DIVA metabolomics: Differentiating vaccination status following viral challenge using metabolomic profiles.

Bovine Respiratory Disease (BRD) is a major source of economic loss within the agricultural industry. Vaccination against BRD-associated viruses does not offer complete immune protection and vaccine failure animals present potential routes for disease spread. Serological differentiation of infected from vaccinated animals (DIVA) is possible using antigen-deleted vaccines, but during virus outbreaks DIVA responses are masked by wild-type virus preventing accurate serodiagnosis. Previous work by the authors has established the potential for metabolomic profiling to reveal metabolites associated with systemic immune responses to vaccination. The current study builds on this work by demonstrating for the first time the potential to use plasma metabolite profiling to differentiate between vaccinated and non-vaccinated animals following infection-challenge. Male Holstein Friesian calves were intranasally vaccinated (Pfizer RISPOVAL®PI3+RSV) and subsequently challenged with Bovine Parainfluenza Virus type-3 (BPI3V) via nasal inoculation. Metabolomic plasma profiling revealed that viral challenge led to a shift in acquired plasma metabolite profiles from day 2 to 20 p.i., with 26 metabolites identified whose peak intensities were significantly different following viral challenge depending on vaccination status. Elevated levels of biliverdin and bilirubin and decreased 3-indolepropionic acid in non-vaccinated animals at day 6 p.i. may be associated with increased oxidative stress and reactive oxygen scavenging at periods of peak virus titre. During latter stages of infection, increased levels of N-[(3α,5ß,12α)-3,12-dihydroxy-7,24-dioxocholan-24-yl]glycine and lysophosphatidycholine and decreased enterolactone in non-vaccinated animals may reflect suppression of innate immune response mechanisms and progression to adaptive immune responses. Levels of hexahydrohippurate were also shown to be significantly elevated in non-vaccinated animals from days 6 to 20 p.i. These findings demonstrate the potential of metabolomic profiling to identify plasma markers that can be employed in disease diagnostic applications to both differentially identify infected non-vaccinated animals during disease outbreaks and provide greater information on the health status of infected animals.

Identification of candidate protein markers of Bovine Parainfluenza Virus Type 3 infection using an in vitro model.

Bovine Parainfluenza Virus Type 3 (BPI3V) infections are often asymptomatic, causing respiratory tissue damage and immunosuppression, predisposing animals to severe bacterial pneumonia, the leading cause of Bovine Respiratory Disease (BRD) mortality. As with many pathogens, routine BPI3V serology does not indicate the presence of damaged respiratory tissue or active infection. In vitro proteomic marker screening using disease relevant cell models could help identify markers of infection and tissue damage that are also detectable during in vivo infections. This study utilised a proteomic approach to investigate in vitro cellular responses during BPI3V infection to enhance the current understanding of intracellular host-virus interactions and identify putative markers of in vivo infection. Through 2D gel electrophoresis proteomic analysis, BPI3V Phosphoprotein P and host T-complex Protein 1 subunit theta were found to be accumulated at the latter stages of infection within bovine fibroblasts. These proteins were subsequently detected using targeted multiple reaction monitoring (MRM) mass spectrometry in the plasma of animals challenged with BPI3V, with differential protein level profiles observed dependant on animal vaccination status. Potential mechanisms by which BPI3V overcomes host cellular immune response mechanisms allowing for replication and production of viral proteins were also revealed. Assessment of circulating protein marker levels identified through an in vitro approach as described may enable more effective diagnosis of active viral infection and diseased or damaged respiratory tissue in animals and allow for more effective utilisation of preventative therapeutic interventions prior to bacterial disease onset and significantly aid the management and control of BRD.

A potentiometric biosensor for rapid on-site disease diagnostics.

Quantitative point-of-care (POC) devices are the next generation for serological disease diagnosis. Whilst pathogen serology is typically performed by centralized laboratories using Enzyme-Linked ImmunoSorbent Assay (ELISA), faster on-site diagnosis would infer improved disease management and treatment decisions. Using the model pathogen Bovine Herpes Virus-1 (BHV-1) this study employs an extended-gate field-effect transistor (FET) for direct potentiometric serological diagnosis. BHV-1 is a major viral pathogen of Bovine Respiratory Disease (BRD), the leading cause of economic loss ($2 billion annually in the US only) to the cattle and dairy industry. To demonstrate the sensor capabilities as a diagnostic tool, BHV-1 viral protein gE was expressed and immobilized on the sensor surface to serve as a capture antigen for a BHV-1-specific antibody (anti-gE), produced in cattle in response to viral infection. The gE-coated immunosensor was shown to be highly sensitive and selective to anti-gE present in commercially available anti-BHV-1 antiserum and in real serum samples from cattle with results being in excellent agreement with Surface Plasmon Resonance (SPR) and ELISA. The FET sensor is significantly faster than ELISA (<10 min), a crucial factor for successful disease intervention. This sensor technology is versatile, amenable to multiplexing, easily integrated to POC devices, and has the potential to impact a wide range of human and animal diseases.

Identification of systemic immune response markers through metabolomic profiling of plasma from calves given an intra-nasally delivered respiratory vaccine.

Vaccination procedures within the cattle industry are important disease control tools to minimize economic and welfare burdens associated with respiratory pathogens. However, new vaccine, antigen and carrier technologies are required to combat emerging viral strains and enhance the efficacy of respiratory vaccines, particularly at the point of pathogen entry. New technologies, specifically metabolomic profiling, could be applied to identify metabolite immune-correlates representative of immune protection following vaccination aiding in the design and screening of vaccine candidates. This study for the first time demonstrates the ability of untargeted UPLC-MS metabolomic profiling to identify metabolite immune correlates characteristic of immune responses following mucosal vaccination in calves. Male Holstein Friesian calves were vaccinated with Pfizer Rispoval® PI3 + RSV intranasal vaccine and metabolomic profiling of post-vaccination plasma revealed 12 metabolites whose peak intensities differed significantly from controls. Plasma levels of glycocholic acid, N-[(3α,5ß,12α)-3,12-Dihydroxy-7,24-dioxocholan-24-yl]glycine, uric acid and biliverdin were found to be significantly elevated in vaccinated animals following secondary vaccine administration, whereas hippuric acid significantly decreased. In contrast, significant upregulation of taurodeoxycholic acid and propionylcarnitine levels were confined to primary vaccine administration. Assessment of such metabolite markers may provide greater information on the immune pathways stimulated from vaccine formulations and benchmarking early metabolomic responses to highly immunogenic vaccine formulations could provide a means for rapidly assessing new vaccine formulations. Furthermore, the identification of metabolic systemic immune response markers which relate to specific cell signaling pathways of the immune system could allow for targeted vaccine design to stimulate key pathways which can be assessed at the metabolic level.