The ontogeny of humoral immunity in rainbow trout, Salmo gairdneri

The ontogeny of the humoral immune response to a 'thymus dependent' and a 'thymus independent' antigen, human gamma globulin (HGG) and Aeromonas salmonicida (AS) respectively, was investigated in the fry of rainbow trout, Salmo gairdneri, by direct immersion vaccination in the antigens (dose 5 mg/L HGG; 10(8) cells/ml AS; 30 minutes) at known ages/weights from 7 days post hatch, and at 1, 2, 3, and 4 months post hatch. Half the fry in each group were tested for antibodies 4 weeks after vaccination, the remainder were reimmunised and tested again after a further 4 weeks. Appropriate controls to test for tolerance induction and memory responses were included. The results indicate that fry are capable of mounting a humoral immune response very early in ontogeny. There is a period of 'unresponsiveness' which persists for longer against HGG, than against AS, though it was not thought to be tolerance as such. Memory could be detected to HGG in fry given a first immunisation at 2 months. The results are compared with preliminary experiments in which fry were first thymectomised 4 weeks before the first immunisation. In fry thymectomised at 1 month post hatch, and tested for primary and secondary responses at 2 and 3 months, the primary response to HGG is unaltered, but the secondary response is reduced. Both the primary and secondary response to AS is unaltered. When thymectomy is performed later, the effect on the secondary response to HGG is no longer apparent, but the primary response to AS is slightly reduced.     

Comparison of the immunogenic effects of covalent-bonded immune complexes in mice and sheep

Covalent antigen-antibody complexes containing the protein antigen ovo-transferrin primed both mice and sheep to give an enhanced antibody response to a subsequent single injection of soluble ovo-transferrin. Complexes prepared using horse, sheep or rabbit antibody had a priming effect in mice, although rabbit antibody-antigen complexes were the most effective. In sheep, only rabbit antibody-antigen complexes significantly enhanced antibody levels.     

Blood changes in carp (Cyprinus carpio) induced by ulcerative Aeromonas salmonicida infections

Only recently Aeromonas salmonicida has been recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. Our attempts to formulate a vaccine based on bacterial surface antigens were unsuccessful in conferring reliable protection against lethal challenge. This lead us to study pathological changes in the humoral defense system during ulcerative A. salmonicida infection in carp. High numbers of opportunist pathogens such as A. hydrophila and Pseudomonas sp. were frequently recovered from the internal organs of moribund fish, in addition to A. salmonicida. These findings together with leucopenia in moribund fish suggest that pathogenesis is characterized by a state of immune suppression. In addition, fish which had sustained a sublethal infection were not protected against a subsequent lethal challenge. However, fish previously injected with a concentrated and inactivated culture supernatant showed protection. Differential blood cell counts did not differ between experimental and control groups during sublethal infection in contrast to serum proteins. Furthermore infected non-immune carp showed a progressive decrease of immunoglobulin and total serum protein levels before the day of peak mortality whereas protected carp maintained the immunoglobulin concentration despite a decrease in protein. Our observations suggest the involvement of multiple pathogenic events, affecting different parts of the humoral defense system during ulcerative A. salmonicida infection. The immunosuppressive effects can be minimized by prior vaccination with culture supernatant.     

Antibody response in salmonids against the 70 kDa serine protease of Aeromonas salmonicida studied by a monoclonal antibody-based ELISA

An antigen-capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies (mAb) was set up and evaluated for selective detection of salmonid antibody responses to the antigen P1, which is a weakly immunogenic exoprotease of typical Aeromonas salmonicida. This new assay permits a specific determination of anti-protease-antibodies, without antigen purification. Serum antibodies induced by the strongly immunogenic lipopolysaccharide could reliably be discriminated from anti-P1-antibodies. Antibody titres of 45 experimental antisera recorded by cELISA were moderately correlated with titres determined by routinely used indirect ELISA (iELISA) by detecting partially different antibody populations (r=0.753). Substitutions of immunoreactants and confirmatory immunoblotting strongly suggest that the mAb-based assay selectively recognises antibodies directed to epitopes of native protease. A conjugate of inhibited protease and cationized bovine serum albumin (cBSA) was found to engender a significant anti-protease-response in three salmonid species (P<0.05), whereas the unconjugated antigen and Apoject 1-Fural were proved to be ineffective. Recorded specific antibody titres were as high as 1:381,400, indicating a considerable enhanced immunogenicity of cBSA-conjugated P1 and high assay sensitivity. The established cELISA offers a promising approach to further improvement of monitoring fish humoral immune response to surface accessible epitopes of the immunosuppressive exoprotease, P1, and to scrutinize its protective significance.     

