Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep

Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs. In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep. The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep. Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5). The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio. In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT. Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups. IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group. Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.     

Serum antibody response of Indian major carp, Labeo rohita to three species of pathogenic bacteria; Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens

The immune response to mixed whole cell antigens of Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens, the common Gram negative bacterial pathogens associated with diseases of Indian major carps were evaluated for their efficacy in triggering antibody responses in rohu, Labeo rohita (Ham.). The rohu yearlings were either immunized with antigens from single bacterial strain, A. hydrophila, E. tarda and P. fluorescens or a combination of all three. An antibody response was detected at 1st week post immunization that rose significantly (p<0.05) at 4th week post immunization in all the immunized groups. The antibody level started declining after 8th week but persisted up to 10th week post immunization in all the immunized groups. Similarly, no significant difference (p>0.05) in the antibody level was found between groups immunized with single and mixed bacterial antigens. Moreover, the use of mixed bacterial antigens did not jeopardize the specific immune response to the vaccine components. Upon challenge with single pathogen, a high relative percent survival was recorded in the group immunized with mixed bacterial antigens and was comparable to those fish immunized with the single bacteria.     

Immune Response of Rainbow Trout to Extracellular Products of Mycobacterium spp.

Abstract

A primary intraperitoneal (IP) vaccination of extracellular products (ECP) from Mycobacterium spp. (strains TB40, TB267, or Mycobacterium marinum) mixed with Freund's incomplete adjuvant and followed by a secondary IP injection at 8 weeks resulted in the elevation of both the nonspecific and the specific immune responses of rainbow trout Oncorhynchus mykiss. Increased nitroblue tetrazolium and phagocytosis activity were observed in these fish; peaks in activity occurred at weeks 2 and 6 after primary immunization with a third peak at week 10. Lysozyme activity, on the other hand, peaked at weeks 2 and 8 after primary immunization except in the TB40-immunized group. A third peak of lysozyme activity was observed at week 10 after primary immunization. The activity of the specific immune response was monitored by an enzyme-linked immunosorbent assay and Western blot. The results indicate that antibodies to the ECP of Mycobacterium spp. were present in rainbow trout serum and that they reacted with major ECP antigens at 65 and 16 kDa (kilodaltons) as well as with some minor antigens at 48, 46, and 40 kDa.     

Effect of Low Dietary Magnesium on Immune Response and Osmoregulation of Atlantic Salmon

Abstract

The influences of dietary magnesium on immune response and on osmoregulation in parr of Atlantic salmon Salmo salar were determined. Groups of fish were fed a casein–gelatin diet unsupplemented (containing about 200 mg Mg/kg) or supplemented with either 300 or 500 mg Mg/kg dry diet (as MgSO₄) for 12 weeks before vaccination to produce fish with different Mg levels, and the feeding regime was continued throughout the study. No differences were observed between the treatment groups in serum-specific antibody levels every second week for 8 weeks after vaccination against Vibrio anguillarum serotypes O1 and O2. Both lysozyme levels and spontaneous hemolytic activities in serum were elevated in vaccinated fish compared with unvaccinated fish. Neither lysozyme activity, complement hemolytic activity, total protein in serum nor blood hemoglobin were affected by dietary Mg. The spontaneous hemolytic activity in serum was lower in fish fed the unsupplemented diet and highest in fish fed the diet supplemented with 500 mg Mg/kg. After 28 weeks on the diets supplemented with graded levels of Mg, a salinity tolerance test (32.5 g/L, 24 h) was performed. High mortality and elevated serum chloride concentrations in all groups after 24 h reflected a general salinity intolerance, but the highest serum chloride level was observed in fish fed the unsupplemented diet. This indicates that low dietary Mg affects the osmoregulation of Atlantic salmon.     

