[show abstract][hide abstract] ABSTRACT: Aortic vascular prosthetic graft infection (AVPGI) with Staphylococcus aureus is a feared post-operative complication. This study was conducted to evaluate the clinical signs and potential biomarkers of infection in a porcine AVPGI model. The biomarkers evaluated were: C-reactive protein (CRP), fibrinogen, white blood cells (WBC), major histocompatibility complex II (MHC II) density, lymphocyte CD4:CD8 ratio and tumour necrosis factor-alpha (TNF-α) in vitro responsiveness. Sixteen pigs were included in the study, and randomly assigned into four groups (n = 4): "SHAM" pigs had their infra-renal aorta exposed by laparotomy; "CLEAN" pigs had an aortic graft inserted; "LOW" and "HIGH" pigs had an aortic graft inserted and, subsequently, S. aureus were inoculated on the graft material (5 × 10(4) colony-forming units [CFU] and 1 × 10(6) CFU, respectively). Biomarkers were evaluated prior to surgery and on day 2, 5, 7, and 14 post-operatively in blood samples. Of all biomarkers evaluated, CRP was superior for diagnosing S. aureus AVPGI in pigs, with a sensitivity of 0.86 and a specificity of 0.75.
European Journal of Clinical Microbiology 11/2010; 29(11):1453-6. · 3.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pathogen-associated molecular patterns (PAMPs) are conserved molecules of microorganisms inducing innate immune cells to secrete distinct patterns of cytokines. In veterinary species, due to a lack of specific antibodies, cytokines are often monitored as expressed mRNA only. This study investigated the induction of IFN-α, IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10 by PAMP-molecules [CpG oligonucleotide D19 (CpG), peptidoglycan (PGN), lipopolysaccharide (LPS), Pam(3)Cys and poly-U] in porcine blood mononuclear cells (BMC) within a 24 h period. As expected, cytokine responses were PAMP-specific, CpG inducing IFN-α and IL-12 p40, and PGN, LPS and Pam(3)Cys inducing varying amounts of IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10. Surprisingly, the ssRNA-mimic poly-U induced IL-6 and IL-1β only. Using CpG, PGN and LPS, the kinetics of cytokine production measured as mRNA (reverse transcription (RT)-qPCR) and protein (ELISA), respectively, correlated well, mRNA responses preceding protein responses. With the exception of IL-1β and IL-6, mRNA-responses were transient, whereas protein responses, except for TNF-α, followed saturation kinetics. Remarkably, LPS-induced TNF-α mRNA was not followed by a protein response. These results provide guidelines concerning the timing and use of protein and mRNA determinations for the characterization of porcine cytokine responses to PAMPs, although given the low number of animals used here results are preliminary and need confirmation in a larger study.
Veterinary Immunology and Immunopathology 10/2010; 139(2-4):296-302. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nine pigs were inoculated intravenously once or twice with 10(8) Staphylococcus aureus per kilogram body weight and sacrificed 12, 24 and 48 h after inoculation. Three sham-infected pigs served as controls. Blood samples were taken for bacteriology, haematology and clinical chemistry. A necropsy was carried out and tissue samples were collected for bacteriology and histology. The onset of clinical disease was seen at 7-8 h after inoculation. The blood bacterial counts remained low throughout the study. All infected pigs developed sepsis characterized by fever, neutrophilia, increased levels of C-reactive protein (CRP) and IL-6, and decreased levels of serum iron. The CRP and IL-6 levels peaked at 36 h, whereas IL-1beta and tumour necrosis factor-alpha showed no obvious changes. Thromboelastography showed increasing hypercoagulability from 12 h and onwards, whereas the platelet numbers declined slightly throughout the experiment. The levels of serum aspartate aminotransferase and bilirubin were elevated at 24 and 36 h. In conclusion, sepsis and severe sepsis were induced as evidenced by dysfunction of the blood clotting system and the liver.
[show abstract][hide abstract] ABSTRACT: The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range , . Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide . A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations . We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic .
