Bringing Science to the Field: Innovative Molecular Tools for the Benefit of Farmers

Aquaculture is the fastest growing fresh food sector at 8% p.a. As an example, the global supply of salmonids has increased ca. 40% over the past decade following the global demand for healthy and sustainable protein. Sustainable intensification of aquaculture is a necessity sector-wide to contribute meaningfully to global food security. Growth is however limited by viral and bacteriological diseases which cause large economic losses globally to fish farming industries therefore a key component will be reducing losses to disease. Central to this effort will be to understand the meaning of ‘good’ health and the development of robust health biomarkers that facilitate management in the diverse aquaculture environments found worldwide. Thus informing the early symptoms of impaired animal welfare. In parallel early diagnosis of pathogens is crucial to prevent the spread of disease causing reduced growth.

An integrated approach to health research in aquaculture requires significant inter-disciplinary effort where diagnostics (pathogen), the host (immunology) and individuals and population (epidemiology) play central roles. Our capacity to identify pathogens using molecular methods fueled by increased genomic resources is rapidly changing the diagnostic landscape with significant changes in technology expected in the near future fostering increased opportunity. A suite of technologies including mobile PCR technology, isothermal amplification and sequencing approaches will significantly impact upon our capacity to identify pathogens under different environmental and culture conditions. On-site rapid diagnostics will facilitate point of care diagnostics that lead to solutions being agreed upon directly with the farmer. Currently we are developing and evaluating mobile PCR diagnostic systems in two distinct and challenging fields; the West coast of Scotland and Egypt.

Today’s diagnostic tools, where available, on the whole are based primarily upon PCR screening, both qualitative and quantitative, of samples sent from fish farms to a central laboratory, in addition to histological information for pathology. In the UK, the process of shipping and analysis often takes more than 2 days that significantly reduces time to making an informed decision upon mitigation measures. However, in Egypt, such a network is not in place therefore epidemiological studies are complex and often hampered by poor data availability. In Scotland PCR is used for routine screening and testing for disease in the salmon industry. The method is also used for measuring the smolt status (i.e. seawater readiness) of salmon. Currently all farm samples are packed and shipped from farm to lab, with results are at best provided by the end of the day the samples are received. The target tissue is the gill that has a major osmoregulatory role during smoltification that is well documented. To this end a set of biomarkers based upon changes in gene expression during smoltification have been developed and validated for absolute qRT-PCR analysis. NaKATPase alpha-1-alpha mRNA transcripts have been validated as target biomarkers for the progression of the smoltification process. In comparison to the classic NaKATPase enzyme activity assay the QPCR assay shows a more robust performance providing increased resolution (Figure 1).

Parallel biomarker research using transcriptomics has been used to mine for novel biomarkers to increase the value of biomarker-based decision-making. Ideally a triad of biomarkers would be applied on-site providing increased robustness to analyses. Several smoltification biomarkers have been field-tested using a portable laboratory system containing an extraction robot for nucleic acids and portable QPCR. Interestingly the choice of tissue sample is crucial to provide a reliable source for marker evaluation. Choice and function of tissue can be significantly different from terrestrial agriculture due to evolutionary trajectory and environment. We will discuss progress and development in this area.

In Scottish salmon aquaculture, the detection of the following viral agents currently appears to be most attractive for onsite diagnostics. The symptoms elicited by SAV (salmon alphavirus), PRV (piscine reovirus) and PMCV (piscine myocarditis virus) are very similar as these viruses infect similar organs and disease outbreaks occur in similar areas. It is therefore very difficult to distinguish and identify the infectious agent actually causing the disease on site. A rapid and effective detection platform assay utilizing quantitative real time reverse transcriptase PCR (qRT-PCR) will increase specificity and detection rates. qRT-PCR is currently used for detection and quantification of many viruses, however only a limited number of assays have been developed for viruses infecting fish, with infectious salmon anemia virus (ISAV), piscine nodavirus (PNV), and SAV 1, 2 and 3 being some of the few developed so far. In reference to SAV these qRT-PCR assays have shown to increase detection sensitivity from that of RT-PCR. Here we describe a multiplex qRT-PCR assay for the simultaneous detection of SAV, PRV and PMCV three individual qPCR assays fused into a multiplex assay. However, increased prognostic capacities remain elusive with critical gaps in knowledge remaining. For example, a quantitative understanding of the relationship between pathogen load, health status and disease remains elusive for many important pathogens thus limiting predictive modelling.

Significant advances are continually being made toward understanding host-pathogen interaction albeit in a limited number of fish species and at the level of the individual. The addition of the zebrafish as a model organism to our research toolkit has greatly increased our understanding of fish immunology for example. Recent studies have shown that fish when infected will increase their body temperature by several degrees by moving to significantly warmer water, this is known as behavioral fever. We have combined both behavioral prophylaxis and molecular diagnostics in a study aimed at mitigating disease in Egyptian tilapia production. Egypt is the second largest tilapia producer globally providing affordable protein for millions of low-income people based on low cost earth pond systems concentrated in the upper Nile Delta. In the last 4 years, mortalities during the summer months have become a growing issue for the sector. Maintaining fish health is problematic in a context where farmers depend on communally accessed agricultural drainage water, on-farm electrification is limited and services offering diagnostic capacity and general technical support are undeveloped. Sustainable intensification of aquaculture is a necessity for the sector to contribute meaningfully to food security. A key component of this is enhancing productivity and reducing losses to disease. Behavioral prophylaxis, the concept of fish challenged by common pathogens being capable of improving their immune response and survival if they can select an optimal temperature regime has been demonstrated in both Zebrafish and Nile tilapia on a laboratory scale. Adapting these principles to commercial aquaculture is now a major priority and the objective of a recently launched Newton-funded collaboration in Egypt. The consortium will test how modifications in the design of hatchery and pond systems and their management can improve water quality and allow behavioral adaptation by fish in order to enhance health outcomes and system productivity. Impact pathways are expected to result in benefits to tilapia value chains locally in Egypt, but also throughout areas of Sub Saharan Africa promoting aquaculture.

In conclusion, the diversity, value and scale of aquaculture systems strongly impacts upon the focus of health research. Environments in which fish are grown range from intensive closed systems to extensive pond based aquaculture each with a different set of variables. The application of affordable mobile and robust diagnostic systems will facilitate new levels of data collection that if matched to population level models will allow researchers and health workers to translate these advances into real benefits for producers and consumers.