal significant alterations of the ASF-slope, neither after 5 nor 6 h post-ingestion. Thus, the results do not support the assumption of the ASF-slope reflecting central serotonergic function.

In the absence of data, qualitative risk assessment frameworks have proved useful to assess risks associated with animal health diseases. As part of a scientific opinion for the European Commission (EC) on African Swine Fever (ASF), a working group of the European Food Safety Authority (EFSA) assessed the risk of ASF remaining endemic in Trans Caucasus Countries (TCC) and the Russian Federation (RF) and the risk of ASF becoming endemic in the EU if disease were introduced. The aim was to develop a tool to evaluate how current control or preventive measures mitigate the risk of spread and giving decision makers the means to review how strengthening of surveillance and control measures would mitigate the risk of disease spread. Based on a generic model outlining disease introduction, spread and endemicity in a region, the impact of risk mitigation measures on spread of disease was assessed for specific risk questions. The resulting hierarchical models consisted of key steps containing several sub-steps. For each step of the risk pathways risk estimates were determined by the expert group based on existing data or through expert opinion elicitation. Risk estimates were combined using two different combination matrices, one to combine estimates of independent steps and one to combine conditional probabilities. The qualitative risk assessment indicated a moderate risk that ASF will remain endemic in current affected areas in the TCC and RF and a high risk of spread to currently unaffected areas. If introduced into the EU, ASF is likely to be controlled effectively in the production sector with high or limited biosecurity. In the free range production sector, however, there is a moderate risk of ASF becoming endemic due to wild boar contact, non-compliance with animal movement bans, and difficult access to all individual pigs upon implementation of control measures. This study demonstrated the advantages of a systematic framework to assist an expert panel to carry out a risk assessment as it helped experts to disassociate steps in the risk pathway and to overcome preconceived notions of final risk estimates. The approach presented here shows how a qualitative risk assessment framework can address animal diseases with complexity in their spread and control measures and how transparency of the resulting estimates was achieved.

Understanding the complexity of live pig trade organization is a key factor to predict and control major infectious diseases, such as classical swine fever (CSF) or African swine fever (ASF). Whereas the organization of pig trade has been described in several European countries with indoor commercial production systems, little information is available on this organization in other systems, such as outdoor or small-scale systems. The objective of this study was to describe and compare the spatial and functional organization of live pig trade in different European countries and different production systems. Data on premise characteristics and pig movements between premises were collected during 2011 from Bulgaria, France, Italy, and Spain, which swine industry is representative of most of the production systems in Europe (i.e., commercial vs. small-scale and outdoor vs. indoor). Trade communities were identified in each country using the Walktrap algorithm. Several descriptive and network metrics were generated at country and community levels. Pig trade organization showed heterogeneous spatial and functional organization. Trade communities mostly composed of indoor commercial premises were identified in western France, northern Italy, northern Spain, and north-western Bulgaria. They covered large distances, overlapped in space, demonstrated both scale-free and small-world properties, with a role of trade operators and multipliers as key premises. Trade communities involving outdoor commercial premises were identified in western Spain, south-western and central France. They were more spatially clustered, demonstrated scale-free properties, with multipliers as key premises. Small-scale communities involved the majority of premises in Bulgaria and in central and Southern Italy. They were spatially clustered and had scale-free properties, with key premises usually being commercial production premises. These results indicate that a disease might spread very differently according to the production system and that key premises could be targeted to more cost-effectively control diseases. This study provides useful epidemiological information and parameters that could be used to design risk-based surveillance strategies or to more accurately model the risk of introduction or spread of devastating swine diseases, such as ASF, CSF, or foot-and-mouth disease.

BACKGROUND

This study aimed to characterize the glycophenotype of osteoarthritic cartilage and human chondrocytes.