Effect of Edwardsiella tarda immunization on systemic immune response,mucosal immune response and protection in catla (Catla catla)

The effect of immunization on systemic and cutaneous mucosal immune responses of fish and their possible relation with protection has not been fully assessed. In this study, healthy catla (Catla catla) were immunized against Edwardsiella tarda using two antigenic preparations namely, whole cell bacterin (B) and bacterin mixed with Freund’s complete adjuvant in a 1:1 (v/v) ratio (B+A) followed by a booster dose after 3 weeks of first injection. Different systemic and cutaneous mucosal immune responses were measured at weekly interval upto 8th week post vaccination (pv). Fish were challenged 8 weeks pv with live E. tarda to study vaccine induced protection. The result showed that although there were strong systemic as well as mucosal immune responses, particularly after booster dose, the challenge produced low to moderate protection in terms of relative percent survival (RPS). The maximum RPS (50 %) was recorded in the adjuvanted bacterin group after 8 weeks pv. Low to moderate protection after challenge, which may be attributed to the intracellular nature of E. tarda and/or use of crude antigenic preparation, accounts for new strategy to be developed for immunization programme against such intracellular pathogen. The results collectively suggest possible involvement of systemic as well as mucosal immune responses in inducing protective immunity in catla.     

Humoral response of cattle to aerosolized Micropolyspora faeni

Calves (7) were exposed to antigens of Micropolyspora faeni by the aerosol route for 9 weeks. The humoral immune response of calves to M faeni antigens was studied; immunoglobulins (Ig) E, G1, G2, A, and M were measured weekly in serum and nasal secretions by enzyme-linked immunosorbent assay (ELISA). Intradermal injection of antigen was performed during the 6th and 9th weeks; responses were evaluated at 30 minutes, 6 to 8 hours, 24, and 48 hours after injection. Total IgE levels in serum and nasal secretions, evaluated weekly, did not show any elevation. Micropolyspora faeni-specific IgE, IgA, IgG1, and IgG2, but not IgM, were produced by calves exposed to the antigen by the aerosol route; individual variability in magnitude of the response was marked. Thirty-minute skin tests were positive for cytotropic antibody in 2 of 3 aerosol-exposed calves by the 9th week, but delayed-type reactivity was not present. The ELISA test results were compared with those from sera of saline solution aerosol-exposed calves and from a parenterally immunized calf. Comparison of isotype-specific ELISA results obtained from M faeni aerosol-exposed calves with ELISA results from calves exposed to aerosolized ovalbumin according to a similar procedure indicated inherent problems in evaluating immune responses to environmental antigens. Aerosolized M faeni elicited a substantial antibody response. In particular, it is noteworthy that antigen-specific IgE responses were detected.     

Moritella viscosa bypasses Atlantic salmon epidermal keratocyte clearing activity and might use skin surfaces as a port of infection

Moritella viscosa is considered the main causative agent of winter ulcer disease in salmonid fish. In order to obtain more details on route of infection, we challenged Atlantic salmon (Salmo salar) epidermal keratocytes with M. viscosa and performed an Atlantic salmon immersion challenge. Although keratocytes were able to remove M. viscosa from surfaces, their engulfment capability appeared inefficient with reduced ability to reepithelialise superficial wounds (scale less skin surfaces) challenged with the bacterium. The immersion challenge revealed a significant connection between exposure area and mortality. Enhanced invasion ability and mortality was observed by M. viscosa exposure of the head and gill region compared to exposure of: the right side of the body; the left side of the body; or the body from pectoral to caudal fin (p=0.04). Ulcer development corresponded to area exposed (p=0.002), suggesting skin ulcer formation to result primarily from direct skin surface colonization. Ulceration of surfaces exposed to M. viscosa in parallel with occurrence of septicaemia suggests that both skin and gills may act as possible initiation sites for M. viscosa infections.     

Demonstration of the Specificity of the Salmonid Hurnoral Response to Renibacterium salmoninarum with a Monoclonal Antibody against Salmonid Immunoglobulin

Abstract

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytscha was compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarum preparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarum antibody.     

