FORMULATIONS FOR IMPROVED ORAL DELIVERY OF IMMUNOACTIVE RIBONUCLEOTIDES AND LPS TO RAINBOW TROUT (Oncorhynchus mykiss)  

F. C. Thomas Allnutt*1, Kennard Roy1, Scott LaPatra2, Mariam Ashraf1 and Arun K. Dhar3
 
BrioBiotech LLC, P.O. Box 26, Glenelg, MD 2173
tom@briobiotech.com

Protein and oligonucleotide based products that deliver improved health to aquacultured species face hurdles to their expanded use in aquaculture - particularly high cost of production, ineffective oral delivery, and regulatory burden for their approval. This project is developing inexpensive formulations that provide improved stability and oral delivery while basing the formulations on materials that are generally regarded as safe (GRAS) or approved dietary ingredients to lower the regulatory burden for approval.

Formulations were developed to better deliver a ribonucleotide-based immunostimulant product, LactORN®, to rainbow trout. In addition, our formulations were used to better deliver Yersinia ruckeri LPS (high molecular weight) as both a control and possible immunostimulant in its own right. Our M12 formulation was used to produce particles containing LactORNs at the recommended dosage as well as 20% of this dosage. LPS was also encapsulated for delivery at rates on the low end of those shown to stimulate the immune system in the literature.  Controls were LactORNs & LPS in unprotected form as well as empty M12 particles as a negative control. The efficacy of our formulations for delivery of LacORNs and LPS was evaluated by evaluation of the expression of candidate immune genes by PCR.

Juvenile rainbow trout (~25 g/5 fish per treatment) were weighed and placed 5-gallon buckets containing 15˚C UV-treated water. Fish were fed commercial diets supplemented with the appropriate additive (see Figure). Fish were fed three days on the test feeds then on Day 4, head kidney and gill arches (from the left side) were sampled. RNA was isolated using RNAzol as described by the manufacturer then cDNA produced.  Real-time RT PCR was run using SYBR-Green (Life Technologies) to measure expression of IL-1, TNFα, IRF-1, LYS, SOD, IFN γ, IL2, TL5, NAD and MX3. EF1α was the housekeeping gene. The results show that the M12 encapsulation provided improved efficiency of delivery of the LactORN and LPS as assessed by the expression data. These data show that less ORN was needed when supplied in the M12 formulation than when not protected for the same gene expression enhancement.