Jiwan Kumar Chettri

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 β-glucan). Transcript of cytokines related to inflammation (IL-1β, 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 β-glucan, genes encoding IL-1β, 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 β-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.

Tissue specific uptake of inactivated and live Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss): Visualization by immunohistochemistry and in situ hybridization

Understanding of uptake and invasion routes of Yersinia ruckeri, causing Enteric Red Mouth Disease (ERM) in rainbow trout (Oncorhynchus mykiss), is essential for improved understanding of the pathogenicity and immune response mechanisms associated this disease. The present work shed light on areas of invasion in rainbow trout by the use of immunohistochemistry and in situ hybridization techniques. Fish were exposed to live or formalin inactivated bacteria and samples were subsequently taken for histology from various outer and inner surfaces. We applied a specific monoclonal antibody and specific oligonucleotide probes binding to Y. ruckeri (serotype O1, biotype 2) in tissue sections and were able to demonstrate a tissue specific uptake of this bacterium (both formalin inactivated and live form). Uptake and subsequent translocation dynamics at various surfaces demonstrated different site specific propensities between the formalin inactivated and live bacterial organisms. Lateral lines, dorsal fin, epidermis and gastro-intestinal tract mucosal tissue were the primary areas where bacterial uptake was demonstrated readily after exposure. The fate of internalized bacterial organisms within the host suggested that central immune organs are involved in the final antigen processing.

Comparative protection of two different commercial vaccines against Yersinia ruckeri serotype O1 and biotype 2 in rainbow trout (Oncorhynchus mykiss).

Differentially extended specific protection by two commercial vaccines against Yersinia ruckeri serotype O1 biotype 2 was studied following 30s immersion exposure. Rainbow trout were challenged intra-peritoneally (i.p.) with Y. ruckeri serotype O1, biotype 2 (≈10(6) to 10(7)CFU/fish) at 4, 6 and 8 months after vaccination with vaccines containing either biotype 1 (AquaVac(®) ERM) or both biotypes 1 and 2 (AquaVac(®) RELERA™). The specific pattern of vaccine-mediated protection was evaluated by relative percentage survival (RPS) analysis at 4 and 6 months post-vaccination and by obtaining gross pathological observations at 4 and 8 months respectively. We determined specific significant and superior protection in terms of increased survivability in AquaVac(®) RELERA™ vaccinated fish and observed correspondingly fewer pathological changes. The challenge trials indicated a longer protection for at least 6 months without any booster vaccination. A specific and adaptive response induced by AquaVac(®) RELERA™ vaccine against Y. ruckeri biotype 2 was clearly indicated. In addition, some degree of cross protection rendered by AquaVac(®) ERM containing biotype 1 during infection with Y. ruckeri biotype 2 was also noted.

Insight from Molecular, Pathological, and Immunohistochemical Studies on Cellular and Humoral Mechanisms Responsible for Vaccine-Induced Protection of Rainbow Trout against Yersinia ruckeri

ABSTRACT 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.

Factors influencing in vitro respiratory burst assays with head kidney leucocytes from rainbow trout, Oncorhynchus mykiss (Walbaum).

Abstract Head kidney leucocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to several factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, exposure time and immune-modulatory molecules on the respiratory burst activity (RBA) of rainbow trout head kidney leucocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to phorbol 12-myristate 13-acetate, zymosan and beta-glucan. Temperature was shown to have a significant effect on production of reactive radicals as illustrated by a high activity in cells maintained at 15-20 degrees C and a reduced activity at temperature extremes (1, 4 and 30 degrees C). Highest activity was found at a cell concentration of 1 x 10(7) cells mL(-1). Reactivity showed a clear decline when cells were exposed for more than 4 h. Moreover, incubation of cells with inhibitory substances viz., DiMePE2, cortisol and superoxide dismutase decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leucocytes for elucidation of rainbow trout immune responses.