Paradoxical effects of cadmium exposure on antibacterial antibody responses in two fish species: inhibition in cunners (Tautogolabrus adspersus) and enhancement in striped bass (Morone saxatilis)

Previous work in a marine fish, the cunner (Tautogolabrus adspersus), showed that endocytosis of bacteria by cells in the liver and spleen was affected by 96-h exposure of the fish to cadmium at a concentration of 12 micrograms/ml; however, antibody response to sheep erythrocytes was not affected. Since the latter finding was questionable because of short immunization times, and data from more than a single fish species were desirable, both the cunner and an anadromous fish, the striped bass (Morone saxatilis), were examined for antibody responses against the bacterium Bacillus cereus in Freund's complete adjuvant during a 6- to 8-wk time period. Exposure to 12 micrograms/ml cadmium caused significant inhibition of serum antibody titers (P less than 0.007) in cunners. Paradoxically, antibody response in striped bass exposed to 10 micrograms/ml cadmium was enhanced sixfold. This enhancement was weaker but still evident when the antigen was injected 48 d after cadmium exposure. Peritoneal exudate cells from cadmium-exposed striped bass also showed more active migration through microporous filters than cells from non-exposed fish. Since the 96-h cadmium LC50 was 26 micrograms/ml for cunners and 20 micrograms/ml for striped bass, the differences in antibody response could not be explained on the basis of differences in cadmium toxicity. Although geometric mean liver cadmium levels after exposure at 15 degrees C were higher in cunners (163.8 +/- 1.2 micrograms/gm) than in striped bass (64.4 +/- 1.2 micrograms/g), cunners exposed to cadmium at 2 degrees C had lower cadmium levels (46.4 +/- 1.2 micrograms/g) which were still effective in inhibiting antibody production at 8 degrees C. On the other hand, striped bass not exposed to cadmium showed a strong, exponential rise in serum antibody when the temperature at immunization, 14 degrees C, was reduced to 9 degrees C; whereas cunners held in the same tanks exhibited a weaker, biphasic serum antibody response. Regardless of the cause (innate differences in cellular response, or better stress adaptability in an anadromous fish), the data show that chemical-stress effects on the immune system of one fish species cannot be extrapolated to another species.     

Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis

Control of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE and AdjuPrime. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE or AdjuPrime resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE in efficacy field trials against canine VL.     

Comparison of immune responses in parenteral FaeG DNA primed pigs boosted orally with F4 protein or reimmunized with the DNA vaccine

We previously showed that an intradermal (i.d.) FaeG DNA prime (2x)-oral F4 protein boost immunization induces a systemic response and weakly primes a mucosal IgG response in pigs, especially when plasmid vectors encoding the A and B subunit of the E. coli thermo-labile enterotoxin (LT) are added to the DNA vaccine. In the present study, we evaluated whether addition of 1alpha,25-dihydroxyvitamin D(3) (vitD(3)) to the DNA vaccine could further enhance this mucosal priming and/or modulate the antibody response towards IgA. To further clarify priming of systemic and mucosal responses by the i.d. DNA vaccination, we firstly compared the localization of the F4-specific antibody response in pigs that were orally boosted with F4 to that in pigs that received a third i.d. DNA immunization and secondly evaluated cytokine mRNA expression profiles after i.d. DNA vaccination. The i.d. DNA prime (2x)-oral F4 boost immunization as well as the 3 i.d. DNA vaccinations induced mainly a systemic response, with a higher response observed following the heterologous protocol. Co-administration of vitD(3), and especially of the LT vectors, enhanced this response. Furthermore, only the heterologous immunization resulted in a weak mucosal priming, which appeared to require the presence of the LT vectors or vitD(3) as adjuvants. In addition, the LT vectors strongly enhanced the FaeG-specific lymphocyte proliferation and this was accompanied by the absence of a clear IL-10 response. However, despite two DNA immunizations in the presence of these adjuvants and an oral F4 boost, we failed to demonstrate the secretory IgA response needed to be protective against enterotoxigenic E. coli.     