Oral immunisation of pigs with fimbrial antigens of enterotoxigenic E. coli: an interesting model to study mucosal immune mechanisms

The intestinal mucosal immune system can discriminate actively between harmful pathogenic agents and harmless food antigens resulting in different immune responses namely IgA production and oral tolerance, respectively. Recently, a pig model has been developed for studying intestinal mucosal immune responses in which F4 fimbrial antigens of enterotoxigenic Escherichia coli (F4 ETEC) are used as oral antigens. A unique feature of this model is that soluble F4 antigens can be administered to pigs which have a receptor for this fimbriae (F4R(+)) on their small intestinal villous enterocytes and pigs which do not have this receptor (F4R(-)). Oral administration of F4 to the F4R(+) pigs results in an intestinal mucosal immune response that completely protects the pigs against a challenge infection. In F4R(-) pigs such an intestinal mucosal immune response does not occur. However, a priming of the systemic immune system can be seen similar to the priming in pigs fed with the same dose of a food antigen, suggesting that F4 in F4R(-) pigs behaves as a food antigen. The fact that different mucosal immune responses can be induced with soluble F4, makes it an interesting model to study mucosal immune mechanisms in the pig.     

Effects of Feeding Spirulina on Specific and Nonspecific Immune Responses of Channel Catfish

Abstract

Administration of various immunostimulants to fish has resulted in enhanced immune responses. The purpose of this study was to determine if feeding Spirulina, a processed form of the blue-green alga Spirulina platensis, enhanced specific and nonspecific immunity and resistance against Edwardsiella ictaluri infection in channel catfish Ictalurus punctatus. Peritoneal phagocytes from fish fed Spirulina showed enhanced phagocytosis to zymosan and increased chemotaxis to E. ictaluri exoantigen. No significant difference in mortality due to E. ictaluri existed between fish fed Spirulina and fish fed a basal diet. No significant difference in antibody titer or in the percentage of fish positive for E. ictaluri antibody was found between the groups after immunization with formalin-killed E. ictaluri. Spirulina-fed fish had significantly higher antibody titers to key hole limpet hemocyanin (KLH) on day 22, and a greater percentage of these fish were positive for KLH antibody on days 15 and 36. Feeding Spirulina enhanced nonspecific cellular immune responses such as chemotaxis and phagocytosis but did not provide protection against infection with E. ictaluri. The use of Spirulina in feed resulted in enhanced antibody responses to KLH, a thymus-dependent antigen, but not to E. ictaluri, a thymus-independent antigen. These results indicate that stimulation of the nonspecific immune system of channel catfish does not provide enhanced protection from E. ictaluri.     

Protection against atypical Aeromonas salmonicida infection in common carp, Cyprinus carpio L., by oral administration of a mixed microbial culture of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae

A microbial culture was prepared by co-cultivation of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae for 48 hr at 30°C in rice bran extract medium, supplemented with dextrose. Oral administration of the resulting non-viable heat-inactivated microbial culture to common carp, Cyprinus carpio L., delivered in feed for four weeks, induced effective protection against experimental atypical Aeromonas salmonicida infection which causes "ulcer disease". After challenge of the carp by immersion, fish mortality and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with mixed microbial culture adsorbed on dry pellets relative to carp treated with medium or without extract. Atypical A. salmonicida was re-isolated from ulcerative lesions in parts of dead and surviving fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying microbial population changes during the progression of skin lesions. Among interleukin-1β (IL-1β), tumor necrosis factor-α, as well as CXC-α and CXC-β chemokines, gene expression of IL-1β was up regulated in the spleen and head kidney three weeks after administration of the mixed microbial culture. These results clearly show that this mixed microbial culture, delivered in feed, is effective in preventing A. salmonicida disease in carp.     