PLoS ONE 01/2010; 5(2):e9068. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The local transcriptional response was studied in different locations of lungs from pigs experimentally infected with the respiratory pathogen Actinobacillus pleuropneumoniae serotype 5B, using porcine cDNA microarrays. This infection gives rise to well-demarcated infection loci in the lung, characterized by necrotic and haemorrhagic lesions. Lung tissue was sampled from necrotic areas, from visually unaffected areas and from areas bordering on necrotic areas. Expression pattern of these areas from infected pigs was compared to healthy lung tissue from un-infected pigs. Transcription of selected genes important in the innate defence response were further analysed by quantitative real-time reverse-transcriptase PCR. A clear correlation was observed between the number of differentially expressed genes as well as the magnitude of their induction and the sampling location in the infected lung, with the highest number of differentially expressed genes, and the most highly induced genes found in necrotic areas. Interestingly, a group of differentially regulated genes was represented in all three areas, comprising genes encoding cytokines, acute phase proteins, and factors related to regulation of apoptosis and the complement system. Interferon-γ was downregulated in both necrotic and bordering areas. Evidence of neutrophil recruitment was seen by the up-regulation of chemotactic factors for neutrophils. In conclusion, we found subsets of genes expressed at different levels in the three selected areas of the infected lung as compared to the control group. Thus it is demonstrated that an infection with clearly defined infected loci leads to a rapid disseminated intra-organ response in neighbouring seemingly unaffected tissue areas of the infected organ. Within the lung, we found a clear division of induced genes as, in unaffected areas a large part of differently expressed genes were involved in systemic reactions to infections, while differently expressed genes in necrotic areas were mainly concerned with homeostasis regulation.
[show abstract][hide abstract] ABSTRACT: Dendrimers are well-defined (monodisperse) synthetic globular polymers with a range of interesting chemical and biological properties. Chemical properties include the presence of multiple accessible surface functional groups that can be used for coupling biologically relevant molecules and methods that allow for precise heterofunctionalization of surface groups. Biologically, dendrimers are highly biocompatible and have predictable biodistribution and cell membrane interacting characteristics determined by their size and surface charge. Dendrimers have optimal characteristics to fill the need for efficient immunostimulating compounds (adjuvants) that can increase the efficiency of vaccines, as dendrimers can provide molecularly defined multivalent scaffolds to produce highly defined conjugates with small molecule immunostimulators and/or antigens. The review gives an overview on the use of dendrimers as molecularly defined carriers/presenters of small antigens, including constructs that have built-in immunostimulatory (adjuvant) properties, and as stand-alone adjuvants that can be mixed with antigens to provide efficient vaccine formulations. These approaches allow the preparation of molecularly defined vaccines with highly predictable and specific properties and enable knowledge-based vaccine design substituting the traditional empirically based approaches for vaccine development and production.
[show abstract][hide abstract] ABSTRACT: Prebiotics are non-digestible food ingredients believed to beneficially affect host health by selectively stimulating the growth of the beneficial bacteria residing in the gut. Such beneficial bacteria have been reported to protect against pathogenic infections. However, contradicting results on prevention of Salmonella infections with prebiotics have been published. The aim of the present study was to examine whether S. Typhimurium SL1344 infection in mice could be prevented by administration of dietary carbohydrates with different structures and digestibility profiles. BALB/c mice were fed a diet containing 10% of either of the following carbohydrates: inulin, fructo-oligosaccharide, xylo-oligosaccharide, galacto-oligosaccharide, apple pectin, polydextrose or beta-glucan for three weeks prior to oral Salmonella challenge (107 CFU) and compared to mice fed a cornstarch-based control diet.
The mice fed with diets containing fructo-oligosaccharide (FOS) or xylo-oligosaccharide (XOS) had significantly higher (P < 0.01 and P < 0.05) numbers of S. Typhimurium SL1344 in liver, spleen and mesenteric lymph nodes when compared to the mice fed with the cornstarch-based control diet. Significantly increased amounts (P < 0.01) of Salmonella were detected in ileal and fecal contents of mice fed with diets supplemented with apple pectin, however these mice did not show significantly higher numbers of S. Typhimyrium in liver, spleen and lymph nodes than animals from the control group (P < 0.20).The acute-phase protein haptoglobin was a good marker for translocation of S. Typhimurium in mice. In accordance with the increased counts of Salmonella in the organs, serum concentrations of haptoglobin were significantly increased in the mice fed with FOS or XOS (P < 0.001). Caecum weight was increased in the mice fed with FOS (P < 0.01), XOS (P < 0.01), or polydextrose (P < 0.001), and caecal pH was reduced in the mice fed with polydextrose (P < 0.001). In vitro fermentation in monocultures revealed that S. Typhimurium SL1344 is capable of fermenting FOS, beta-glucan and GOS with a corresponding decline in pH.
