3.3.5.1 Optimisation of the GFP-ELISA
No conclusive positive anti-GFP antibody responses were detected, despite numerous attempts, mainly due to high background levels from control fish resulting in very high cut off ODs (i.e. >0.7) (Fig. 3.11 A & B). However, greater antibody values were detected in the GK vaccine group compared to all other groups (Fig 3.11 A), and although K vaccine group fish also produced high antibody values to rGFP, this group were also positive to BSA antigen suggesting a greater presence of cross-reacting non-specific antibodies (Fig 3.11 B).
Interestingly, the GK vaccinated group were still the strongest responders with a positive antibody titre of 1/256 during optimisation involving intensive blocking, although there were only minimal differences compared to responses from non-GFP immunised fish in the K vaccine group and controls, which were also always higher than the sensitivity threshold of the assay (results not shown), thus producing false positive results.
3.3.5.2 Anti-GFP antibody screening from GFP + KHV marker vaccine trial
All fish from the trial were finally screened using internal sera blocking with 1% casein as a diluent, which minimised the non-specific binding, but revealed a lack of GFP-specific antibodies in any of the vaccinated fish in this trial (results no shown) suggesting the results observed during optimisation may have been associated with natural antibody responses.
Figure 3.11 Optimisation of green fluorescent protein (GFP) enzyme linked immunosorbent assay (ELISA) with sera from carp immunised with GFP. (A) ELISA plate coated with 3µg mL-1 recombinant GFP and screened with pooled sera from each vaccine group (n=2 – 4) from a GFP + KHV marker vaccine trial. (B) ELISA plate coated with 3µg mL-1 BSA and screened with the same serum samples as ‘A’. Colour coded legend indicates the dilution of serum used for screening: red = 1/64; blue = 1/128. C = Control, G = GFP inoculated only, GK = GFP + KHV vaccinated, K = KHV vaccinated only. Black line indicates cut-off.
3.3.5.3 Optimisation of KHV ELISA
In contrast to responses observed to GFP antigen, there were strong antibody responses to KHV, up to titres of 1/3200, from pooled sera of all fish in KHV vaccinated groups (results not shown). However, there were also very high backgrounds noted with negative control sera, especially at lower dilutions (1/100), whereby pooled fish sera from the G vaccinated group and control group produced ODs as high as 0.59 resulting in false positive results (results no shown). Therefore further optimisation was carried out and 10% casein proved to be the most effective blocking reagent for post-coating ELISA plates, similar to that utilised by St-Hilaire et al. (2009) for KHV serology. Intensive blocking using 5% casein within the sera, that was similar to the method employed by Kim et al. (2007a), but more stringent than that applied in the KHV ELISA by Adkison et al. (2005), reduced absorbance ODs in both negative and positive control sera, however, while the negative control sera remained negative at a 1/200 dilution, the end point antibody titre remained at 1/1600 for positive
control sera (results not shown). Therefore reduced ODs from blocking of positive sera was not detrimental to the assay as the highest affinity IgM remained bound to the antigen, even at higher dilutions.
Other ELISA parameters that were varied in an attempt to optimise the assay included altering the concentration of the conjugate, anti-carp MAb and the KHV coating antigen by screening with 2-fold dilutions of carp sera. Variation in antigen concentration had the biggest impact on improving the assay, which resulted in reduction in absorbance of approximately OD450nm 0.1 per 0.1 µg well-1 KHV protein. Due to the limited amount of purified KHV and the difficulty in generating purified virus, as described in Section 2.5.2, a final working assay concentration of 0.4 µg mL -1 was utilised for carp antibody screening, whereby an OD450nm of 0.6 could still be obtained at a dilution of 1/800. Limiting the concentration of anti-carp MAbs also resulted in little variation of positive signal. Controls were consistently negative at all dilutions with all assay variations attempted during optimisation, further verifying the effectiveness of the intensive serum blocking.
3.3.5.4 Anti-KHV antibody screening from GFP + KHV marker vaccine trial
The mean response from KHV vaccinated fish was significantly higher (p = 0.01) than controls at titres from 1/200 to 1/3200 (Fig. 3.12 A). There was no significant difference between the GK vaccine group and the K vaccine group (p = >0.05), although the mean response of the K vaccine group was consistently higher than GK group. However, 6/8 carp from the control group (sham vaccinated) were also sero-positive for KHV at a 1/200 sera dilution and 1/8 were positive at a 1/400 dilution (Fig. 3.12 B). The naïve carp sera (negative control) remained negative and the positive control (pooled anti-KHV sera from experimentally challenged carp, CEFAS) (Fig 3.12 A) was consistently positive. Six
vaccinated fish were positive at a titre of 1/800 from both the K vaccine group and GK vaccine group and 5 of these fish produced even higher titres (Fig. 3.12).
Figure 3.12 Optimised koi herpesvirus (KHV) enzyme linked immunosorbent assay (ELISA): Final screening of carp sera from KHV marker vaccine trial, 6 weeks post vaccination (wpv). ELISA was undertaken with the optimised protocol for KHV antibody screening. (A) Values are mean ± SE (n = 7 - 8) of antibody responses from each vaccine group. Colour coded legend indicates the different vaccine groups; C = Control, K = KHV vaccinated only, GK = GFP + KHV vaccinated. Note GFP only group was not tested against KHV antigen. Different letters indicate significant differences within groups for each antigen (Mann Whitney-U pairwise comparison test; p<0.05); (B) Number of serologically positive fish from from each vaccine group at each titre above the cut-off. Note the high number of false positive fish from C group at 1/200 dilution despite protocol optimisation. Black broken line indicates cut-off from background controls.
3.4 Discussion
Vaccines containing positive markers, foreign to the host, have been shown to enable differentiation between vaccinated and non-vaccinated mammals and birds using serology (Walsh et al., 2000a; b; Mebatsion et al., 2002; James et al., 2007; 2008, Fang et al., 2008).
Marker vaccination approaches like these may enable the implementation of control and eradication programmes for notifiable diseases.
A model exogenous marker approach was undertaken in this study using inactivated vaccines for ISA and KHVD by administering foreign antigens found previously to be immunogenic in mammals, avians and fish (Hodgins et al., 1967; Gonzalez et al., 1988;
Killie and Jørgensen, 1994; Jones et al., 1999a; Cain et al., 2002; Companjen et al., 2006;
James et al., 2007; Swan et al., 2008; Lu et al., 2009; Valdenegro-Vega et al., 2013). The principle objective was to determine if an antibody response to the marker antigens could be produced and detected by serology using ELISA, thus enabling the distinction between vaccinated and unvaccinated Atlantic salmon and Mirror carp to ISAV and KHV, respectively.
3.4.1 Exogenous marker vaccination for infectious salmon anaemia in Atlantic