Isolation and partial characterization of immunoglobulin from cod (Gadus morhua L.) (2023)

Burbot (Lota lota) are an ideal candidate for cool or cold-water aquaculture and are gaining interest because of their high economic value, low temperature requirements, and fast growth rate. Limited information exists on the innate and adaptive immune systems of this species. This is partly due to the lack of species-specific tools to determine antibody responses following disease or vaccination or to characterize the immune response in general. An anti-IgM monoclonal antibody (mAb 27C) was developed and characterized via enzyme-linked immunosorbent assay (ELISA) and Western blot for species specificity, affinity to the heavy chain of burbot IgM, and cross-reactivity to other reagents used in the analysis. The 27C monoclonal antibody was further utilized to develop an ELISA protocol to measure the specific antibody response of burbot following exposure to two pathogenic strains of Aeromonas sp. (A141 and IR004). This ELISA confirmed that vaccinated burbot that survived the challenge with either strain developed statistically higher titers of anti-Aeromonas antibodies specific for the relative strain when compared to fish that were not vaccinated or challenged. Western blot analysis further demonstrated that burbot surviving challenge had serum IgM that recognized distinct antigens specific to the strain they were challenged with, A141 bound to antigens in the 50-250Kda range and IR004 bound to a distinct 150Kda antigen. Western blots further indicated that each strain shared antigenic regions regardless of experimental Aeromonas strain exposure. Finally, immunofluorescent staining confirmed that mAb 27C binds to membrane-bound IgM (presumably B cells) on burbot head kidney cells. Taken together, results from this study demonstrate that mAb 27C specifically recognized burbot IgM and will be an important tool to further characterize the adaptive and cellular immune responses of this fish species.

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F.noatunensis compared to saline formulation. The antigen epitope was a 20–25kDa component of F.noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1β and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1β expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1β and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F.noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F.noatunensis in cod.

Natural antibodies are present in the serum of vertebrates regardless of antigenic stimulation. Characteristic activity is commonly detected against haptenated proteins, single stranded DNA and thyroglobulin. Natural antibodies are believed to provide an instant protection against pathogens of a broad specificity and to participate in homeostasis. Cod is a poor antibody responder but shows a relatively high level of natural antibodies against haptenated proteins. In this project the specificity, activity and affinity of natural antibodies was studied in different groups of cod and the effects of age/size, environmental temperature, immunisation and infection examined. Antigen driven selection of natural antibodies was also studied in one group of cod. The results demonstrated a broad and yet characteristic specificity, primarily directed against haptenated proteins and possible food antigens. The antibody activity increased with increasing age and at higher temperature whereas immunostimulation by immunisation or infection resulted in variable response. The affinity index of natural antibodies of cod generally did not correlate with changes in the antibody activity but it was in the same range as the affinity index of acquired cod antibodies and that of some mammalian monoclonal acquired antibodies. Analysis of antigen driven antibody selection showed that the natural antibody repertoire of individual cod was heterogeneous with respect to its affinity for haptenated protein.

Bacterial diseases such as vibriosis, atypical furunculosis and francisellosis, are registered as an increasing problem in cod farming in Norway. In order to develop efficient vaccines against diseases it is of interest to investigate if the cod immune system differentiates between various serotypes of Vibrio anguillarum and variants of Aeromonas salmonicida associated with the diseases by raising specific antibody responses. Cod of the same origin were shown to raise significant responses to V. anguillarum, A. salmonicida and the intracellular bacteria Francisella sp. Individual responses to V. anguillarum or A. salmonicida varied from none to high responses, while all individuals immunised with Francisella revealed a significant response. The cod immune system appeared in some degree to distinguish between V.anguillarum serotypes and A. salmonicida variants. Although all bacteria had induced significant antibody responses detectable in whole cell ELISA, only some had induced antibodies with specificity to linear O-polysaccharide epitopes on blot.

Indian major carps (IMC), rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were immunized with bovine serum albumin and the serum immunoglobulin M (IgM) was purified by affinity chromatography. The heavy and light chain of IgM of all the three species of IMC were about 88 and 26kDa, respectively. Anti-fish IgM antibody against all the three species were raised in mice and the reaction of anti-fish IgM antibodies with IgM of all the three species of IMC were studied by Western blot. The anti-fish IgM antibodies reacted strongly with the heavy chain of the same species against which it was raised while the reactions with the heavy chain of other species were milder indicating some degree of epitope sharing among the heavy chains of IgM of IMCs. However, there was no cross-reaction with the light chain of any of the IgM.

Materials Chemistry and Physics, Volume 171, 2016, pp. 346-351

Nano-sized CePO₄@NaCe(SO₄)₂(H₂O) and CePO₄ with hexagonal phase have been prepared by simply varying the reactant P/Ce molar ratio in bacterial liquid. The phase composition of two samples was checked via Fourier transform infrared spectroscopy (FTIR), energy dispersive analysis of X-rays (EDS) and X-ray diffraction (XRD) analyses, displaying the presence of CePO₄@NaCe(SO₄)₂(H₂O) and CePO₄ with average crystallite size are 32.34 and 15.61nm, respectively. The scanning electron microscopy (SEM) images show that nano-clusters and sphere-like in shape with a narrow diameter distribution were observed in two samples. The transmission electron microscopy (TEM) photographs further indicate obtained CePO₄@NaCe(SO₄)₂(H₂O) and CePO₄ nanoparticles correspond to nanosheets and nanorods, respectively. The emission spectra of CePO₄@NaCe(SO₄)₂(H₂O) and CePO₄ display a broad band of 300–380nm range with the strongest emission at 342nm in the violet region.

