The effective control of porcine reproductive and respiratory syndrome virus (PRRSV), which causes substantial economic losses to the pig industry worldwide, is challenged by complicated host-pathogen interactions and delayed immunity. This study aimed to characterise myeloid immune cells associated with PRRSV (monocytes, macrophages, dendritic cells), and to assess their susceptibility to infect ion, in order to help identify underlying mechanisms that might facilitate PRRSV success. Cellular expression of putative PRRSV receptors CD 163 (scavenger receptor) and CDl69 (sialic-acid binding lectin), was central to investigations. Porcine monocytes isolated from peripheral blood were treated with various cytokines and macrophage activating factors before infection with Eastern European PRRSV strain Lena. IL-10 and dexamethasone (dexa) significantly up-regulated PRRSV replication, which correlated with increased CD163/CD169. M-CSF differentiated monocyte derived macrophages (MoM0S) were stimulated with activators for classical (U'SIIFN-r) or alternative (IL-4) activation. GM-CSF and IL-4 generated monocyte derived dendritic cells (MoDCs) were activated with a maturation cocktail containing LPS, IFN-γ, IL-Iβ, TL-6, TNF-α and PGE2• Dexa and IL-10 were added to MoMos and MoDCs to further assess their significance. Cells were characterised by morphology, phenotype and function, and PRRSV replication measured using flow cytometry and RT-qPCR. Analysis of porcine macrophage subsets highlighted some divergence from described human counterparts. MoDCs, however, appeared similar to mouse and human DCs, showing a MHC11hiCD80/86hiCDI41ow phenotype upon maturation. Infection studies revealed similar replication across activation states, and dexa and IL- 10 significantly increased MoMo susceptibility: MoDCs showed low replication, which was independent of CD 163/CD 169. These novel findings demonstrate the high variability of porcine myeloid cells. PRRSV tropism was not restricted to macrophages, and not always dependent on CD163/CD1 69. Cortisol appeared not to be associated with immunosuppress ion of myeloid ce ll s, but supported PRRSV rep lica0 ion in monocytcs and macrophages, a finding significant for future PRRSV control strategies and perhaps relevant to other porcine infectious diseases.

Enzymes belonging to the CTX-M family are now the most prevalent extended spectrum p-lactamases, conferring resistance to multiple β-lactams. In 2009 an outbreak of CTX-M harbouring Enterobacteriaceae occurred at UK pig farm in which the pigs had received multiple rounds of antibiotic treatment. The aim of this study was to characterise the outbreak CTX-M strains and to identify the fitness contribution of the outbreak CTX-M plasmids beyond antimicrobial resistance. CTX-M producing Escherichia coli, Klebsiella pneumoniae and Salmonella enterica of serovars 4,5 ,12,i:- and Bovismorbificans were isolated indicating an instance of non-clonal spread of resistant organisms. Plasmid characterisation revealed that a CTX-M-14 plasmid of 50kb and unknown incompatibility group was harboured by two E. coli strains. A second plasmid type was a CTX-M-1, sul2 and floR Incll plasmid of I04kb found in all three species of bacteria. Sequencing of the CTX-M-I plasmid revealed that it was a novel plasmid that had arisen by the acquis ition of two resistance islands by a Collb-p9-like plasmid. A curing method specific for IncI I plasmids was developed and successfully used to cure lab strains harbouring the outbreak plasmids, outbreak K. pneumoniae strain 83791 and wild type E. coli strains harbouring unrelated Incll plasmids. Additionally, a plasmid free Salmonella 4,5,12,i:-strain was obtained from the outbreak farm and used, together with B3791, to investigate the phenotypic contribution of the outbreak plasmids. Competitive fitness studies showed that carriage of the outbreak plasmid imposed a burden on K pneumoniae strain B3791 whilst being neutral to the growth of Salmonella 4,5,12,i:-S348/11. No effect was seen in the virulence or biofilm formation in either strain. In conclusion, the outbreak of CTX-M with in this farm was caused by the incurs ion and spread of a successful, novel CTX-M-I –IncI-1 plasmid, p1FM3804. Relating to the prevalence of this plasmid, carriage of multiple resistance genes together with the use of multiple antibiotics could partly explain its widespread presence within this farm.