Supplementing a cornstarch-based rodent diet with 10% FOS or XOS was found to increase the translocation of S. Typhimurium SL1344 to internal organs in mice, while 10% apple pectin was found to increase the numbers of S. Typhimurium in intestinal content and feces.
[show abstract][hide abstract] ABSTRACT: Knowledge on gene expression in the liver during respiratory infections is limited although it is well-established that this organ is an important site of synthesis of several systemic innate immune components as response to infections. In the present study, the early transcriptional hepatic response of genes associated with innate immune responses was studied in pigs 14-18 h after intranasal inoculation with Actinobacillus pleuropneumoniae, using innate immune focused microarrays and quantitative real-time PCR (qPCR). The microarray analysis of liver tissue established that 51 genes were differentially expressed. A large group of these genes encoded proteins involved in the acute phase response, including serum amyloid A, C-reactive protein, fibrinogen, haptoglobin and tumor necrosis factor-α the expression of which were all found to be up-regulated and glutathione S-transferase, transthyretin, transferrin and albumin which were down-regulated. Additional genes associated with innate immune responses were investigated using qPCR; genes encoding interleukin-(IL-)1, IL-6, IL-8, lipopolysaccharide binding protein, lactotransferrin, and PigMAP were up-regulated and interferon 1α, α₁-acid glycoprotein, mannan-binding lectin A, surfactant protein D, and surfactant protein A1 were down-regulated in the liver of infected animals. Down-regulation of α₁-acid glycoprotein during infection has not been described previously in any species. These results confirm that the liver plays an important role in initiating and orchestrating the innate immune response to A. pleuropneumoniae infection.
[show abstract][hide abstract] ABSTRACT: The serum concentration of acute phase proteins (APPs) increases dramatically in response to inflammation and tissue injury. APPs are clinically useful in a range of domesticated mammals; however, knowledge is limited in nondomesticated mammals. The detective ability of two assays for each of three potential APPs--serum amyloid A (SAA), C-reactive protein (CRP), and haptoglobin (Hp)--was evaluated in eight species. For SAA, a turbidimetric immunoassay (TIA) demonstrated significant detective abilities in the Asian elephant (Elaphas maximus), impala (Aepyceros melampus), musk ox (Ovibos moschatus), and chimpanzee (Pan troglodytes), as did an SAA enzyme-linked immunosorbent assay (ELISA) in the impala. For CRP, both TIA and ELISA had significant detective abilities in the chimpanzee. For Hp, a colorimetric assay demonstrated significant detective abilities in impala, musk ox, sitatunga (Tragelaphus spekeii), and chimpanzee, as did the Hp ELISA in the impala, musk ox, and sitatunga. In conclusion, these results suggest that assays for detection of relevant APPs in several nondomesticated animals are available.
Journal of Zoo and Wildlife Medicine 04/2009; 40(1):199-203. · 0.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of the present longitudinal study was to assess the evolution of two acute phase proteins (APPs), pig-major acute phase protein (pig-MAP) and haptoglobin (HPT), in serum from pigs that developed postweaning multisystemic wasting syndrome (PMWS) in comparison to healthy and wasted non-PMWS affected pigs. In addition, evidence of infection with other pathogens and its relation with variations in APPs concentrations was also assessed. Fourteen independent batches of 100-154 pigs were monitored from birth to PMWS outbreak occurrence in 11 PMWS affected farms. Pigs displaying PMWS-like signs and age-matched healthy controls were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted criteria and pigs were classified as: (i) PMWS cases, (ii) wasted non-PMWS cases and (iii) healthy pigs. At the moment of PMWS occurrence, pig-MAP and HPT concentration in PMWS affected pigs were higher than in healthy ones (p<0.0001). No differences in APPs serum concentrations between subclinically PCV2-infected pigs and healthy non-PCV2-infected pigs (based on quantitative PCR on serum results) were detected. Results showed a significant correlation between PCV2 loads and both pig-MAP (R=0.487-0.602, p<0.0001) and HPT (R=0.326-0.550, p<0.05-0.0001) concentrations in serum of PMWS affected pigs, indicating that the acute phase response in PMWS affected pigs occurred concomitantly to PCV2 viremia. No other pathogen, apart from PCV2, was consistently related with variations in APPs concentrations. A ROC analysis, made to determine the capacity of discrimination of both APPs between PMWS affected and non-affected pigs, showed higher sensitivity and specificity values using pig-MAP compared to HPT. These results suggest that pig-MAP might be a better indicator of PMWS status than HPT. Moreover, the fact that APR occurred some weeks before the start of clinical signs suggests that APPs could provide valuable prognostic information for PMWS development.
[show abstract][hide abstract] ABSTRACT: C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP for phosphocholine. In this design synthetic globular polymers (dendrimers) are used as scaffolds for the multivalent display of phosphocholine molecules. CRP present in a sample binds to the phosphocholine moiety presented at high density in the coating layer and is detectable by specific antibodies. The ELISA was applied to determination of pig and human CRP using commercially available antibodies against human CRP. The assay was shown to be more sensitive than previously published immunoassays employing albumin-coupled cytidine diphosphocholine. The coating was stable for at least 30 days at room temperature and the assay showed high intra- and interassay reproducibility. Results were compared with an immunoturbidimetric method and with a commercial ELISA kit and there was very good agreement with the immunoturbidimetric method, however not with the commercial assay, probably due to a calibration discrepancy. The assay is applicable to other species by providing an adequate detection antibody having the desired species specificity.
Journal of immunological methods 03/2009; 343(2):112-8. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Trans-tracheal aspirations from 56 apparently healthy calves and 34 calves with clinical signs of pneumonia were collected in six different herds during September and November 2002. The 90 samples were cultivated and investigated by PCR tests targeting the species Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Mycoplasma dispar, and Mycoplasma bovirhinis. A PCR test amplifying the lktC-artJ intergenic region was evaluated and shown to be specific for the two species M. haemolytica and Mannheimia glucosida. All 90 aspirations were also analyzed for bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus, and bovine corona virus by antigen ELISA. Surprisingly, 63% of the apparently healthy calves harbored potentially pathogenic bacteria in the lower respiratory tract, 60% of these samples contained either pure cultures or many pathogenic bacteria in mixed culture. Among diseased calves, all samples showed growth of pathogenic bacteria in the lower respiratory tract. All of these were classified as pure culture or many pathogenic bacteria in mixed culture. A higher percentage of the samples were positive for all bacterial species in the group of diseased animals compared to the clinically healthy animals, however this difference was only significant for M. dispar and M. bovirhinis. M. bovis was not detected in any of the samples. BRSV was detected in diseased calves in two herds but not in the clinically healthy animals. Among the diseased calves in these two herds a significant increase in haptoglobin and serum amyloid A levels was observed compared to the healthy calves. The results indicate that haptoglobin might be the best choice for detecting disease under field conditions. For H. somni and M. haemolytica, a higher percentage of the samples were found positive by PCR than by cultivation, whereas the opposite result was found for P. multocida. Detection of P. multocida by PCR or cultivation was found to be significantly associated with the disease status of the calves. For H. somni a similar association with disease status was only observed for cultivation and not for PCR.
[show abstract][hide abstract] ABSTRACT: Post-mortem diagnosis of transmissible spongiform encephalopathies (prion diseases) is primarily based on the detection of a protease resistant, misfolded disease associated isoform (PrP(Sc)) of the prion protein (PrP(C)) on neuronal cells. These methods depend on antibodies directed against PrP(C) and capable of reacting with PrP(Sc)in situ (immunohistochemistry on nervous tissue sections) or with the unfolded form of the protein (western and paraffin embedded tissue (PET) blotting). Here, high-affinity monoclonal antibodies (mAbs 1.5D7, 1.6F4) were produced against synthetic PrP peptides in wild-type mice and used for western blotting and immunohistochemistry to detect several types of human prion-disease associated PrP(Sc), including sporadic Creutzfeldt-Jakob Disease (CJD) (subtypes MM1 and VV2), familial CJD and Gerstmann-Sträussler-Scheinker (GSS) disease PrP(Sc) as well as PrP(Sc) of bovine spongiform encephalopathy (bovine brain), scrapie (ovine brain) and experimental scrapie in hamster and in mice. The antibodies were also used for PET-blotting in which PrP(Sc) blotted from brain tissue sections onto a nitrocellulose membrane is visualized with antibodies after protease and denaturant treatment allowing the detection of protease resistant PrP forms (PrP(RES)) in situ. Monoclonal antibodies 1.5D7 and 1.6F4 were raised against the reported epitope (PrP153-165) of the commercial antibody 6H4. While 1.5D7 and 1.6F4 were completely inhibitable by PrP153-165, 6H4 was not, indicating that the specificity of 6H4 is not defined completely by PrP153-165. The two antibodies performed similarly to 6H4 in western blotting with human samples, but showed less reactivity and enhanced background staining with animal samples in this method. In immunohistochemistry 1.5D7 and 1.6F4 performed better than 6H4 suggesting that the binding affinity of 1.5D7 and 1.6F4 with native (aggregated) PrP(Sc)in situ was higher than that of 6H4. On the other hand in PET-blotting, 6H4 reached the same level of reactivity as 1.5D7 and 1.6F4. This shows that 6H4 needs denatured PrP(RES) to reach maximal reactivity, confirming earlier results. As an exception, human PrP(RES) still reacted relatively poorly with 6H4 in PET-blotting, while 1.5D7 and 1.6F4 reacted well with PrP(RES) from most human CJD types. Taken together this implies that the binding epitope of 1.5D7 and 1.6F4 is accessible in the aggregates of undenatured PrP(Sc) (IHC) while the binding site of 6H4 is at least partly inaccessible. In techniques incorporating a denaturing and/or disaggregating step 6H4 showed good binding indicating increased accessibility of the binding site. An exception to this is human samples in PET-blotting suggesting that huPrP(RES) might not be as easily unfolded by denaturation as BSE and scrapie PrP(RES). Also of interest was the ability of 1.5D7 and 1.6F4 to discriminate between two allelic variants of PrP CJD(Sc) (VV vs. MM) in immunohistochemistry as opposed to the normally used antibody 3F4.
Journal of Immunological Methods 09/2008; 337(2):106-20. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dendrimers are well-defined chemical polymers with a characteristic branching pattern that gives rise to attractive features such as antibacterial and antitumor activities as well as drug delivery properties. In addition, dendrimers can solubilize prion protein aggregates at very low concentrations, but their mode of action is unclear. We show that poly(propylene imine) dendrimers based on di-aminobutane (DAB) and modified with guanidinium surface groups reduce insulin thermostability and solubility considerably at microgram per microliter concentrations, while urea-modified groups have hardly any effect. Destabilization is markedly generation-dependent and is most pronounced for generation 3, which is also the most efficient at precipitating insulin. This suggests that proteins can interact with both dendrimer surface and interior. The pH-dependence reveals that interactions are mainly mediated by electrostatics, confirmed by studies on four other proteins. Ability to precipitate and destabilize are positively correlated, in contrast to conventional small-molecule denaturants and stabilizers, indicating that surface immobilization of denaturing groups profoundly affects its interactions with proteins.
[show abstract][hide abstract] ABSTRACT: Stressors such as weaning, mixing and transportation have been shown to lead to increased blood concentrations of acute phase proteins (APP), including serum amyloid A (SAA) and haptoglobin, in calves. This study was therefore undertaken to assess whether SAA and haptoglobin levels in blood mirror stress in adult cattle. Six clinically healthy Holstein cows and two Holstein heifers were transported for four to six hours to a research facility, where each animal was housed in solitary tie stalls. Blood samples for evaluation of leukocyte counts and serum SAA and haptoglobin concentrations were obtained before (0-sample) and at 8, 24 and 48 hours after the start of transportation. Upon arrival the animals gave the impression of being anxious, and they appeared to have difficulty coping with isolation and with being tied on the slippery floors of the research stable. Serum concentrations of SAA and haptoglobin increased significantly in response to the stressors (P < 0.01 and 0.05 at 48 hours, respectively). Additionally, the animals had transient neutrophilia at 8 and 24 hours (P < 0.05). In conclusion, the results of the study suggest that SAA and haptoglobin may serve as markers of stress in adult cattle.
Veterinary Research Communications 05/2008; 32(7):575-82. · 1.08 Impact Factor