Attachment 1 - Oyster Phenotyping Services - SOW.pdf

Statement of work for Pacific Shellfish Research Unit OsHV-1 disease challenge

Background:

The Pacific Shellfish Research Unit operates a Pacific oyster breeding program. The current goal of the breeding program is to increase resistance and survival to Ostreid Herpes Virus 1 microvariants (hereafter, OsHV1). OsHV1 is linked with catastrophic losses in Pacific oyster aquaculture globally. When disease outbreaks occur up to 90% of animals are lost. OsHV1 is present in two waterbodies in California but remains isolated and is not widespread along the US West Coast. Because of the limited distribution of OsHV1 and the desire to keep the pathogen isolated, a laboratory-based disease challenge is required for phenotyping breeding program animals. Without a bio-secure, laboratory challenge for OsHV1 the PSRU breeding program will not be able to increase resistance to OsHV1.

Objective:

To experimentally expose cohorts of Pacific oyster families to OsHV1 for phenotyping. PSRU scientists will utilize the phenotypic data in combination with genetic data and statistical analysis to increase resistance to OsHV1.

Scope of work:

The contractor shall provide all resources necessary to accomplish the tasks and deliverables described in this document. The contractor shall receive animals from the PSRU and conduct a disease challenge experiment, record experimental data, collect tissues, and return that data and tissue (or extracted DNA) to PSRU scientists.

Period of Performance:

The first period of performance is 1 Apr 2023 – 30 Sept 2023. There is a need to conduct disease challenges on each PSRU breeding program cohort produced. Because of that I expect 1-2 disease challenge experiments to be needed each FY. Because this effort must be repeated to achieve breeding program goals, an option year will be included from 1 Oct 2023 – 30 Sept 2024.

Specific requirements:

The following experimental protocol is required for the OsHV1 disease challenge.

OsHV1 replication using donor oysters:

Sub-adult Pacific oysters are needed to replicate the virus so that juveniles from the breeding program can be exposed to infective seawater using an immersion experimental design.

Preserved viral stock(s) in possession of the contractor must be prepared and injected into sub-adult animals. These donor animals must be monitored and allowed to shed virus into the surrounding seawater. Before the juvenile exposure begins the infective seawater must be analyzed to determine the concentration of OsHV1. A dose of approximately 1 x 106 copies per mL of seawater is prepared for each OsHV1 treatment. Once the required volume of infective seawater is determined the experimental immersion exposure can begin.

Juvenile immersion experiment.

Each family must be tested in triplicate, with 3 treatment and 3 controls. A total of 450 rearing units are required (225 control, 225 OsHV1 treatment). Up to 20 animals per family will be in each replicate rearing unit. A single OsHV1 dose will be used for the treatment.

Juvenile animals will be shipped overnight to the contractor. There will be 1 bag of animals per family. The contractor will need to divide each family into the correct number of treatment and control rearing units. The contractor will maintain the families in conditioned seawater, feed using algae for up to 14 days before the immersion challenge occurs.

The OsHV1 treatment will be performed via immersion, with families held in modified plates containing ca. 10 individuals per family per well and at minimum 3 replicate wells per family, at maximum 6 wells per family. During the immersion challenge, daily environmental monitoring, water exchanges, and feeding will be performed. Mortalities will be removed and will be shucked with tissues preserved individually in 1.5mL centrifuge tubes with 95% ethanol (or other suitable DNA preservative). Data for all mortalities will be recorded according to well and plate at least 1x per day. All surviving animals at the end of the experiment will be sampled individually, using the same protocol as was done for mortalities. All surviving animals will have plate and well recorded for data as well.

Deliverables to PSRU scientists:

After conclusion of the experiment the contractor shall supply a datasheet that includes the following for all challenged oysters: individual ID, plate, well, phenotype (alive/dead), date and notes. An additional experimental summary document will be supplied that describes the protocols used (including viral replication in donor oysters), and supplies information on the precise dose of OsHV1 used in the immersion experiment, and environmental parameters recorded throughout the experiment. The contractor will also prepare all tissue samples for shipment to PSRU scientists or will perform DNA extractions and ship extracted DNA to PSRU scientists. The shipment of tissue/DNA will be dependent on contractor’s capabilities to perform such service in the bio-secure setting.