Parasitology International, Volume 64, Issue 2, 2015, pp. 167-172

Intestinal mucous cell numbers and their glycoconjugate composition were investigated by histochemical methods in uninfected chub, Squalius cephalus, and in conspecifics naturally parasitised with the acanthocephalan Pomphorhynchus laevis. A sub-population of 42 chub from the River Tiber (Perugia, Italy) were sampled and screened for ecto and endoparasites. No parasites were found in gills and in other visceral organs of chub and P. laevis appeared to be the only enteric worm encountered. In all infected chub (twenty-eight out of 42) this acanthocephalan was encountered mainly in the mid-gut. In situ, an excessive yellowish mucus or catarrh was observed around each acanthocephalan. Hyperplasia and hypertrophy of the mucous cells were only evident near the site of P. laevis attachment where the total number of mucous cells and the number of those containing acidic, particularly non-sulphated mucins, or mixed glycoconjugates were significantly higher. In intestinal regions of infected fish far away from the point of parasite attachment, there were no statistical differences in the density of mucous cells in comparison to uninfected fish. Interestingly, in parasitised chub, the length of intestinal folds was significantly larger close to the sites at which P. laevis attach when compared to the length of the intestinal folds located further away from the acanthocephalans and/or in uninfected intestines. The effect of P. laevis on intestinal mucous cells of S. cephalus was compared to other parasite–host systems and the role of enhanced mucus production in parasitized intestines was discussed.

Vaccine, Volume 33, Issue 30, 2015, pp. 3526-3532

The highly conserved extracellular domain of Matrix protein 2 (M2e) of influenza A virus has been previously investigated as a potential target for an universal influenza vaccine. In this study we prepared four lipopeptide influenza vaccine candidates in which the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine, (Pam2Cys) was attached to either the N- or C-terminus of the M2e consensus sequence SLLTEVETPIRNEWGCRCNDSSDP and its analogue sequence with the two cysteine residues replaced with serine residues. The results of animal study show that each of these lipopeptides induced strong M2e-specific antibody responses in the absence of extraneous T helper cell epitope(s) which are normally incorporated in the previous studies or addition of extraneous adjuvant and that these antibodies are protective against lethal challenge with influenza virus. Comparison of different routes of inoculation demonstrated that intranasal administration of M2e lipopeptide induced higher titers of IgA and IgG2b antibodies in the bronchoalveolar lavage than did subcutaneous vaccination and was better at mitigating the severity of viral challenge. Finally, we show that anti-M2e antibody specificities absent from the antibody repertoire elicited by a commercially available influenza vaccine and by virus infection can be introduced by immunization with M2e-lipopeptide and boosted by viral challenge. Immunization with this lipidated form of the M2e epitope therefore offers a means of using a widely conserved epitope to generate protective antibodies which are not otherwise induced.

Aquaculture, Volume 456, 2016, pp. 76-82

Acta Tropica, Volume 152, 2015, pp. 56-59

The human Pegivirus (HPgV, also known as GBV-C virus or hepatitis G virus) is a lymphotropic RNA-virus phylogenetically related to the Hepatitis C virus, which infects approximately 5% of the world’s human population. Recently, two novel, presumably hepatotropic, pegiviruses, designated as equine Pegivirus (EPgV) and Theiler’s Disease Associated Virus (TDAV), were discovered in horses with clinical and laboratory evidence of hepatic disease. To verify the occurrence of pegiviruses infection in horses from Pará State, northern Brazil, serum samples from 114 horses located in four cities (Acará, Belém, Dom Eliseu and Ananindeua) were submitted for the molecular analysis of EPgV by nested RT-PCR. The results of nucleotide sequencing and phylogenetic analysis of EPgV NS3 and NS5B genomic regions confirmed one positive sample among 114 tested samples (1/114; 0.8%). No evidence of TDAV infection was found, but despite the low prevalence and unknown clinical significance among the studied population, these results represent the first molecular detection of EPgV in horses in South America.

International Dairy Journal, Volume 47, 2015, pp. 46-51

Interactions between bovine β-lactoglobulin (β-Lg) genetic variants (A, B and a mixture thereof) and α-ionone, β-ionone and vanillin were studied by tryptophan fluorescence spectroscopy under various conditions of pH and ionic strength. When β-ionone was added to β-Lg, we found progressive increase in quenching from pH 3.0 to pH 8.0 and relative decrease at pH 11.0. Fluorescence quenching progressively increased from pH 3.0 through to pH 11.0 when vanillin was added. Small differences in quenching of variants β-Lg variants A and B were observed at pH 8.0 for β-ionone, but not for vanillin at any pH. NaCl affected the fluorescence quenching of β-Lg by β-ionone at pH 7.0 and 8.0 and by vanillin at pH 8.0 and 11.0. No apparent interaction between α-ionone and β-Lg was observed under the conditions studied. The results suggest that β-ionone and vanillin bind at different domains of β-Lg, possibly with different binding mechanisms.