Protection against atypical Aeromonas salmonicida infection in carp (Cyprinus carpio L.) by oral administration of humus extract

Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms have been present. In the present study, oral administration of humus extract to common carp (Cyprinus carpio L.) induced effective protection against experimental atypical Aeromonas salmonicida infection. Mortality of fish and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with 10%, 5% or 1% humus extract adsorbed on dry feeding pellets. The median surviving days was also greater in fish treated with 10% or 5% humus extract than in untreated fish. Atypical A. salmonicida was isolated from ulcerative lesions of part of dead fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying bacterial population changes during the progression of skin lesions. These results clearly show that treatment of fish with humus extract is effective in preventing A. salmonicida disease.     

甲硝唑对鸡回忆性体液免疫应答反应的作用

本试验系统观察了甲硝唑对鸡回忆性体液免疫应答反应的作用,并与咪唑类化合物左旋咪唑进行了比较。28日龄鸡,免疫注射绵羊红细胞 牛血清白蛋白(SRBC BSA)悬液,或稀释的布氏杆菌(BA)抗原。56日龄时,以10.0、100.0mg/kg甲硝唑或10.0mg/kg 左旋咪唑饮水给药,连用3d,给药第二天同时进行第二次免疫注射。结果表明甲硝唑(10.0~100.0mg/kg)可使鸡体内对颗粒性胸腺依赖性抗原SRBC和非胸腺依赖性抗原BA抗体滴度回忆性免疫应答反应明显升高;但使鸡对可溶性胸腺依赖性抗原BSA抗体滴度回忆性免疫应答反应明显降低。与甲硝唑比较,左旋咪唑(10.0mg/kg)也存在着非常类似的作用。     

Comparison of antibody and cell-mediated immune responses in horses following feeding of a novel dietary antigen, ovalbumin, and rotavirus.

Adult ponies which were fed ovalbumin (OVA) daily for 2 weeks had significantly greater serum anti-OVA IgG (P = 0.001) and antigen specific lymphocyte responses (P = 0.031) after intramuscular injection with OVA given with saponin than control ponies which had not been fed the antigen. This suggests that, despite the lack of evidence of B- or T-cell activation in peripheral blood during the period of OVA feeding, the animals were primed for an active secondary immune response. Adult ponies were challenged with equine rotavirus, strain H-2, but no statistically significant differences were found in serum IgG-associated antibody responses or antigen-specific lymphocyte responses between the rotavirus-challenged group and the control group, either following rotavirus challenge or intramuscular injection of rotavirus antigen given with saponin. Our findings, that feeding the non-replicating protein antigen OVA appeared to prime for an increased immune response rather than inducing oral tolerance, may be of relevance to future studies on the way the equine gastrointestinal tract handles usually harmless antigens.     

Presence of antigen sensitized leukocytes in carp (Cyprinus carpio L) following bath immunization against Flexibacter columnaris

Bath immunization of carp (Cyprinus carpio L) resulted in protection of fish at natural challenge. Stimulation of leukocytes derived from thymus, spleen, anterior kidney and mid-kidney of fish immunized with Flexibacter columnaris bacterin revealed the presence of antigen sensitized cells in all lymphoid tissues except the anterior kidney. After 28 days a response was obtained in thymus and spleen leukocyte cultures.     

Age related changes in humoral immune response of hybrid striped bass (Morone chrysops x Morone saxatilis)

A number of factors can influence immune function affecting the magnitude and duration of specific immune responses. One factor which has been noted to affect immune responses is age of animal. In mammals, juveniles have a lower immune response when compared to adults. In fish, fry have a lower immune response than adults; however, few studies have investigated the immune response in juveniles, the age when most fish are reared in aquaculture settings. The humoral immune responses of hybrid striped bass (Morone saxatilis x Morone chrysops) at five different ages were compared to determine any maturational changes. Fifty fish were bath immunized with a commercial Vibrio vaccine at 4, 6, 9, 15, and 19 months of age. The antibody response in these fish was monitored by an enzyme-linked immunosorbent assay (ELISA) for 106 days post-vaccination. The magnitude, duration, and time to peak level were compared to controls and between the different ages using analysis of variance (ANOVA). Younger fish exhibited significantly lower antibody levels indicating that juvenile fish may not be able to mount as effective an immune response as young adult fish.     

