Although the immune responses after vaccination with several E. tarda vaccine candidates have been investigated in different fish species (Cheng et al. 2010; Jiao et al. 2009, 2010; Sun et al. 2010, 2011; Tang et al. 2010), information regarding OMPs of E. tarda as a vaccine candidate in pangas catfish is limited. Previous reports suggested that OMPs of E. tarda are highly conserved, immunogenic and could confer broad protection (Kumar et al. 2007; Tang et al. 2010; Dubey et al. 2019; LiHua et al. 2019). In this study, we used crude OMPs of E. tarda CGH9 as a vaccine candidate. Usually, OMPs of bacterial species contain many immunogenic components that elicit protective immunity (Dubey et al. 2019). In the T1 group without AH adjuvant, the ROB activity was significantly high on 1-wpv (0.455 OD) compared to the control. Whereas, in the T2 group with AH adjuvant, a gradual increase in ROB activity was observed up to 3-wpv, indicating stimulation of immune responses for longer protection. Contrarily, in an earlier report, the stimulating effect of a recombinant subunit vaccine candidate along with AH adjuvant on the ROB activity of P. olivaceus was observed to be significantly high only on 1- and 7-day post-vaccination (pv) but not on 14- and 21-day pv (Sun et al. 2011). It has been demonstrated that the vaccine, when conjugated with AH adjuvant, helps in long-term antigen exposure (Gupta et al. 1995; Gupta 1998). Possibly, the crude E. tarda OMPs vaccine-AH adjuvant complex might have resulted in a gradual increase in the ROB activity of the T2 group. Surprisingly, no further increase in ROB activity of catfish was observed when administering a booster dose of E. tarda CGH9 OMPs with or without AH adjuvant at the same concentration.
The lymphocyte proliferation of HK leukocytes of the T1 group upon stimulation with Con-A showed a significant increase on 1-wpv compared to control and reduced thereafter to the level of control on 4-wpv. A similar trend was observed in the proliferative responses even with E. tarda CGH9 whole cells as the test antigen. On the other hand, when the E. tarda CGH9 OMPs were used as the antigen, the proliferative responses of HK leukocytes of the T1 group increased up to 2-wpv and were reduced thereafter. Contrastingly, the T2 group showed a significant increase in the proliferative responses up to 3-wpv in all the tested antigens, viz., Con-A, E. tarda CGH9 OMPs and E. tarda CGH9 whole cells. Our results corroborate the observations of LiHua et al. (2019), who reported an increased proliferation of whole blood cells on 21, 28 and 42 days post-immunization when Anguilla japonica was vaccinated with outer membrane protein A (OmpA) of E. anguillarum and Freund's incomplete adjuvant (FIA). In our study, the observed levels of proliferative responses in all the tested antigen groups were high on 2-, 3- and 4-wpv when compared to the T1 group. This suggested that the naïve lymphocytes of catfish administered with OMPs-adjuvant complexes are effectively stimulated for a longer period than the group-administered only the OMPs as a vaccine.
The T1B group showed no significant increase in lymphocyte proliferation with all tested antigens. Similarly, in the T2B group, the lymphocyte proliferation with all tested antigens showed no significant changes on 1-wpbv and 2-wpbv when compared to 2-wpv from the T2 group. Interestingly, the proliferative responses of the T2B group when tested with E. tarda CGH9 OMPs as the antigen displayed a significant increase on 1-wpbv (2.766 OD) compared to 2-wpv from the T2 group. However, the proliferative responses observed on 1-wpbv (2.766 OD) was almost similar to that of 3-wpv (2.762 OD) from the T2 group. Furthermore, the T1B and T2B groups showed no additional increase in lymphocyte proliferation when compared respectively to the T1 and T2 groups. Unfortunately, it was not possible to compare and discuss the observed results of the present study with the report of other authors, because similar reports assessed the immunoprotective efficacy of OMPs of E. tarda with or without AH as an adjuvant by in-vitro lymphocyte proliferation were scanty.