METHODS

Articular knee cartilage was obtained from nine osteoarthritis (OA) patients. mRNA levels for 27 glycosyltransferases were analyzed in OA chondrocytes using RT-qPCR. Additionally, N- and O-glycans were quantified using mass-spectrometry. Histologically, two cartilage areas with Mankin scores (MS) either ≤ 4 or ≥ 9 were selected from each patient representing areas of mild and severe OA, respectively. Tissue sections were stained with (1) a selected panel of plant lectins for probing into the OA glycophenotype, (2) the human lectins galectins-1 and -3, and (3) the glycoprotein asialofetuin (ASF) for visualizing β-galactoside-specific endogenous lectins.

RESULTS

We found that OA chondrocytes expressed oligomannosidic structures as well as non-, mono- and disialylated complex-type N-glycans, and core 2 O-glycans. Reflecting B4GALNT3 mRNA presence in OA chondrocytes, LacdiNAc-terminated structures were detected. Staining profiles for plant and human lectins were dependent on the grade of cartilage degeneration, and ASF-positive cells were observed in significantly higher rates in areas of severe degeneration.

CONCLUSIONS

In summary, distinct aspects of the glycome in OA cartilage are altered with progressing degeneration. In particular, the alterations measured by galectin-3 and the pan-galectin sensor ASF encourage detailed studies of galectin functionality in OA.

African swine fever is a devastating viral disease of domestic and wild pigs against which no vaccine or therapy is available. Therefore, we applied the CRISPR (clustered regularly interspaced short palindromic repeats) - Cas9 nuclease system to target the double-stranded DNA genome of African swine fever virus (ASFV). To this end, a permissive wild boar lung (WSL) cell line was modified by stable transfection with a plasmid encoding Cas9 and a guide RNA targeting codons 71 to 78 of the phosphoprotein p30 gene (CP204L) of ASFV. Due to targeted Cas9 cleavage of the virus genome, plaque formation of ASFV was completely abrogated and virus yields were reduced by four orders of magnitude. The specificity of these effects could be demonstrated by using a natural ASFV isolate and escape mutants possessing nucleotide exchanges within the target sequence, which were not inhibited in the Cas9-expressing cell line. Growth of the cell line was not affected by transgene expression which, as well as virus inhibition, proved to be stable over at least 50 passages. Thus, CRISPR-Cas9 mediated targeting of the ASFV p30 gene is a valid strategy to convey resistance against ASF infection, which may also be applied in its natural animal host.

Aberrant host immune responses to bacterial components of the resident microflora may initiate and perpetuate gastrointestinal inflammation. To investigate how microbial perturbation promotes host immunological responsiveness to commensal bacteria and contributes to the development of typhlocolitis, we selectively colonized defined (altered Schaedler) flora C3H mice with either Helicobacter bilis or Brachyspira hyodysenteriae. Following selective colonization, tissues were analyzed for gross/histopathologic lesions and bacterial antigen-specific B- and T-cell responses. Gnotobiotic mice colonized with H. bilis or B. hyodysenteriae developed typhlocolitis of varying severity, with the most severe gross and histopathogical lesions observed in B. hyodysenteriae-colonized mice. Antigen-specific IgG1 and IgG2a responses to the resident microflora were increased in both H. bilis-and B. hyodysenteriae-colonized mice. The greater antibody responses were associated with less severe cecal inflammation in H. bilis-colonized mice. Altered Schaedler flora (ASF)-stimulated mesenteric lymphocytes from B. hyodysenteriae-colonized mice produced higher levels of interferon-gamma and interleukin (IL)-4 than did lymphocytes from H. bilis-colonized mice. However, ASF-stimulated mesenteric and splenic lymphocytes from both H. bilis and B. hyodysenteriae-colonized mice secreted higher amounts of IL-10 compared to similarly stimulated lymphocytes recovered from control mice. These results indicate that microbial perturbation may induce differential immune responses to nonpathogenic resident bacteria that can lead to intestinal inflammation.