Classical swine fever virus (CSFV) causes a serious disease of pigs that is a major threat to pork production industries worldwide. Due to the limitations of available vaccines there is a need for alternative strategies to aid (SF outbreak control. One such alternative could be through the use of type I interferon (IFN ). During the acute phase of CSFV infection, high levels of systemic porcinelFNα (Po IFNα) are detected in pigs and one recombinant Pol FNα subtype has been reported to have anti-viral activities against CSFV in vitro. The aim of this study was to express and characterize the full panel of recombinant PoIFN-α subtypes and to establish an in vitro model to assess their anti-CSFV activity if given prophylactically. All 17 PoIFN-α subtypes were cloned, expressed in Chinese hamster ovary cells and purified. The biological activity of each subtype was confirmed using a VSV bioassay. Following the optimisation of an infection model using a commercial preparation of PoIFNα, differential activity was observed between subtypes following treatment of porcine monocytes prior to infection with CSFV. The subtypes were subsequently grouped as having high, medium or low anti-CSFV activity. The potency of representative members of the active subtypes was investigated over a 72hr pre-treatment assay which confirmed PoIFNα12 as the most potent subtype against CSFV in vitro. An additive effect was observed when equal ratios of subtypes with in an activity group were used in combination. The expression of each subtype was then assessed du ring the course of a primary CSFV infection in vivo. When comparing the relative patterns of expression of the PolFNα subtypes in individual pigs, several subtypes appeared to be expressed at higher levels. However, the only consistent increase post CSFV infection was that of PoIFNα12, In conclusion, this study has shown that PolFNα subtypes possess differential anti-CSFV activities in vitro, and that PoIFNα12 represents a possible candidate for investigation as a me◦taphylactic treatment against CSFV infection.

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A cross sectional study of 103 indoor and outdoor British pig farms was carried out in 2003-2004. Over 12,000 pigs aged from 3 days up to multiparious breeding sows were examined. Prevalence of foot, limb and body lesions and lameness was recorded using clear case definitions. Detailed data were also collected on the pen or paddock that the pigs were housed in with particular reference to the floor design, material and condition. Associations between prevalence of disease and the environment the pig was housed in were analysed using multilevel regression models. Post-mortem examination of a small sample of foot and limb lesions was carried out to better understand the pathology. There was a lower prevalence of body and limb lesions in pigs of all ages, and foot lesions in preweaning piglets, housed outdoors compared with indoors. However, there was little difference in the prevalence of foot lesions and lameness in gilts and pregnant sows kept indoors compared with outdoors. In most pigs housed indoors, there was a trend for an increased risk of limb and body lesions and lameness in pigs housed on hard and slatted floors compared with solid concrete floors with bedding. Although, in contrast to this the prevalence of wounds on the limbs in piglets was lower on slatted floors compared with solid concrete floors. The associations between foot lesions and indoor floor type varied with the age of the pig and the type of lesion. In piglets, sole bruising was associated with housing on slatted floors while sole erosion was associated with housing on solid concrete floors without bedding. In gilts and sows, heel flaps were associated with housing on slatted floors while toe erosion was associated with solid floors with deep bedding. In conclusion, this study has provided the most accurate estimates of the prevalence of foot, limb, body lesions and lameness in the English pig herd to date and generated useful hypotheses regarding the aetiology of these lesions. To further understand this topic cohort and intervention studies are now needed.

African swine fever (ASF) is a fatal haemorrhagic disease of domestic pigs. The etiological agent of ASF is African swine fever virus (ASFV), a large DNA virus and only member of the Asfarviridae family. The virus replicates in macrophages and infection has previously been suggested to correlate with expression of cell surface markers, including CD163, which is a characteristic marker of intermediate and late stages of macrophage differentiation. ASFV cell tropism and host factors which modulate infection have not previously been studied extensively in macrophages. Macrophages retain a plasticity of function in vivo and in vitro and alter their phenotype in response to various stimuli. This allows them to respond to a variety of situations. The impact of priming macrophages by different stimuli on ASFV infection rates was investigated. The results provide information relevant to identifying cellular factors which modulate infection in vitro and in vivo. To characterise the impact of macrophage activation on ASFV replication, adherent porcine bone marrow cells were stimulated to form regulatory, classically or alternatively activated macrophages using media supplemented with IL10, IFNγ or IL4 respectively. Cell surface marker expression was assessed using FACS analysis to confirm the predicted macrophage phenotypes were induced. The percentage of macrophages infected was shown to vary significantly, dependent upon virus isolate, treatment and duration of infection. Differences were also observed in production of infectious progeny virus and cell survival following infection of macrophages in different activation states. The results indicate that the activation state of macrophages is important for ASFV infection and that treatment with IL4 to stimulate alternative activation may increase persistence of ASFV infection. Infection of differentially activated cells with porcine reproductive and respiratory syndrome virus (PRRSV) was also investigated to allow comparison to ASFV infection. Interestingly, despite similar cell tropisms of ASFV and PRRSV, PRRSV infection of alternatively activated macrophages was severely inhibited unlike ASFV infection. The requirement of the cell surface marker CD163 for ASFV infection was investigated as it is a marker of alternatively activated macrophages and has also been suggested to be a receptor for ASFV. However, ASFV infection rate did not correlate with its presence on the cell surface and the data indicated that ASFV infection rates were not increased in cells stably transformed to express CD163.

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No description available.