Estimation of immunity in the developing calf: cellular and humoral responses to keyhole limpet haemocyanin

The efficacy of keyhole limpet haemocyanin (KLH) as a test antigen was determined in calves. Humoral and cellular (in vivo and in vitro) responses were compared. Calves were immunized with KLH at either 3 weeks or up to 5 months of age and immune responses were subsequently tested. Class and subclass antibody responses were detected by ELISA, lymphocyte blastogenesis was measured using a whole blood culture technique (LTT) and skin sensitivity responses were measured as an increase in skin thickness following intradermal injection. In young calves, skin test responses were maximal at 24 h and were found to correlate with IgG1 and IgG2 responses (P less than 0.01), with IgA (P less than 0.05) but not with IgM or LTT. Histological examination of skin swellings found a sequence of cellular events, with polymorphonuclear cells dominating until 48 h after intradermal injection, when mononuclear cells became involved. However, in older calves, skin test responses correlated not only with IgG1 and IgA responses but also with lymphocyte transformation (P less than 0.05). These findings suggest that, while immune responses to KLH may be a useful indicator of immune competence in calves, interpretation should be made with caution particularly in young calves.     

多头绦虫抗原特性的研究──应用IHA观察绵羊免疫及感染后的抗体消长规律

为了观察绵羊用多头蚴抗原免疫及感染后的抗体消长规律,为羊脑多头蚴病的免疫预防和免疫诊断提供依据,本试验应用多头蚴原头节可溶性抗原、囊壁可溶性抗原、囊液粗抗原致敏绵羊红细胞对绵羊免疫3次及虫卵攻击感染后的血清抗体进行间接血凝试验（IHA）检测。结果表明,原头节抗原免疫组、囊壁抗原免疫组、囊液抗原免疫组及原头节ES抗原免疫组在首次免疫后1周,抗体滴度迅速升高,第3次免疫后1周达到峰值,虫卵感染后开始下降,到感染后30周接近正常水平。多头蚴3种抗原对同种抗原免疫组血清检测敏感性、特异性优于其它抗原,原头节免疫组、囊壁免疫组、囊液免疫组抗体水平明显高于原头节ES抗原免疫组。     

Adjuvant and immunostimulatory effects of beta-glucan administration in combination with lipopolysaccharide enhances survival and some immune parameters in carp challenged with Aeromonas hydrophila

Combined effects of beta-glucan and lipopolysaccharide (LPS) on survival and immune response were studied in Cyprinus carpio that were challenged with the pathogen Aeromonas hydrophila. beta-Glucan from Saccharomyces cervisiae and LPS from a virulent strain of A. hydrophila were used in this study. Different concentrations of beta-glucan+LPS mixture were administered on days 1, 7, and 14 through different routes (intraperitoneal injection, bathing, and oral administration). Control and test fish were challenged by intraperitoneal injection of LD50 concentration of A. hydrophila on day 16 and subsequently, mortality and relative percent survival (RPS) were recorded. Intraperitoneal injection elicited 100% RPS even at the lowest concentration (100 microg beta-glucan+10 microg LPS); whereas, oral administration improved RPS rate of carps at higher concentration (1% beta-glucan+0.25% LPS). Bathing did not improve the RPS. Test animals injected with even the minimum dose of the immunomodulators (100 microg beta-glucan+10 microg LPS/fish) had a significant increase in total blood leukocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by macrophages was also elevated, which presumably aided the efficient killing of bacterial pathogen. Lower concentration of beta-glucan+LPS had an adjuvant effect on antibody production as pretreatment by injection of 100 microg beta-glucan+10 microg LPS/fish resulted in higher antibody titer against A. hydrophila following vaccination. RT-PCR analyses showed that the expression of interleukin-1beta mRNA did not increase in test fish when compared with the control. Classical and alternative complement pathways were not affected by either the dose or the route of administration of the compounds. It may be concluded that intraperitoneal injection and oral administration, and not the bathing, of beta-glucan+LPS mixture in carp could enhance resistance to challenge by A. hydrophila through changes in several non-specific and specific immune responses.     

Vaccination Response of Young Foals to Keyhole Limpet Hemocyanin: Evidence of Effective Priming in the Presence of Maternal Antibodies

The ability to produce antibodies is essential for protection from infectious disease; however, in the neonate, maternal antibodies have been proposed to interfere with the foal's ability to respond to vaccination. In species other than the equid, keyhole limpet hemocyanin, a high-molecular weight protein, is used in vivo as an experimental vaccine component because of its high intrinsic immunogenicity. In this study, we show that young foals are able to produce a primary antibody response to vaccination at an early age. Thus, foals, like human infants, are capable of responding to antigenic exposure to a novel antigen (keyhole limpet hemocyanin) during the neonatal period. Although vaccinating foals in the presence of maternal antibodies failed to induce a primary serological response, priming occurred as comparable anamnestic responses were detected upon subsequent exposure to the antigen. There was no evidence of tolerance induction.     

