Serum IgM and IgG to HA were measured by ELISA at 10dpi in rabbits inoculated with UrHA, or coinfected with UrHA/Uriarra, or UrHA/SLS (Figure 2.4, Table 2.2). Serum was titrated in duplicate in 2 fold serial dilutions. The endpoint titre was defined as the reciprocal of the final dilution giving a positive result at least 0.1 OD units above the OD of the 1:100 negative control. Differences in titres between groups of individual rabbits were tested using a one-way Mann Whitney U test. There were no significant differences in IgM titres between any of the groups of animals infected (Figure 2.4A). The group of rabbits coinfected with UrHA/SLS, and the group coinfected with UrHA/Uriarra, had significantly depressed levels of circulating IgG to HA at 10dpi compared to the group of rabbits inoculated with UrHA alone (PO.05) (Figure 2.4B).
2.3,1.5 Proportion o f B cells secreting specific IgM and IgG to HA at 10dpi in the popliteal lymph node and spleen
The proprotion of B cells secreting specific IgM or IgG to HA at 10dpi in the spleen and popliteal lymph node of infected rabbits was determined by ELISPOT. Differences in numbers of positive cells between groups were tested using a one-way Mann Whitney-U test (Table 2.2). The group of rabbits coinfected with UrHA/Uriarra had significantly lower numbers of cells secreting IgM and IgG in both the popliteal lymph node and spleen when compared to the group of rabbits inoculated with UrHA alone (P<0.05) (Figure 2.5; 2.6). The group of rabbits coinfected with UrHA/SLS also had significantly lower numbers of IgM and IgG secreting cells in the spleen but not the popliteal lymph node, compared to the group of rabbits inoculated with UrHA alone (Figure 2.5; 2.6). However, of the group of rabbits coinfected with SLS, one animal (identification number 197) produced considerably more IgM and IgG antibody secreting cells (ASC) in the popliteal lymph node than the other five animals in that, or any other, group. The omission of this animal from analysis resulted in a significant decrease in the number of both IgM and IgG ASC in the lymph node compared to animals infected with UrHA alone (Figure 2.5). There was no correlation between circulating serum IgM or IgG with the number of IgM or IgG ASC in the lymph node or spleen, or both combined, within individual rabbits (test of correlation coefficient, r).
A
UrHA Ur/UrHA SLS/UrHA
Infection group
B
MV titre o o CO CM t: r* Öin E 1 <n c o C <5 (0 2 £UrHA Ur/UrHA SLS/UrHA
Infection group
Figure 2.3: ELISA IgM (A) and IgG (B) to myxoma virus at 10dpi. Rabbits were infected with UrHA alone (n=6) and with UrHA on one flank and coinfected on the opposite flank with Uriarra (Ur/UrHA, n = 5) or SLS (SLS/UrHA, n = 6). Serum from 10dpi was diluted in duplicate in 2 fold serial dilutions, where 1 = 1:50. The endpoint titre was defined as the final dilution which was positive at least 0.1 OD units above the OD of the 1:100 negative control. Each column represents the titre for an individual rabbit.
A
9
UrHA Ur/UrHA SLS/UrHA
Infection group
B
9UrHA Ur/UrHA SLS/UrHA
Infection group
Figure 2.4: ELISA IgM (A) and IgG (B) to HA at 10dpi. Rabbits were infected with UrHA alone (n = 6) or with UrHA on one flank and coinfected on the opposite flank with Uriarra (Ur/UrHA, n = 5) or SLS (SLS/UrHA, n = 6). Serum from 10dpi was diluted in duplicate in 2 fold serial dilutions, where 1 = 1:50. The endpoint titre was defined as the final dilution which was positive at least 0.1 OD units above the OD of the 1:100 negative control. Each column represents the titre for an individual rabbit.
UrHa Ur/UrHa Infection group SLS/UrHA B 1200 UrHa Ur/UrHa Infection group SLS/UrHA
Figure 2.5: Antibody Secreting Cells (ASC) per 106 lymphoid cells from the popliteal lymph node of rabbits adjacent to the UrHA inoculation site, secreting specific IgM (A) and IgG (B) to HA at 10dpi, measured by ELISPOT. Rabbits were infected with UrHA on the left flank (UrHA, n = 6). Rabbits were coinfected on the right flank with either Uriarra (Ur/UrHA, n = 5) or SLS (SLS/UrHA, n = 6). Each column represents the triplicate sample from an individual rabbit (^SE). 1747 refers to the number of
A B 600 - 200 - Ur/UrHa SLS/UrHA Infection group
Figure 2.6: Antibody Secreting Cells (ASC) per 106 lymphoid cells from the spleen of rabbits, secreting specific IgM (A) and IgG (B) to HA at 10dpi, measured by ELISPOT. Rabbits were infected with UrHA on the left flank (UrHA, n = 6). Rabbits were coinfected on the right flank with Uriarra (Ur/UrHA, n = 5) or SLS (SLS/UrHA, n = 6). Each column represents the triplicate sample from an individual rabbit (^SE).
Whitney U test, P<0.05) to HA at 10dpi measured by ELISA or ELISPOT between groups of rabbits infected with Uriarra and UrHA (Ur/UrHA), SLS and UrHA (SLS/UrHA) or UrHA alone. (ND) no difference, (L) significantly less than. (*) indicates significance when rabbit 197 (identification number) was omitted from the analysis (see 2.3.1.4).
ELISA ELISPOT
Lymph Node Spleen
Ur/UrHA with respect to UrHA IgM