Secondly, increased stress on the pig as an individual can lead to increase in serum levels of the stress hormone cortisol. Our data suggest that there is no need for an additional pathogen to develop PCVAD in LY2452473 conventional status pigs, and growth retardation and clinical signs can be induced in PCV2 infected pigs that are exposed to environmental stressors alone. can exacerbate PCV2 infection and can lead to PCVAD. This co-infection has been shown to increase PCV2 replication in the host and also to modify cytokine production and profile (Opriessnig and Halbur, 2012; Segales et al., 2013). Recently, the role of environmental factors and their contribution to the onset of PCVAD has been explored. Housing conditions, hygiene, biosecurity and husbandry have all been linked to PCVAD development (Segales et al., 2013). A recent study demonstrated that reduced pen size and cross-fostering in farrowing crates alter the course of PCV2 infection, favouring earlier infections and therefore possibly exacerbating disease (Andraud et al., 2009). It seems that PCVAD is a truly multifactorial disease and disease progression may not only be dependent on PCV2 infection and one other contributing factor but could depend on a multitude of factors. Currently, little is known about the impact of these different co-factors in the outcome and severity of disease. During their life, pigs are exposed to many environmental stressors in addition to weaning; these include changes in temperature, mixing, noise and shipping. Many of these have been shown to LY2452473 suppress the immune system and therefore increase susceptibility to disease (Kelley, 1980; McGlone et al., 1993). However, whether these environmental stressors affect progression of PCV2 infection is currently poorly understood. Using recently identified environmental risk-factors for occurrence of PCVAD in a herd (Alarcon et al., 2011a,b), we investigated the role of these potential co-factors on PCV2 infection with Rabbit Polyclonal to 53BP1 the aim of developing a disease model for PCVAD which does not rely on gnotobiotic pigs. 2.?Material and methods 2.1. Ethics statement All animal studies were performed according to the regulations and guidance provided under the UK Home Office Animals (Scientific Procedures) Act 1986. Experimental protocols were approved under project licence number PPL 70/7219, as well as the RVC Ethics and Welfare Committee. 2.2. Animals In a cross-sectional study of 114 farms in England in 2008, antibodies against PCV2 were detected in 99.1% of herds and PCV2 was detected by PCR on 90.4% of farms, indicating a nearly endemic infection (Wieland et al., 2010). However, we were able to purchase a total of 54 large white??landrace pigs of a similar age from a commercial farm that tested PCV2 free as by PCR and antibody ELISA before study recruitment. Pigs were randomly allocated to nine groups (for 5?min at 4?C and the supernatant added to the retained media. This virus suspension was then concentrated approximately 10-fold using dialysis tubing (Spectra/Por, Biotech Cellulose Ester membrane; Spectrum Europe B.V.) in polyethylene glycol (PEG 12000 flake; Whyte Chemicals Ltd.) at 4?C. Concentrated virus suspension was subsequently dialysed in MEM overnight and aliquoted. PCV2 stocks were titrated on PK15-ALR-NPro cells as described elsewhere. The titre of the virus stock was determined by qPCR. 2.4. Experimental design In the study design, four treatment groups, one challenged only control group (each with n?=?6 animals, repeated in a 2??2 study design for a total of 12 animals per treatment in two separate rooms), and one unchallenged control group were allocated to nine identical rooms, all of which had an isolated environmental system, allowing for control of airflow, humidity and temperature. At four weeks of age (Day 0) treatment groups and challenged control groups were inoculated intra-nasally with 1??1010 PCV2 particles in 5?ml media. Non challenged controls (C) were inoculated with 5?ml of virus free media. One treatment group was inoculated with virus (V) but not subjected to LY2452473 other environmental stressors. The remaining groups, all inoculated with virus, were subjected to either high stocking density (V SD), high environmental temperature (V T) or both, high stocking density and high temperature combined (V SD T). High stocking density was calculated as DEFRA guidelines (https://www.gov.uk/pig-welfare-regulations) minus 25% for the average weight of pig in that group. Areas were altered weekly, after weekly weights.