At present, the eradication of African swine fever (ASF) in affected countries is based only on an efficient diagnosis program because of the absence of an available vaccine. The highly antigenic ASF virus proteins p54 and p30, encoded by genes E183L and CP204L respectively, were expressed in baculovirus for diagnostic purposes. A sequence comparison analysis of these genes from different field virus strains which are geographically diverse and isolated in different years, revealed that both genes are completely conserved among the isolates. Partially purified baculovirus-expressed proteins were used in ELISA and Western blot for ASF antibody detection in sera from experimentally inoculated pigs and field sera from ASF innaparent carriers. These comparative analyses showed that p54 presents better reactivity than p30 in Western blot. However, recombinant p30 was more efficient for antibody detection by ELISA, improving the discrimination between positive and negative sera by this technique. These data suggest the convenience of using p30 as ELISA antigen, while p54 should be the selected antigen for ASF virus antibody detection by Western blot. The combined use of both antigens for serodiagnosis of ASF disease will improve the sensitivity of innaparent carriers detection, facilitating also the interpretation of the tests, and eliminating the use of ASF virus in antigen production.

A detailed study was made in 1983-5 in three villages in Mchinji district in the African swine fever (ASF) enzootic area of Malawi, following an outbreak of ASF which affected all three villages. Ticks of the Ornithodoros moubata complex were collected from both pig sties and houses shortly after the outbreak, and approximately 24% contained ASF virus. The proportion of ticks infected did not differ significantly in the three villages, or more surprisingly in different types of premises, and was equivalent in all stages of ticks. The proportion infected decreased with the passage of time, but infected ticks were still present in all three villages 8 months after the outbreak, some with high titres of virus. The proportion of seropositive pigs in the three villages approached 100% following the outbreak, with many apparently healthy pigs being seropositive. It is suggested that Malawian isolates of ASF virus may be less virulent in African than European breeds of domestic pig.

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% has positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5 IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of ASFV-infected pigs.

African swine fever (ASF) has caused the swine industry of the Russian Federation substantial economic losses over the last 7 years, and the disease spread from there to a number of neighbouring countries. Wild boar has been involved in the spread of the disease both at local and at transboundary levels. Understanding ASF dynamics in wild boars is prerequisite to preventing the spread and to designing and applying effective surveillance and control plans. The reproductive ratio (R0 ) is an epidemiological indicator commonly used to quantify the extent of disease spread. Here, it was estimated in nine spatio-temporal clusters of ASF in wild boar cases in the Russian Federation (2007-2013). Clusters were defined by exploring the maximum distance of association of ASF cases using K Ripley analysis and spatio-temporal scan statistics. A maximum spatial association of 133 km in wild boar cases was identified which is within de the conventional radius of surveillance zone (100-150 km). The mean range value of R0 = 1.58 (1.13-3.77) was lower compared to values previously estimated for ASF transmission within farms but similar to early estimates between farm (R0 = 2-3), in domestic pigs using notification data in the Russian Federation. Results obtained provide quantitative knowledge on the epidemiology of ASF in wild boars in the Russian Federation. They identify the ASF transmission rate value in affected natural wild populations, for the first time, which could provide basis for modelling ASF transmission and suggest that current surveillance radius should be reviewed to make surveillance in wild nature more targeted and effective.

Aging is the major risk factor per se for the development of cardiovascular diseases. The senescence of the endothelial cells (ECs) that line the lumen of blood vessels is the cellular basis for these age-dependent vascular pathologies, including atherosclerosis and hypertension. During their lifespan, ECs may reach a stage of senescence by two different pathways; a replicative one derived from their preprogrammed finite number of cell divisions; and one induced by stress stimuli. Also, certain physiological stimuli, such as transforming growth factor-β, are able to modulate cellular senescence. Currently, the cellular aging process is being widely studied to identify novel molecular markers whose changes correlate with senescence. This review focuses on the regulation of alternative splicing mediated by the serine-arginine splicing factor 1 (SRSF1, or ASF/SF2) during endothelial senescence, a process that is associated with a differential subcellular localization of SRSF1, which typically exhibits a scattered distribution throughout the cytoplasm. Based on its senescence-dependent involvement in alternative splicing, we postulate that SRSF1 is a key marker of EC senescence, regulating the expression of alternative isoforms of target genes such as endoglin (ENG), vascular endothelial growth factor A (VEGFA), tissue factor (T3), or lamin A (LMNA) that integrate in a common molecular senescence program.