2.2. Trial 1: Disease Periods Trial

To study the factors influencing viral transmission it was first necessary to determine the duration of disease periods for KHVD in naïve juvenile carp. The disease periods evaluated in this study were the pre-infectious period (i.e., latent period), incubation period, prodromal period, clinical period, and infectious period, which are defined in Table 1 [67,68]. Note that the pre-infectious period of disease is described as the time from exposure to infectiousness and does not refer to latent infections of CyHV-3 occurring in convalescent carp [69].

CyHV-3 disease periods.

Disease periods definitions were obtained from Thomas et al. (2001) and Mueller et al. (2008).

Sixteen carp were anesthetized in a solution of 100 µL/L of clove oil (90% Eugenol; Velona, Elk Grove Village, IL, USA) and inoculated with CyHV-3 via swabbing of the caudal fin/peduncle (three swab strikes across either side of the caudal fin) with CyHV-3 cell culture supernatant (TCID50 = 100/mL, qPCR copy number = 5.89 × 106/mL) using a sterile cotton swab (Dynarex, Orangeburg, NY, USA) (Figure 1). This method of inoculation was chosen to disrupt the mucus layer of carp [52] and to directly inoculate the skin with CyHV-3, allowing us to use gill swabs as evidence of viral shedding and infectiousness. Each fish was uniquely marked with colored injectable elastomer (Northwest Marine Technology, Anacortes, WA, USA) to observe the progression of disease periods in each fish individually, then moved into a 60 L aquarium with flow through well water (flow rate = 3–4 tank volumes/h) at 23 °C.

Trial 1 schematic and disease periods. Average values, standard deviations, and definitions are given for each CyHV-3 disease period.

To identify the timepoints of viral shedding at each day post exposure (1 dpe, 2 dpe, etc.), all fish were collected with a soft net and swabbed with sterile cotton swabs to collect mucus from the gills (i.e., one swab strike across gills, alternating left or right side each day), and then returned to the tank. Swab tips were aseptically broken off into 1.5 mL microcentrifuge tubes (Globe Scientific, Mahwah, NJ, USA) and frozen at –20 °C until nucleic acid extraction and qPCR could be performed (described below). The appearance of early clinical signs (e.g., discolored skin, loss of the mucosal layer, or frayed fins) was noted for each fish and moribund or dead fish were removed from the tank. A control group of sixteen additional carp were inoculated with sterile cell culture medium and sampled and monitored identically to the experimental group to differentiate morbidity and mortality caused by the sampling protocol from the development of KHVD.