Saving Pink gold!

Scientific Opportunities often present themselves as Adversities

Selva Kumar, a shrimp farm owner, and several hundred others in the coastal state of Andhra Pradesh, had no idea that their crop was harboring a viral disease, not until their ponds had turned into graveyards merely 5 days into the crop cycle. It cost them their entire produce and additional thousands of rupees to identify the cause in the first place. Such incidents resurface time and again, calling our attention to the depressed state of an otherwise booming industry.

Can shrimp farm owners, like Selva Kumar, avert this kind of blow in future? What can they possibly do, to check and contain a disease outbreak? Are farm conditions responsible for disease outbreaks? Are innocent farmers being fooled into buying poor quality seed crops leading to such disasters?

Many of us would ponder on the plight of these farmers and leave it at that. But there are others who have seen this as an opportunity to develop simple tools to help minimize losses from disease outbreaks.

The Bigger Picture: Aquaculture Harnessing India’s Marine Wealth

With an estimated 61% cultivable land, as recognized by World Bank, agriculture is a strong pillar of India’s GDP. But, is land the only resource India is banking upon? The answer is certainly not. India has the world’s third longest coastline spanning over 8000 Km, in addition to rivers and canals spread over 190,000 Km. This valuable natural resource has spawned a billion-dollar industry in India known as Aquaculture.

If you are wondering how and when did aquaculture become so huge in our country, the answer is: it has been here for ages and has slowly and steadily carved its niche as a forerunner in global exports.According to Food and Agriculture Organization, we were the second largest country in terms of Aquaculture produce in 2016. In 2017, we became the 5th largest exporter of Fish and Fishery Products, worth USD 7.2 billion.

Pink Gold (Shrimps) Exports Raking in the Moolah

It is noteworthy that Shrimps, or Pink Gold, as they are rightly called, has the highest export value in Indian aquaculture. We, at the Nanobioscience Department of Agharkar Research Institute (ARI), are developing a field usable rapid diagnostic test to identify predominant viruses like White Spot Syndrome Virus (WSSV) affecting Shrimps. The need to develop such a tool is compelled by inherent drawbacks of existing procedures, which require:

  • Extensive sample preparation
  • Moving sample to a laboratory-setting
  • Following of lab protocols
  • Skilled personnel

Apparently, these procedures have long turn-around times and employ stringent protocols limiting their utility in a field setting. And what happens when a farmer is unable to identify disease outbreaks in the shrimp crop? It compromises the quality of final produce or even worst, results in mass mortality with zero produce.

Shrimp Diseases: A Growing Concern among Aqua farmers

Shrimps are susceptible to infectious diseases throughout the crop cycle primarily from bacteria, protozoa and viruses, of which viruses are extremely challenging to deal with. At field level, even a set of well-trained eyes cannot suspect viral disease onset until it is too late, at a stage where the crop is most likely to succumb to disease. Mortality rates are as high as 70-100% in WSSV infected shrimps. This is because there are no effective preventive measures or treatment options for viral diseases in shrimp. Therefore, early diagnosis and handling of WSSV could be a game changer in shrimp farming business.

Having said that, is it practically possible to identify every virus affecting a shrimp farm with one single test? No, the answer to such a complex problem cannot simple. But one can simplify the approach towards solving it. The solution is in the approach, which is: Rapid Diagnosis.

The aim is not to supersede the existing diagnostic methods but instead confirm or rule out one type of disease and lead the diagnosis in a certain direction. Something like a litmus test, you see a blue or red and you know what to infer. Diagnosis is a skill and given the limited skill-set of workers at the field level they need simple tools and techniques to assess and interpret a situation early on. So the next important question is: How to design a diagnostic test that addresses most, if not all, of the above criteria?

Simplifying Disease Diagnosis: Paper to the Rescue of Pink Gold

Going about this ambitious pursuit, we at ARI made an attempt to develop a paper-based point-of-care (POC) test to identify WSSV. WSSV, a highly virulent DNA virus, known to have the largest genome of all animal infecting viruses. The reason for targeting WSSV is that the most hazardous diseases affecting shrimps in Indian hatcheries are caused by viruses. Unlike bacteria and fungi, the isolation and identification of viral pathogens is laborious and time consuming.