Induction of systemic and mucosal immune response in cattle by intranasal administration of pig serum albumin in alginate microparticles

Biodegradable microparticles are an efficient mucosal delivery system that protect antigens from the harsh mucosal environment and facilitate their uptake by M cells at the epithelium of mucosal-associated lymphoid tissue. In this study, we determined the systemic and mucosal immune response in calves following intranasal and oral immunization with pig serum albumin (PSA) encapsulated in alginate microparticles. The size of the particles ranged from 1 to 50 microm in diameter, with 95% of the particles being smaller than 5 microm. High levels of anti-PSA IgG1 antibodies were found in the serum, nasal secretions, and to a less extent in saliva of calves vaccinated intranasally, but not orally, with PSA-microparticles. There was no significant increase of PSA-specific IgA. A weak lymphocyte proliferative immune response was observed in peripheral blood mononuclear cells (PBMCs), and few anti-PSA antibody-secreting cells (ASC) were detected in the blood of calves immunized intranasally. The combined systemic and mucosal response observed in intranasally immunized animals may be attributed to the wide variation in the size of the alginate microparticles, with smaller particles translocating to regional lymph nodes and inducing a systemic immune response, and larger particles being retained in the NALT and inducing a mucosal immune response. The procedure presented here may be useful as an intranasal vaccine against respiratory diseases in cattle.     

Infectious salmon anemia virus: causative agent, pathogenesis and immunity

Infectious salmon anemia (ISA) virus (ISAV), an economically important new pathogen in marine aquaculture, is classified in the family Orthomyxoviridae, genus Isavirus. The main structural properties of this genus include enveloped virions 90-140 nm in diameter with surface projections of a combined receptor-binding hemagglutinin and receptor-destroying enzyme activity demonstrated to be an esterase, hence recently designated HE, and a genome composed of eight segments of linear, single-stranded, negative sense RNA ranging in length from 1.0 to 2.4 kb, with a total size of approximately 14.3 kb. The viral genome encodes at least ten proteins, of which nine are structural and one is non-structural. Examination of more than 160 ISAV isolates has led to the identification of two hemagglutinin subtypes of ISAV, one North American and one European. The immune response against ISAV after infection or vaccination does not provide full protection against the infection. The recent discovery of antibody-mediated uptake and replication of ISAV in macrophage-like fish cell lines suggests that Fc receptor-mediated antibody-dependent enhancement of the ISA virus infection might also occur in vivo, as the virus in Atlantic salmon (Salmo salar) targets endothelial cells lining blood vessels and macrophage-like cells. Cumulative mortalities in Atlantic salmon during natural ISA outbreaks and experimental infections range from 0 to 100%. ISAV causes fatal systemic infections in marine-farmed Atlantic salmon and asymptomatic infections in feral fish. Experimentally induced fatal clinical disease in rainbow trout (Oncorhynchus mykiss) has identified a correlate of virulence of ISAV that may explain its emergence as a fish pathogen.     

Differing Resistance of Atlantic Salmon Strains and Rainbow Trout to Gyrodactylus salaris Infection

Abstract

Laboratory studies were conducted on the susceptibility of different strains of Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss to infection with the monogenean Gyrodactylus salaris. This parasite, probably originating from the Baltic Sea region, is known to minimally affect Neva River (Baltic Sea) Atlantic salmon. However, following its introduction into Norway, G. salaris has caused severe mortality and morbidity among Norwegian Atlantic salmon, which are considered a highly susceptible strain. The cohabitation experiment included one stock of rainbow trout and four different strains of Atlantic salmon from the Baltic Sea region (Mörrum River, Sweden), Europe (Skjern River, Denmark; Conon River, Scotland), and North America (Bristol Cove River, Canada). Fish were exposed to a Norwegian strain of G. salaris, and parasite population development and distribution were monitored for 7 weeks. Rainbow trout exhibited low susceptibility to G. salaris infection, whereas Conon River and Skjern River Atlantic salmon were highly susceptible and exhibited high mortality rates. Mörrum River Atlantic salmon exhibited intermediate susceptibility and low mortality. Bristol Cove River Atlantic salmon harbored relatively low parasite numbers, but fish mortality was high. Our experiment showed that the Danish Skjern River strain of Atlantic salmon is highly susceptible to G. salaris infection, further supporting the hypothesis that Atlantic Ocean strains are more susceptible to G. salaris infection than are Baltic strains.     

