Differential expression of genes encoding CD30L and P-selectin in cattle with Johne's disease: progress toward a diagnostic gene expression signature.

Mycobacterium avium subspecies paratuberculosis (Mycobacterium paratuberculosis), the causative agent of paratuberculosis (paraTB) or Johne's disease in ruminants, is a health problem for the global cattle industry with significant economic losses related to decreased milk production and reduced fertility. Commonly paraTB in cattle is diagnosed by antibody detection by serum enzyme-linked immunosorbent assay (ELISA), by detection of the pathogen by cultivation of individual faecal samples, or by in vitro measurement of cell mediated immune responses using the IFN-gamma test. There is an ongoing need for developing new diagnostic approaches as all currently available diagnostic tests for paraTB may fail to detect sub-clinical infection. We used cDNA microarrays to simultaneously measure expression of over 1300 host genes to help identify a subset of gene expression changes that might provide a unique gene expression signature for paraTB infection. In the present study, non-stimulated leukocytes isolated from 10 sub-clinical paraTB infected cows were examined for genes being expressed at significantly different levels than in similar cells from control cows with the same herd background. We included cattle (Holstein) from two locations (Denmark and USA) for the microarray experiment. Our results indicate that expression profiles of at least 52 genes are different in leukocytes from M. paratuberculosis infected cattle compared to control cattle. Gene expression differences were verified by quantitative real-time reverse transcriptase polymerase chain reactions (qRT-PCR) on the same group of cattle (Holstein) used for the microarray experiment. In order to assess the generality of the observed gene expression, a second and different group of cattle (Jersey) was also examined using qRT-PCR. Out of the seven genes selected for qRT-PCR, CD30 ligand (CD30L) and P-selectin were consistently differentially expressed in freshly isolated leukocytes from paraTB infected and control animals of both breeds of cattle. Although further work is clearly needed to develop a more complete gene expression signature specific for paraTB, our results demonstrate that a subset of genes in leukocytes are consistently expressed at different levels, depending upon M. paratuberculosis infection status.

Marked induction of IL-6, haptoglobin and IFNgamma following experimental BRSV infection in young calves.

Bovine respiratory syncytial virus (BRSV) has been identified worldwide as an important pathogen associated with acute respiratory disease in calves. An infection model has been developed reflecting accurately the clinical course and the development of pathological signs during a natural BRSV-infection. In the experiments described in the present study, calves were infected at 13-21 weeks of age and reinfected 14 weeks later. Blood samples from the entire infection period were analysed for acute phase protein (haptoglobin) by ELISA and for expression (mRNA level in peripheral blood mononuclear cells) of the cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-gamma (IFNgamma) by quantitative real-time reverse transcribed polymerase chain reaction (RT-PCR). IFNgamma, interleukin-6 and haptoglobin were markedly induced together with development of clinical signs in response to the first infection with BRSV. The IFNgamma response was biphasic, with an early peak at day 1-3 post infection (p.i.) and a later increase between day 5 and 8 p.i. Reinfection also resulted in an induction of IFNgamma, but without induction of clinical signs, IL-6 and haptoglobin. These results indicate that early mediators connected with the innate responses are induced on a first encounter with the pathogen, but not on a second encounter (reinfection) where the adaptive immune system may act as the first line defence.