Immunostimulants added to injected Aeromonas salmonicida bacterin enhance the defense mechanisms and protection in rainbow trout (Oncorhynchus mykiss).

Immunostimulants were given to rainbow trout for assaying effects on modulating non-specific defense mechanisms, specific immune response, and protection levels against pathogen challenge with Aeromonas salmonicida. Three drugs, levamisole (an approved veterinary drug in the USA), a quaternary ammonium compound (QAC), and a short-chain polypeptide (ISK) were found to affect the non-specific defense mechanism activities, which were measured by changes in circulatory neutrophil and phagocytic activity levels, and the specific immune response factors, which were measured by numbers of plaque-forming cells, and circulatory antibody levels. When given alone, the immunostimulants elevated the non-specific factors. When injected in combination with an A. salmonicida O-antigen bacterin, the non-specific factors were further elevated, and the specific response was raised over samples taken from fish given the bacterin without the immunostimulants. Challenge tests with the virulent pathogen, A. salmonicida, showed a 5-6 day delay in the onset of mortalities in the fish given the immunostimulants alone, and a 12-14 day delay when immunostimulants given were combined with the bacterin. In the groups given the QAC or ISK with the bacterin, there was a 20% and 40% survival rate, respectively.     

蛋氨酸对幼建鲤疾病抵抗能力及免疫应答的影响

本试验旨在研究蛋氨酸对幼建鲤疾病抵抗能力及免疫应答的影响.选择体重为(12.34±0.02)g健康建鲤864尾,平均分成6组,每组144尾(每组设3个重复,每个重复48尾),分别饲喂蛋氨酸水平为0.39%、0.70%、1.00%、1.30%、1.60%和1.90%的饲料,饲养60 d后用嗜水气单胞菌攻毒17 d,考察蛋氨酸对幼建鲤免疫功能的影响.结果表明:适宜蛋氨酸水平极显著提高了攻毒前头肾体指数、后肾体指数、脾体指数、血液红、白细胞数量和攻毒后红细胞数量(P<0.01);显著提高了攻毒前白细胞吞噬率和攻毒后成活率、白细胞数量、白细胞吞噬率、血清酸性磷酸酶活力、溶菌酶含量、补体C3含量、凝集素水平和抗嗜水气单胞菌抗体效价(P<0.05).由此得出,蛋氨酸通过增强白细胞吞噬能力和提高特异性抗体效价从而提高幼建鲤的非特异性和特异性免疫力,增强疾病抵抗力.     

F4 receptor-independent priming of the systemic immune system of pigs by low oral doses of F4 fimbriae

Oral administration of F4 fimbriae of Escherichia coli induces intestinal mucosal immune responses in F4 receptor-positive (F4R(+)) pigs, but not in F4R(-) pigs. We examined whether F4 fimbriae in F4R(-) animals behave like a food antigen and can induce oral tolerance. Therefore, F4R(+) and F4R(-) pigs were fed 2mg of F4 and challenged i.m. to evaluate the effect of oral F4 on the systemic immune system. As control antigen, two different oral doses (2 and 600 mg) of OVA were used. Thirty days after the i.m. OVA challenge, the OVA-specific serum IgG titre in 600 mg-fed pigs was lower than that in non-fed animals, indicating that tolerance was induced. Conversely, in the 2mg-fed pigs a rapid increase of OVA-specific IgG with higher titres than those in non-fed pigs was seen following challenge, indicating a priming of the systemic immune system. A similar priming was seen in both F4-fed F4R(-) and F4R(+) pigs. Upon challenge, non-fed pigs displayed a primary immune response with a slow increase of F4-specific serum IgG, whereas F4-fed F4R(-) and F4R(+) pigs showed secondary responses with a rapid increase of serum IgG. This was expected in F4R(+) pigs, as in these animals oral F4 induces F4-specific antibody-secreting cells in the spleen, suggesting a priming of the systemic immune system. However, also the F4-fed F4R(-) pigs displayed a secondary response, despite the failure to detect a response upon oral F4 administration. These findings suggest that the F4 antigen, at a dose of 2 mg, behaves like a common food antigen in F4R(-) pigs, namely it induces a systemic priming.     

Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells

ABSTRACT: Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry.