Protective effect of in-feed specific IgM towards Yersinia ruckeri in rainbow trout.

Tightened regulations and an environmentally friendly approaches in fish production have greatly reduced the use of antibiotics but green solutions are continuously being explored. The use of functional feed may have a potential in the aquaculture sector in securing biomass and minimizing the loss from disease. In the present study, we tested the concept that blood from the fish slaughterhouse can be used for mass purification of specific antibodies which subsequently can be used for feeding fish and thereby confer protection against diseases. IgM was purified from serum from Yersinia ruckeri vaccinated rainbow trout and an IgM sandwich ELISA was developed for quantification of rainbow trout IgM. The purified IgM was encapsulated in alginate microparticles and top-coated in fish feed. IgM re-extracted from the alginate microparticles was shown to retain high reactivity towards Y. ruckeri antigens indicating that its bioactivity remained intact after encapsulation. IgM release from the alginate microparticles was only observed at high pH (pH 8.2) and minimal at low pH, indicating protection of IgM at low pH in the fish stomach during passage. In a feeding - challenge experiment (feeding 1 week before Y. ruckeri challenge and for two weeks following challenge), a statistically non-significant 10% lower mortality was observed in the high dose (400 µg IgM/fish/day fed over 3 weeks) group.

Effects of adjuvant Montanide™ ISA 763 A VG in rainbow trout injection vaccinated against Yersinia ruckeri.

Enteric redmouth disease (ERM) caused by the fish pathogen Yersinia ruckeri is a major threat to freshwater production of rainbow trout (Oncorhynchus mykiss) throughout all life stages. Injection vaccination of rainbow trout against Y. ruckeri infection has been shown to confer better protection compared to the traditionally applied immersion vaccination. It may be hypothesized, based on experience from other vaccines, that adjuvants may increase the protective level of ERM injection vaccines even more. Controlled comparative vaccination studies have been performed to investigate effects of the oil adjuvant Montanide™ ISA 763 A VG (Seppic) when added to an experimental Y. ruckeri bacterin (containing both biotype 1 and 2 of serotype O1). A total of 1000 fish with mean weight 19 g was divided into five different groups (in duplicated tanks 2 × 100 fish per group) 1) non-vaccinated control fish (NonVac), 2) fish injected with a commercial vaccine (AquaVac(®) Relera™) (ComVac), 3) fish injected with an experimental vaccine (ExpVac), 4) fish injected with an experimental vaccine + adjuvant (ExpVacAdj) and 5) fish injected with adjuvant alone (Adj). Injection of the experimental vaccine (both adjuvanted and non-adjuvanted) induced a significantly higher antibody (IgM) level, increased occurrence of IgM(+) cells in spleen tissue and significant up-regulation of several immune genes. Additional experiments using a higher challenge dosage suggested an immune enhancing effect of the adjuvant as the challenge produced 100% mortality in the NonVac group, 60% mortality in both of ComVac and Adj groups and only 13 and 2.5% mortalities in the ExpVac and the ExpVacAdj groups, respectively.

Characterization of serum amyloid A (SAA) in rainbow trout using a new monoclonal antibody.

Serum amyloid A (SAA) is an integral part of the innate immune response in mammals and considered to be important during the acute phase response. The present study was undertaken to elucidate the role of SAA protein in the innate immune response of rainbow trout. A monoclonal antibody raised against a recombinant peptide of rainbow trout SAA was characterized using Western blot, dot blot, ELISA and immunohistochemistry. SAA association with high density lipoprotein (HDL) complicated band identification in Western blot, but delipidization of the SAA-HDL isolate highly increased the quality of reaction in the western blot. Rainbow trout fry (87 days post hatch) infected with Yersinia ruckeri showed a significant up-regulation of the SAA gene at 72 h post infection with an increase until 96 h post infection. Non-significant up-regulations were seen at earlier time points i.e. 4 and 24 h. The expression pattern of SAA significantly correlated to the immunohistochemical analysis of the infected fry. A weak staining was seen in liver tissue at 4 h post infection which increased in intensity during the course of infection i.e. 24, 72 and 96 h post infection.

Adaptive and innate immune molecules in developing rainbow trout, Oncorhynchus mykiss eggs and larvae: expression of genes and occurrence of effector molecules.

The ontogenetic development of the immune system was studied during the egg phase and the early post-hatch period of rainbow trout. Quantitative real-time PCR (qPCR) was used to assess the timing and degree of expression of 9 important immune relevant genes and EF1-α. Further, immunohistochemical staining using monoclonal antibodies was applied on rainbow trout embryos and larvae in order to localize five different protein molecules (MHCII, CD8, IgM, IgT and SAA) in the developing tissue and immune organs. Maternally transferred transcripts of EF1-α mRNA were detected in the unfertilized egg. Early onset of expression was seen for all immune genes at very low levels. The amount of mRNA slowly increased and peaked around and after hatching. The highest increases were seen for MHCII, C3, C5 and SAA. Immunohistochemistry using five monoclonal antibodies showed positive staining from day 84 post fertilization. Skin, gills, intestine, pseudobranch and thymus showed reactivity for MHCII, thymus for CD8, gill mucus for IgT and pseudobranch and cartilage associated tissue for SAA. The importance of detected factors for early protection of eggs and larvae is discussed.

Insight from molecular, pathological, and immunohistochemical studies on cellular and humoral mechanisms responsible for vaccine-induced protection of rainbow trout against Yersinia ruckeri.

The immunological mechanisms associated with protection of vaccinated rainbow trout, Oncorhynchus mykiss, against enteric redmouth disease (ERM), caused by Yersinia ruckeri, were previously elucidated by the use of gene expression methodology and immunochemical methods. That approach pointed indirectly to both humoral and cellular elements being involved in protection. The present study correlates the level of protection in rainbow trout to cellular reactions in spleen and head kidney and visualizes the processes by applying histopathological, immunohistochemical, and in situ hybridization techniques. It was shown that these cellular reactions, which were more prominent in spleen than in head kidney, were associated with the expression of immune-related genes, suggesting a Th2-like response. Y. ruckeri, as shown by in situ hybridization (ISH), was eliminated within a few days in vaccinated fish, whereas nonprotected fish still harbored bacteria for a week after infection. Vaccinated fish reestablished normal organ structure within a few days, whereas nonprotected fish showed abnormalities up to 1 month postinfection. Protection in the early phase of infection was mainly associated with the expression of genes encoding innate factors (complement factors, lysozyme, and acute phase proteins), but in the later phase of infection, increased expression of adaptive immune genes dominated. The histological approach used has shown that the cellular changes correlated with protection of vaccinated fish. They comprised transformation of resident cells into macrophage-like cells and increased occurrence of CD8α and IgM cells, suggesting these cells as main players in protection. Future studies should investigate the causality between these factors and protection.

Differential immune response of rainbow trout (Oncorhynchus mykiss) at early developmental stages (larvae and fry) against the bacterial pathogen Yersinia ruckeri.

Innate immune factors play a crucial role in survival of young fish especially during early stages of life when adaptive immunity is not fully developed. In the present study, we investigated the immune response of rainbow trout (Oncorhynchus mykiss) larvae and fry at an early stage of development. We exposed 17 and 87° days post hatch larvae and fry (152 and 1118 degree days post hatch; avg. wt. 70 and 770 mg, respectively) to the bacterial pathogen, Yersinia ruckeri for 4h by bath challenge. Samples were taken at 4, 24, 72 and 96 h post exposure for qPCR and immunohistochemical analyses to elucidate the immune response mounted by these young fish. Larvae showed no mortality although infected larvae at 48 h post exposure showed hyperaemia in the mouth region and inflammation on the dorsal side of the body. Gene expression studies showed an up-regulation of iNOS and IL-22 in infected larvae 24h post exposure but most of the investigated genes did not show any difference between infected and uninfected larvae. Immunohistochemical studies demonstrated a high expression of IgT molecules in gills and CD8 positive cells in thymus of both infected and uninfected larvae. Infection of rainbow trout fry with Y. ruckeri, in contrast, induced a cumulative mortality of 74%. A high expression of cytokines (IL-1ß, TNF-α, IL-22, IL-8 and IL-10), acute phase proteins (SAA, hepcidin, transferrin and precerebellin), complement factors (C3, C5 and factor B), antimicrobial peptide (cathelicidin-2) and iNOS was found in infected fry when compared to the uninfected control. IgT molecules and mannose binding lectins in gills of both infected and uninfected fry were detected by immunohistochemistry. The study indicated that early life stages (yolk-sac larvae), merely up-regulate a few genes and suggests a limited capacity of larvae to mount an immune response by gene regulation at the transcriptional level. Based on the observed clearance of bacteria and lack of mortality it could be speculated that larvae may be covered by protective shield of different immune factors providing protection against broad range of pathogens. However, the increased susceptibility of older fry suggests that Y. ruckeri may utilize some of the immune elements to enter the naive fish. The up-regulation of iNOS and IL-22 in the infected larvae implicates an important role of these molecules in immune response at early developmental stages. A dense covering of surfaces of gill filaments by IgT antibody in the young fish suggest a role of this antibody as innate immune factor at early developmental stages.

PAMP induced expression of immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss).

Host immune responses elicited by invading pathogens depend on recognition of the pathogen by specific receptors present on phagocytic cells. However, the reactions to viral, bacterial, parasitic and fungal pathogens vary according to the pathogen-associated molecular patterns (PAMPs) on the surface of the invader. Phagocytic cells are known to initiate a respiratory burst following an exposure to the pathogen, but the underlying and associated specific elements are poorly elucidated in fish. The present study describes the differential response of head kidney leukocytes from rainbow trout (Oncorhynchus mykiss) to different PAMPs mimicking viral (poly I:C), bacterial (flagellin and LPS) and fungal infections (zymosan and ß-glucan). Transcript of cytokines related to inflammation (IL-1ß, IL-6, IL-10 and TNF-α) was highly up-regulated following LPS exposure whereas flagellin or poly I:C induced merely moderate reactions. In contrast, IFN-γ expression was significantly higher in the poly I:C stimulated group compared to the LPS group. When head kidney cells were exposed to zymosan or ß-glucan, genes encoding IL-1ß, TNF-α, IL-6 and IL-10 became up-regulated. Their level of up-regulation was comparable to LPS but the kinetics differed. In particular, TNF-α induction was considerably slower when stimulated with zymosan or ß-glucan. The gene encoding the COX-2 enzyme, a central element during initiation of inflammatory reactions, was significantly higher in stimulated cells although a depressing effect of high concentrations of LPS and zymosan became evident after 4h exposure. This study suggests that rainbow trout leukocytes respond differently to viral, bacterial and fungal PAMPs, which may reflect activation of specific signaling cascades eventually leading to activation of different immune effector molecules.