Rickettsial infection in farmed Atlantic salmon in eastern Canada

The cause of death in a postsmolt, Atlantic salmon population with elevated levels of mortalities was investigated. Diagnosis of a rickettsia-like organism was based on gross pathology, histopathology, differential staining, electron microscopy and fluorescent antibody tests. The course of the infection and response to treatment are discussed. This is the first reported occurrence of salmon rickettsias in the Atlantic coast of North or South America.     

The efficacy of a single intraperitoneal injection of oxolinic acid in the treatment of bacterial infections in goldsinny wrasse (Ctenolabrus rupestris) and corkwing wrasse (Symphodus melops) studied under field and laboratory conditions

The efficacy of a single intraperitoneal injection of oxolinic acid to control an outbreak of atypical Aeromonas salmonicida infection in goldsinny wrasse (Ctenolabrus rupestris) and in the treatment of systemic vibriosis in corkwing wrasse (Symphodus melops) was examined. In addition a field study was performed to examine the effect of medication on the survival rate of goldsinny wrasse in Atlantic salmon cages. Four groups of wild caught goldsinny wrasse, each of 50 fish, were treated with an intraperitoneal injection of propylene glycol:saline (50:50) (control) or 50 mg/kg oxolinic acid at a concentration of 50 mg/mL. Three days after medication the fish in all groups were treated by an intraperitoneal injection of prednisolone acetate and an increase in seawater temperature from 9.0 to 11.5 degrees C. Cumulative mortalities were 18% in the two groups treated with oxolinic acid and 94 and 100% in the unmedicated control groups, giving a 'relative percentage survival' (RPS) value of 82%. A laboratory maintained population of originally wild caught corkwing wrasse experiencing high daily mortality was treated with oxolinic acid (50 mg/kg) or propylene glycol:saline (control). Cumulative mortalities were 84% (control) and 42% (oxolinic acid medicated group) giving an RPS value of 50%. In a field investigation using goldsinny wrasse approximately 30% were medicated with oxolinic acid (50 mg/kg) prior to stocking in cages with Atlantic salmon. In two of three cages the cumulative mortality was significantly lower (P = 0.025 and P < 0.001) in the medicated groups.     

Metabolism and residue depletion of albendazole and its metabolites in rainbow trout, tilapia and Atlantic salmon after oral administration

Metabolic and residue depletion profiles of albendazole (ABZ) and its major metabolites in three fish species, rainbow trout, tilapia and Atlantic salmon are reported. Based on these profiles, similarities (or dissimilarities) between species will determine the potential to group fish species. ABZ at 10 mg/kg body weight was incorporated into fish food formulated in a gelatin base or in gel capsule and fed as a single dose to six fish from each species. Rainbow trout were held three each in a partitioned 600-L tank. Tilapia and Atlantic salmon were housed in separate 20-L tanks. Samples of muscle with adhering skin were collected at 8, 12, 18, 24, 48, 72, and 96 h postdose from trout kept at 12 degrees C, at 4, 8, 12, 24, 48, 72, 96, 120, and 144 h postdose from tilapia kept at 25 degrees C and at 8, 14, 24, 48, 72, and 96 h postdose from Atlantic salmon kept at 15 degrees C. The samples were homogenized in dry ice and subjected to extraction and cleanup procedures. The final extracts were analyzed for parent drug ABZ and its major metabolites, albendazole sulfoxide (ABZ-SO), albendazole sulfone (ABZ-SO2) and albendazole aminosulfone using high-performance liquid chromatography with fluorescence detection. ABZ was depleted by 24 h in trout and tilapia and by 48 h in salmon; ABZ-SO, a pharmacologically active metabolite, was depleted by 48 h in tilapia, by 72 h in rainbow trout and was present until 96 h in salmon; and low levels of ABZ-SO2 and albendazole aminosulfone, both inactive metabolites, were detectable at least till 96 h in all three fish species.