To address these limitations, we have developed a rapid diagnosis prototype to detect the presence or absence of WSSV, in as less as 15 minutes. That is not all; the prototype is paper-based, making it economic, easy to use and interpret results. Lateral Flow Assays (LFA) was the ideal choice for designing a handheld prototype for WSSV detection. The best-known example of LFA in diagnostics is ‘at-home pregnancy test’ that gives a definite positive or negative result in minutes. We then went about identifying and incorporating key components into the assay.

Identifying the Right Match: Choosing Peptides over Antibodies

Typically, a rapid diagnostic test is based on identification of highly specific antigen-antibody interactions. We targeted the WSSV capsid protein: VP28 as our antigen of choice to develop this test. When injected into a host, VP28 generates an antibody response. However, commercial production of VP28 specific antibodies is highly expensive. Determined to design a low-priced test we went for an inexpensive alternative: Peptides! Instead of antibodies, we used Peptide that reacts with the antigen VP28 with high specificity.

There is provision to obtain a desired peptide using what is known as Phage Display technique, which requires intensive screening of numerous peptides from a huge peptide library. We found a peptide labeled pep28 exhibiting high specificity for VP28 and can detect as less as 103 WSSV copies. These findings demonstrated two things:

  1. Pep28 exhibits high specificity and no cross-reactivity
  2. Pep28 shows excellent sensitivity

These results implied that pep28 can be used for successful identification of WSSV.

Using Gold Moieties for Easy Result Interpretation

Once the sensitivity of pep28 for VP28 was established, we needed a means to identify successful reaction between the two biomolecules—a simple readable signal like color change. To achieve this, Pep28 is attached to gold nanoparticles when formulating the prototype. Gold nanoparticles exhibit interesting properties; at a nanoscale their surface chemistry is instrumental in signaling a successful reaction between target antigen and pep28 by displaying red color.

Pep28 and Gold nanoparticles constitute key components of our LFA-based prototype. Farmers can easily incorporate its use in their workflow. To start with, they need a specimen obtained by crushing shrimp tissue in a buffer. A clear “soup” is thus obtained and applied on the proximal end of the test strip onto the sample pad. The soup then flows seamlessly towards the Conjugate Pad impregnated with pep28-gold nanoparticles detector-complex. A reaction occurs between the specimen and detector complex. If the specimen is infected, the virus interacts with the detector complex (Conjugate) and migrates towards the “Nitrocellulose Membrane” of the test strip.

This membrane has a test line and a control line, impregnated with pep28 and a reference protein, respectively. These lines serve to capture the virus and conjugate to enable result interpretation. Irrespective of presence or absence of virus, the control line will always appear, it is an indicator that ‘all is well’ with the kit.

A positive test result is inferred by color change in test and control line, while no color in test line indicates a negative result.

The figures demonstrate WSSV LFA prototype.

A Correct Diagnosis Is Three Fourth The Remedy

This diagnostic tool is likely to be the first of its kind in aquaculture disease management. It will serve as first-level of screening to eliminate WSSV-positive samples in shrimp farms at every stage of the crop cycle. Farmers will then need to process only LFA-negative samples for further testing, which will significantly reduce their cost burden.

The Road Ahead

Our goal is to target dreadful virus posing threat to shrimp farms, and extend the use of a single test to detect multiple viruses. Having laid the ground work for paper diagnostics to enter the aquaculture industry, we are positive that this prototype will materialize into a commercially viable product in the perceivable future. 

Acknowledgements

First & foremost, I’d like to thank my sister Vijeytha Biradar for her invaluable assistance in drafting and narrating this story. I would like to express my deep sense & gratitude to my senior Dr Prabir Kumar Kulabhusan for his scholarly advice and guidance in this work. I greatly acknowledge my guide Dr Jyutika Rajwade and Dr Kishore Paknikar for their insightful comments and constant encouragement. I would like to thank CSIR and DBT funding agencies for financially supporting this work. Finally, I would like to thank Biopatrika and Team for publishing the story so that it can reach out to the masses.

About author: Snehal Jamalpure is currently pursuing Ph.D. (CSIR-SRF) in Microbiology from DST- Agharkar Research Institute, Pune, India. Prior to this, she worked as a Project Fellow for a tenure of 3 years in CSIR Centre for Cellular and Molecular Biology, Hyderabad. She did her post-graduate degree in Microbial gene technology from Madurai Kamaraj University, Tamil Nadu.

Edited by: Nivedita Kamath

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