Serum Amyloid A (SAA) Concentration after Vaccination in Horses and Mules.

Serum amyloid A (SAA) is a sensitive acute-phase response (APR) marker in equids. Prominent APRs with elevations of SAA concentrations ([SAA]) have been reported after vaccination. The authors hypothesized that vaccination with an inactivated EHV-1/-4 vaccine would cause increase in [SAA] and antibody responses and that higher [SAA] would be positively correlated with the antibody titer in both equids. Twelve Haflinger horses and 12 mules were included in this longitudinal prospective study. All horses and mules were vaccinated with a commercially available EHV-1/-4 vaccine. Blood was sampled before and after vaccination to measure [SAA] and virus-neutralizing response (VN-T). In horses and mules, significantly higher [SAA] were measured on days 1, 3, and 5 after EHV-1/-4 vaccination; [SAA] on day 1 after vaccination were only measured in animals that developed fever, where mean [SAA] were significantly higher in horses than in mules (horses: 1,365.75 ± 87.64 mg/L, mules: 615.5 ± 153.444 mg/L) (P > .05). Four horses and 2 mules developed fever after vaccination, lasting for ≤24 hours. Increased antibody responses (VN-T) on days 7 and 14 after vaccination were observed in all animals, whereas mules showed higher overall antibody responses. Nevertheless, [SAA] did not correlate with the intensity of the antibody responses (VN-T) stimulated by the vaccine (P < .05). EHV-1/-4 vaccination caused a prominent APR, higher in horses than in mules, but [SAA] did not correlate with antibody responses. Measuring [SAA] after vaccination could help identify severe APRs that may require longer resting intervals before training or competition.

Pre-conditioning of Equine Bone Marrow-Derived Mesenchymal Stromal Cells Increases Their Immunomodulatory Capacity.

Mesenchymal stem/stromal cells (MSCs) are increasingly explored for the treatment of degenerative and inflammatory diseases in human and veterinary medicine. One of the key characteristics of MSCs is that they modulate inflammation mainly through the secretion of soluble mediators. However, despite widespread clinical use, knowledge regarding the effector mechanisms of equine MSCs, and consequently their effectiveness in the treatment of diseases, is still unknown. The objectives of this study were to determine the mechanisms underlying inhibition of lymphocyte proliferation by equine bone marrow-derived MSCs, and to evaluate the effect of pre-conditioning of equine MSCs with different pro-inflammatory cytokines on inhibition of lymphocyte proliferation. We determined that inhibition of lymphocyte proliferation by equine MSCs depends on activity of prostaglandin-endoperoxide synthase 2 and indoleamine 2,3-dioxygenase. Additionally, pre-conditioning of MSCs with TNF-α, IFN-γ or their combination significantly increased the expression of prostaglandin-endoperoxide synthase 2, indoleamine 2,3-dioxygenase, iNOS and IL-6. This upregulation correlated with an increased inhibitory effect of MSCs on lymphocyte proliferation. In conclusion, pre-conditioning of bone marrow-derived MSC increases their inhibitory effect on lymphocyte proliferation in horses.

Peritoneal fluid immunocytochemistry used for the diagnosis of a possible case of equine gastrointestinal B-cell lymphoma.

After physical examination, ultrasonographic evaluation of thorax and abdomen, and peritoneal fluid analysis, gastrointestinal neoplasia with suspected diffuse peritoneal metastasis was diagnosed in a 17-year-old Arabian gelding. The owner elected euthanasia and declined postmortem examination. Immunocytochemistry analysis of the peritoneal fluid resulted in a diagnosis of B-cell lymphoma.

Immunocytochimie du liquide péritonéal utilisée pour le diagnostic d'un cas possible de lymphome gastro-intestinal à cellules B. Après un examen physique, une évaluation échographique du thorax et de l'abdomen et une analyse du liquide péritonéal, une néoplasie gastro-intestinale avec métastase péritonéale diffuse suspectée a été diagnostiquée chez un hongre arabe âgé de 17 ans. Le propriétaire a choisi l'euthanasie et a refusé l'examen postmortem. L'analyse par immunohistochimie du liquide péritonéal a donné lieu à un diagnostic de lymphome à cellules B.(Traduit par Isabelle Vallières).

Detection of Neorickettsia risticii, the agent of Potomac horse fever, in a Gypsy Vanner stallion from Manitoba.

A horse with colitis from Manitoba referred to the Veterinary Medical Centre, Western College of Veterinary Medicine, was diagnosed with Potomac horse fever (PHF). Polymerase chain reaction analysis of the feces confirmed Neorickettsia risticii infection. This is the first reported case of PHF in Manitoba.

Détection deNeorickettsia risticii,agent de la fièvre du Potomac, chez un étalon Gypsy Vanner du Manitoba. Un diagnostic de fièvre du Potomac a été posé pour un cheval du Manitoba atteint de colite qui avait été recommandé au Veterinary Medical Centre du Western College of Veterinary Medicine. Une analyse des fèces par amplification en chaîne par la polymérase a confirmé l'infection par Neorickettsia risticii. Il s'agit du premier cas de fièvre du Potomac au Manitoba.(Traduit par Isabelle Vallières).

Establishment and evaluation of a bead-based luminex assay allowing simultaneous quantification of equine IL-12 and IFN-γ.

BACKGROUND/AIM: Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are key cytokines in immunemediated equine melanoma therapy. Currently, a method for accurate simultaneous quantification of these equine cytokines is lacking. Therefore, we sought to establish an assay that allows for accurate and simultaneous quantification of equine IL-12 (eIL-12) and IFN-γ (eIFN-γ). MATERIALS AND METHODS: Several antibodies were evaluated for cross-reactivity to eIL-12 and eIFN-γ and were used to establish a bead-based Luminex assay, which was subsequently applied to quantify cytokine concentrations in biological samples. RESULTS: Cytokine detection ranged from 31.5-5,000 pg/ml and 15-10,000 pg/ml for eIL-12 and eIFN-γ, respectively. eIL-12 was detected in supernatants of stimulated peripheral blood mononuclear cells (PBMCs) and supernatants/cell lysates of eIL-12 expression plasmid-transfected cells. Low or undetectable cytokine concentrations were measured in negative controls. In equine serum samples, the mean measured eIL-12 concentration was 1,374 ± 8 pg/ml. The bead-based assay and ELISA for eIFN-γ used to measure eIFN-γ concentrations, showed similar concentrations. CONCLUSION: Results demonstrate, to our knowledge for the first time, that cross-reactive antibody pairs to eIL-12 and eIFN-γ and Luminex bead-based technology allow for accurate, simultaneous and multiplexed quantification of these key cytokines in biological samples.

Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity.

BACKGROUND: Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare.Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. RESULTS: The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect. CONCLUSIONS: This study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered.