Brigitte Sigrist

Quantitative real-time PCR for the measurement of feline cytokine mRNA.

Abstract We have developed real-time PCR systems to quantitate feline cytokine gene expression. The method is based on the cleavage of fluorescent dye-labelled probes by the 5′–3′ exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection System. The feline-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation of complementary DNA versus genomic DNA amplification products. Quantitative analysis of cytokine cDNA concentrations was performed in comparison to feline GAPDH. Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. GAPDH mRNAs were readily detectable in cDNAs prepared from unstimulated feline peripheral blood mononuclear cells (PBMCs) and from frozen cell pellets, while cytokines (Interleukin (IL)-4, IL-10, IL-12 p35, IL-12 p40, IFNγ, IL-16) were expressed at variable amounts. IFNγ transcription was found to be upregulated in stimulated PBMCs and feline cell lines. The synthesis of cDNA and the performance of the PCR in separate tubes proved to be of superior sensitivity compared to a single-tube based system. The assays described are highly reproducible, require no post-PCR manipulation of the amplicons and permit the analysis of several hundred PCR reactions per day. With this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of cells even after storage of samples for at least 5 years.

Feline leukaemia provirus load during the course of experimental infection and in naturally infected cats

Feline leukaemia virus (FeLV) infection in domestic cats can vary in its outcome (persistent, transient, no infection) for reasons that are not entirely known. It was hypothesized that the initial virus and provirus load could significantly influence the course of retrovirus infection. To determine the role of provirus loads, two methods of PCR, a nested PCR and a fluorogenic probe-based (TaqMan) real-time quantitative PCR, which were specific to the U3 region of FeLV-A were established. FeLV provirus in naturally and experimentally infected cats was then measured. Only 3 weeks after experimental FeLV-A infection, persistently infected cats demonstrated higher provirus loads and lower humoral immune responses than cats that had overcome antigenaemia. Lower initial provirus loads were associated with successful humoral immune responses. Unexpectedly, provirus in the buffy-coat cells of two cats that tested negative for the p27 antigen (a marker for viraemia) was also detected. In 597 Swiss cats, comparison of p27 antigen levels with PCR results revealed broad agreement. However, similar to the experimental situation, a significant number of animals (10%) was negative for the p27 antigen and FeLV-positive by PCR. These cats had a mean provirus load 300-fold lower than that of animals testing positive for the p27 antigen. In conclusion, an association between the provirus load and the outcome of FeLV infection was found. Detection of provirus carriers should contribute to further the control of FeLV. In addition, quantification of provirus loads will lead to a better understanding of FeLV pathogenesis and anti-retrovirus protective mechanisms.

Quantitative real-time PCR detection of insulin signalling-related genes in pancreatic islets isolated from healthy cats.

The cat has recently been proposed as a valuable model for type 2 diabetes mellitus (T2DM), because feline diabetes shares several similarities with the disease in humans. Impaired beta-cell function, decreased beta-cell mass, insulin resistance that is often related to obesity, and pancreatic amyloid deposition, are among these common features. In this study, and to further develop the cat as a model of T2DM, feline pancreatic islets were isolated and real-time PCR quantification of mRNA transcripts of genes central to beta-cell function and survival established. In particular, mRNA quantification systems were determined for insulin, the insulin enhancer pancreatic duodenal homeobox-1 (PDX-1), the insulin suppressor CCAAT/enhancer binding protein-beta (C/EBPbeta), glucose transporter isoform 2 (GLUT2), Fas receptor, the caspase-8 inhibitor FLIP (FLICE [caspase-8]-inhibitory protein) and two chemokines, interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1). Pancreatic islets were isolated by collagenase digestion from healthy cat donors. Partial feline mRNA sequences were determined for PDX-1, C/EBPbeta, GLUT2 and FLIP using primers identified from conserved regions of human, dog and rat mRNA. These novel and the previously available sequences (insulin, Fas receptor, IL-8 and MCP-1) were used to design feline-specific primers suitable for real-time PCR in isolated pancreatic islets. The adopted protocol of collagenase digestion yielded pancreatic islets that were frequently surrounded by acinar cells. Quantification of mRNA transcripts was simple and reproducible in healthy cats. Characterisation of genes related to insulin signalling in cats will prove useful to better understand the pathogenesis of feline diabetes and possibly of human T2DM.