The antibody-mediated humoral immunity, an important defence mechanism, was reported in O. niloticus, O. mykiss, P. olivaceus and A. japonica against bacterial infections caused by Streptococcus iniae (Shelby et al. 2002), S. agalactiae (Pasnik et al. 2006), Flavobacterium psychrophilum (LaFrentz et al. 2003), Aeromonas hydrophila (LaPatra et al. 2010) and E. tarda (Jiao et al. 2010; LiHua et al. 2019). In the present study, the serum antibody production in the T1 group reduced up to 2-wpv compared to the control. In contrast, its level was significantly high from 1-wpv when compared to control in the T2 group. The observed reduction up to 2-wpv in the serum antibody levels of the T1 group could be the result of false tolerance or false suppression as vaccination may lead to the substantial decrease of natural antibodies, which cross-reacts with most antigens (Sinyakov and Avtalion 2009). On the other hand, this false tolerance or suppression was not observed in the T2 group, which might probably be the effect of AH adjuvant. Additionally, Whyte (2007) described that the level of natural antibodies may vary among the fish species and it also depends on the environmental conditions in which they are present. The serum antibody production was observed to be the highest from 4-wpv to 6-wpv in both T1 and T2 groups. The observations of the present study confirmed many earlier reports performed in P. olivaceus, in which the antibody levels were observed to be highest at 4–8 weeks after vaccinating the fish with E. tarda antigen Eta21 (Jiao et al. 2009), E. tarda antigen Et49 (Jiao et al. 2010), attenuated E. tarda (Sun et al. 2010), recombinant E. tarda antigen rEta2 (Sun et al. 2011) and E. tarda OMPs (Tang et al. 2010). Furthermore, the T2 group recorded the highest level of serum antibody production on 6-wpv (0.581), which was >1.5-folds higher than those observed in the T1 group. This observation was in agreement with Jiao et al. (2010), which documented a significantly high level of specific antibody in P. olivaceus vaccinated with E. tarda major antigen Et49 and AH adjuvant.
The vaccinated groups with or without AH adjuvant that administered a booster dose of E. tarda CGH9 OMPs, i.e., T1B and T2B, recorded high serum antibody production on all wpbv when compared to 2-wpv. The time points 1-wpbv, 2-wpbv and 3-wpbv in T1B and T2B groups corresponded with the time point 3-wpv, 4-wpv and 5-wpv of the T1 and T2 groups without the booster. On comparing the groups vaccinated with E. tarda CGH9 OMPs and AH adjuvant complex, viz., T2 and T2B, the highest immune response in terms of antibody production was observed in the T2 group on 6-wpv. In the T2B group, the same level of antibody production was observed on 2-wpbv (0.587 OD), which was about two weeks earlier than the T2 group. Likewise, Cheng et al. (2010) reported that the group of P. olivaceus vaccinated with E. tarda ATCC 15947 via injection with boost produced the highest antibody titers. Contrarily, the levels of serum antibody in T1 and T1B groups vaccinated with E. tarda CGH9 OMPs without adjuvant were in the same range. These observations suggested that booster dose with AH adjuvant can able to increase the serum antibody levels quickly. Possibly, it effectively augmented the specific immunity induced by E. tarda CGH9 OMPs.
Many earlier reports by different research groups described the immunoprotective efficacy of different E. tarda vaccine candidates in P. olivaceus by examining RPS (Jiao et al. 2009, 2010; Sun et al. 2010, 2011; Tang et al. 2010). As shown in Table 1, the high RPS in the T2 group (89.00±15.56) could be attributed to the high levels of serum antibodies. Observations similar to the present work was made earlier in which a correlation between the serum antibody levels and protection against infection was observed in P. olivaceus, following intraperitoneal injection with OMPs of E. tarda in FIA (Tang et al. 2010) or with E. tarda antigen Et49 and AH adjuvant mixture (Jiao et al. 2010). The efficacy of E. tarda CGH9 OMPs and AH adjuvant mixture in terms of RPS of the present study was comparatively higher than the RPS (77.70) noted in A. japonica vaccinated with OmpA of E. anguillarum in FIA after the E. anguillarum challenge (LiHua et al. 2019). The present observations are also in close agreement with that of Jiao et al. (2010). The observed RPS in T1B (55.50±31.82) and T2B (78.00±31.11) groups were low when compared respectively to the T1 and T2 groups. A similar observation with low RPS in I. punctatus vaccinated with a booster dose of 12.5 µg/100 µL of total E. ictaluri OMP was reported (Bader et al. 2004). Likewise, Tang et al. (2010) reported a low RPS in P. olivaceus vaccinated with a booster dose of 50 µg OMPs of E. tarda. Interestingly, in the present study, serum antibody levels in the T1B and T2B groups were almost similar to the T1 and T2 groups but still showed a lower RPS. This suggested that the catfish that received a booster dose of 50 µg E. tarda CGH9 OMPs with or without AH adjuvant might be too weak to resist the infection challenge. Because, this group was challenged immediately after 2-wpbv; while the vaccinated group without booster was challenged after 4-wpv.