Comparative evaluation of administration methods for a vaccine protecting rainbow trout against Yersinia ruckeri O1 biotype 2 infections

Numerous outbreaks of enteric red mouth disease (ERM) caused by Yersinia ruckeri O1 biotype 2 in rainbow trout farms are currently being recorded despite established vaccination procedures against this disease. This could indicate that the currently used application of single immersion vaccination (using a commercial vaccine AquaVac(®) RELERA™) does not provide full protection. We elucidated by a controlled duplicated experiment if different vaccine administration methods can improve level and extent of protection. Rainbow trout, Oncorhynchus mykiss were vaccinated by: (1) a single immersion in bacterin diluted 1:10 for 30s (only primary vaccination); (2) two times 30s immersion (primary immersion vaccination followed by booster immersion vaccination 1 month later); (3) a single i.p. injection (only primary vaccination); (4) immersion vaccination followed by injection booster 1 month later; (5) a single 1h bath in bacterin diluted 1:2000; and (6) immersion (30s, 1:10) plus booster (1h in diluted 1:2000 vaccine) 5 months later). Injection challenge experiments were performed 3, 5 and 7 months post primary vaccination with 8.5×10(6) CFU/fish, 10.6×10(6) CFU/fish and 1×10(8) CFU/fish, respectively. In the first challenge trial, control fish exhibited a mortality of 76%, one time immersion vaccination had a mortality of 37%, two times immersion vaccinated fish had a 4% mortality, the one-time injection vaccinated group showed a mortality of 2% and the immersion plus injection boostered fish showed no mortality at all. When rainbow trout were challenged 5 months post primary vaccination, 26% mortality occurred in control fish, 21% in one time immersion group, 12% in two times immersion group, 5% in the one-time injection vaccinated group whereas immersion plus injection boostered fish again showed no mortality at all. When challenged 7 months post vaccination, one-time immersion vaccinated were not protected at all compared to the control group whereas injection vaccinated fish showed lower mortality (17%) compared to booster immersed fish (32% mortality) which was still better than un-vaccinated controls (44% mortality). It was noteworthy that a diluted bacterin (1:2000 for 1h after 5 months post primary vaccination) booster showed the same effect as a booster with 1:10 bacterin dilution for 30s applied 1 month after primary vaccination. Antibody levels showing significant elevations 28 days post challenge in vaccinated fish point to this immune parameter as a protective element. The superior and extended protection offered by booster vaccination or simply injection is noteworthy and may be applied in future vaccination strategies at farm level.

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.

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.

Comparative evaluation of infection methods and environmental factors on challenge success: Aeromonas salmonicida infection in vaccinated rainbow trout

When testing vaccine-induced protection an effective and reliable challenge method is a basic requirement and we here present a comparative study on different challenge methods used for infection of rainbow trout Oncorhynchus mykiss with Aeromonas salmonicida, a bacterial pathogen eliciting furunculosis. Fish were vaccinated with three different adjuvanted trivalent vaccines containing formalin killed A. salmonicida, Vibrio anguillarum O1 and O2a. These were 1) the commercial vaccine Alpha Ject 3000, 2) an experimental vaccine with water in paraffin oil adjuvant, 3) an experimental vaccine with water in paraffin oil in water adjuvant. Fish were then exposed to A. salmonicida challenge using i.p. injection, cohabitation in freshwater, cohabitation in saltwater (15 ppt) or combined fresh/saltwater cohabitation. Cohabitation reflects a more natural infection mode and was shown to give better differentiation of vaccine types compared to i.p. injection of live bacteria. The latter infection mode is less successful probably due to the intra-abdominal inflammatory reactions (characterized in this study according to the Speilberg scale) induced by i.p. vaccination whereby injected live bacteria more effectively become inactivated at the site of injection. Compared to cohabitation in freshwater, cohabitation in saltwater was less efficient probably due to reduced survivability of A. salmonicida in saltwater, which was also experimentally verified in vitro.

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.