Detection of Avian coronavirus infectious bronchitis virus type QX infection in Switzerland

Infectious bronchitis, a disease of chickens caused by Avian coronavirus infectious bronchitis virus (IBV), leads to severe economic losses for the poultry industry worldwide. Various attempts to control the virus based on vaccination strategies are performed. However, due to the emergence of novel genotypes, an effective control of the virus is hindered. In 1996, a novel viral genotype named IBV-QX was reported for the first time in Qingdao, Shandong province, China. The first appearance of an IBV-QX isolate in Europe was reported between 2003 and 2004 in The Netherlands. Subsequently, infections with this genotype were found in several other European countries such as France, Italy, Germany, United Kingdom, Slovenia, and Sweden. The present report describes the use of a new set of degenerate primers that amplify a 636-bp fragment within the S1 gene by reverse transcription polymerase chain reaction to detect the occurrence of IBV-QX infection in Switzerland.

Development of a new real-time polymerase chain reaction assay to detect Duck adenovirus A DNA and application to samples from Swiss poultry flocks

Between 2008 and 2012, commercial Swiss layer and layer breeder flocks experiencing problems in laying performance were sampled and tested for infection with Duck adenovirus A (DAdV-A; previously known as Egg drop syndrome 1976 virus). Organ samples from birds sent for necropsy as well as blood samples from living animals originating from the same flocks were analyzed. To detect virus-specific DNA, a newly developed quantitative real-time polymerase chain reaction method was applied, and the presence of antibodies against DAdV-A was tested using a commercially available enzyme-linked immunosorbent assay. In 5 out of 7 investigated flocks, viral DNA was detected in tissues. In addition, antibodies against DAdV-A were detected in all of the flocks.

Development and validation of a Myxoma virus real-time polymerase chain reaction assay

To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus in fresh and paraffin-embedded rabbit tissues originating from myxomatosis cases from various regions in Switzerland.

Effect of hyperlipidemia on 11β-hydroxysteroid-dehydrogenase, glucocorticoid receptor, and leptin expression in insulin-sensitive tissues of cats

Glucocorticoid (GC) action depends on GC plasma concentration, cellular GC receptor expression, and the pre-receptor hormone metabolism catalyzed by 11β-hydroxysteroid dehydrogenase (11β-HSD). 11β-Hydroxysteroid dehydrogenase exists in 2 isoforms; 11β-HSD1 converts inactive cortisone to cortisol, and 11β-HSD2 converts cortisol to cortisone. Increasing evidence in humans and experimental animals suggests that altered tissue cortisol metabolism may predispose to diabetes mellitus (DM). Once DM is established, hyperglycemia and hyperlipidemia may further maintain the abnormal metabolism of cortisol. To gain further insight in this regard, healthy cats were infused for 10 d with lipids (n = 6) or saline (n = 5). At the end of the infusion period, tissue samples from adipose tissue (visceral, subcutaneous), liver, and muscle were collected to determine mRNA expression of 11β-HSD1, 11β-HSD2, and GC receptor by real-time reverse-transcriptase polymerase chain reaction; blood samples were collected to determine plasma cortisol and leptin concentrations. Lipid infusion resulted in greater 11β-HSD1 expression and lower GC receptor expression in visceral and subcutaneous adipose tissue, and lower 11β-HSD2 expression in visceral adipose tissue and liver. Plasma cortisol did not differ. Leptin and body weight increased in lipid-infused cats. In spite of comparable circulating cortisol levels, up-regulation of 11β-HSD1 and down-regulation of 11β-HSD2 expression may result in increased tissue cortisol concentrations in fat depots of hyperlipidemic cats. Down-regulation of GC receptor may represent a self-protective mechanism against increased tissue cortisol levels. In conclusion, hyperlipidemia has a profound effect on 11β-HSD expression and supports the connection between high lipid concentrations and tissue cortisol metabolism.