Search

A thorough understanding of the immune system, including the role of different cytokines, during inflammatory diseases in ruminants could lead to the development of new diagnostic methods and treatments. Tumour necrosis factor-α (TNF-α) is an important cytokine in the onset of the inflammatory responses. Unfortunately, the number of studies on cytokines, like TNF-α, in ruminants is limited due to a lack of species-specific reagents. As cytokines have remained rather conserved during evolution, cross-reactivity between animal species may occur. Therefore, the aim of the present study was to investigate 5 commercially available antibodies against human TNF-α for their ability to cross-react with ovine and/or bovine TNF-α, using a bead-based flow cytometric method. Two of the antibody clones (Mab 11 and 6401.1111) showed cross reactivity with ovine recombinant TNF-α in concentrations above 2.5 ng/ml. However, none of the antibodies detected TNF-α in bovine milk, or serum containing known concentrations of bovine TNF-α, as earlier determined with ELISA. The results could be due to inability of the antibodies to cross-react between species, but quenching of the signal by matrix proteins might also have lowered the response.

A technical stop in automatic milking systems may result in a severely prolonged milking interval (PMI) with subsequent impact on milk somatic cell count (SCC). This study investigated the inflammatory reaction, milk composition and yield during SCC peak observed in composite milk after exposing cows to a single PMI of 24h. At the first milking after the PMI, a sharply increased proportion of milk polymorphonuclear leukocytes (PMN) but marginally increased SCC were observed. The peak in SCC was not seen until morning milking day 2 after the PMI, notably, concomitantly with a decreased PMN proportion. An increase in blood lactose, milk bovine serum albumin and serum amyloid A (SAA) and a drop in milk alpha lactalbumin (ALA) were seen concomitantly with the peak in PMN. All parameters mentioned, had returned to base line after day 2. The changes in SCC and SAA had the longest duration. Lactate dehydrogenase in afternoon milk was decreased during the whole study as was also afternoon milk yield. Interleukin-1β could not be detected in milk at any time. SAA and ALA, respectively, may influence chemotaxis and the changed concentrations observed after the PMI might have contributed to the increased migration of PMN to milk., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Infectious diseases can cause large health problems in cattle. The infections cause an acute inflammatory response, mediated by pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. By mapping the pattern of cytokines during inflammations, valuable information about the course of an infection is gained. The aim of the present study was to evaluate a particle-based flow cytometric method, the xMAP technique, using ovine/bovine reagents, for quantification of IL-1beta, IL-6 and TNF-alpha, for application in studies on ruminant infectious diseases with emphasis on bovine milk and plasma samples. Singleplex, duplex and triplex xMAP assays were evaluated, and limits of detection (LODs) as well as intra- and inter-assay variabilities were determined for each assay. Cross-reactivity between reagents in multiplex assays was also tested. In addition, presence of cytokines in milk and plasma samples from healthy and mastitic cows was studied. The LODs were significantly lower for singleplex xMAP assays than for duplex and triplex assays. In singleplex assays, the LODs were 0.08, 0.2 and 0.5 ng/ml, for IL-1beta, IL-6 and TNF-alpha, respectively. Corresponding LODs in triplex assays were 2.0, 6.5 and 3.5 ng/ml. Data indicate that the linear ranges of the multiplex assays were narrower than in singleplex assays. The intra-assay coefficients of variation were < or =10.7% for singleplex assays, while they ranged from 6.2 to 23.2% in the triplex assay. The inter-assay variance ranged from 5.1 to 35.8% in singleplex assays, and from 8.8 to 78.4% in triplex assays. Cross-reactivity between reagents was not observed, and all three cytokines were detected in bovine milk and plasma samples collected from cows with clinical mastitis. In conclusion, our results show that the xMAP technique can be used for quantification of IL-1beta, IL-6 and TNF-alpha in bovine samples, and that further work is required to optimize the multiplex assays.

Background: Multiplex flow cytometry is in widespread use for detection of cytokines in human samples. However, no report on the measurement of porcine cytokines using this method has previously been published. We report on the detection of the porcine proinflammatory cytokines TNF-alpha, IL-8, and IL-1beta by the xMap-assay for multiplex flow cytometry., Methods: Commercially available antibodies to porcine cytokines were used as capture antibodies by attaching them to goat anti-mouse IgG coated microspheres with different fluorescent signatures. By the use of biotinylated detection antibodies and SAv-PE the amount of cytokines bound to the spheres were measured. Experiments were performed to determine the limits of detection and the amount of crossreactivity in buffer, serum, and plasma, using spiking with recombinant porcine cytokines., Results: The limit of detection ranged from 0.18 to 12 ng/ml. Generally, the detection limit was higher in serum and plasma, than in buffer. No crossreactivity between reagents was found., Conclusions: Porcine proinflammatory cytokines can be detected utilizing this method with satisfactory detection limits, and no crossreaction between the reagents involved., (Copyright (c) 2006 International Society for Analytical Cytology.)