Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.     

Electrophoretic and Western blot analyses of the lipopolysaccharide and glycocalyx of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS) and it has emerged as one of the most significant bacterial pathogens in salmonid aquaculture worldwide. Previous studies have suggested that the O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) of F. psychrophilum is highly immunogenic and may be involved in eliciting a protective immune response in rainbow trout (Oncorhynchus mykiss Walbaum). In the present study, SDS-PAGE and Western blotting techniques were used to analyse the carbohydrate antigens of F. psychrophilum. Our analysis identified two distinct carbohydrate-banding patterns. One banding pattern corresponds with LPS, and we hypothesise that the other carbohydrate-banding pattern is that of the loosely associated glycocalyx of F. psychrophilum. Electron microscopy of F. psychrophilum cells immunogold labelled with a monoclonal antibody specific for this banding pattern supports this hypothesis as the outermost layer of the bacterium was heavily labelled. This is a significant finding because the immunogenic antigens that have been referred to as the O-PS of LPS, and implicated as potential vaccine candidate antigens, appear to be components of the glycocalyx of F. psychrophilum. This research suggests that the glycocalyx of F. psychrophilum may be an important antigen to consider for the development of a vaccine to control CWD and RTFS.     

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.     

Responses of cloned rainbow trout Oncorhynchus mykiss to an attenuated strain of infectious hematopoietic necrosis virus

The objective of this work was to examine the response of homozygous clones of rainbow trout to vaccination by an attenuated strain (Nan Scott Lake; NSL) of infectious hematopoietic necrosis virus (IHNV). Adult rainbow trout of the Hot Creek Strain (YY males maintained in a recirculating system at 12 degrees C) were injected 3 times with 10(5) to 10(7) plaque forming units (pfu) of NSL. Intraperitoneal injections were given at Day 0 and at 2 and 4 mo post-infection. All fish were nonlethally bled at monthly intervals for 18 mo. Serum from each fish was analyzed by the complement-dependent neutralization assay and by western blot against purified NSL virus. The highest virus neutralization titers were detected 4 mo after the first injection, and peaked at 1280. When sera were analyzed by western blot, the predominating responses of the serum from immunized fish on the reduced western blot were against M1, a matrix protein of the virus and to a 90 kDa stress protein. The 90 kDa protein was identified by a monoclonal antibody as a stress protein derived from the CHSE-214 cells in which the purified IHN virus was grown and which associates with the virus during purification.     

Expressed Sequence Tag Analysis of Kidney and Gill Tissues from Rainbow Trout (Oncorhynchus mykiss) Infected with Infectious Hematopoietic Necrosis Virus

Expressed sequence tags (ESTs) were obtained from the kidney and gill tissues of rainbow trout, Oncorhynchus mykiss, infected with infectious hematopoietic necrosis virus (IHNV). The results of single-pass sequencing of ESTs from 198 clones (AU081027–AU081192) from kidney complementary DNA and 45 clones (AU081193–AU081236) from gill cDNA are reported herein. Sequences of the cDNA clones were compared with sequences in the GenBank database. Fourteen clones (20%) appeared to be completely unknown and may represent newly described genes, whereas 158 clones (80%) were identified on the basis of matches to sequences in the database. Three of the unidentified sequences were isolated from both the kidney and the gill cDNA libraries. However, no sequences were identical between kidney and gill clones. Received December 7, 1999; accepted April 28, 2000.     

Concurrent injection of a rhabdovirus-specific DNA vaccine with a polyvalent,oil-adjuvanted vaccine delays the specific anti-viral immune response in Atlantic salmon,Salmo salar L.

Vaccines are commonly used in salmonid aquaculture as a method of disease prevention. Although there is a substantial amount of published research regarding the immunological and physiological effects following the injection of different polyvalent vaccines and DNA vaccines, there are no published reports examining the physiological and immunological effects of concurrent vaccine injection, which is the situation encountered in aquaculture. Using key immunological parameters such as lysozyme activity and specific antibody titres we examined the short-term activation of the immune response of cultured Atlantic salmon (Salmo salar L.) following concurrent injection with a traditional, polyvalent, oil-adjuvanted vaccine (AV) and an IHNV-specific DNA vaccine (DV). Our results indicate that different aspects of the innate and adaptive immune responses are influenced in either a positive or negative manner. While concurrent vaccine injection elicited an increase in lysozyme activity, changes in antibody titre (Ab) were antigen specific. The production of anti-Aeromonas salmonicida Abs was significantly greater in the combined vaccine group at 296 degree days post-vaccine injection (dd pvi), while the production of anti-Listonella anguillarum Abs was significantly greater at 106 dd pvi in the combined vaccine group. Of even greater interest was the apparent delay in production of IHNV-specific neutralizing antibodies (NAb) when the DV was injected concurrently with the polyvalent AV. The results indicated that concurrent injection of a polyvalent oil-AV and a DV can be beneficial to the production of antibodies; however, the specific anti-viral response may be delayed.     

Evidence from bioinformatics, expression and inhibition studies of phosphoinositide-3 kinase signalling in Giardia intestinalis

Background  

Giardia intestinalis is a parasitic protozoan and major cause of diarrhoeal disease. Disease transmission is dependent on the ability of the parasite to differentiate back and forth between an intestine-colonising trophozoite and an environmentally-resistant infective cyst. Our current understanding of the intracellular signalling mechanisms that regulate parasite replication and differentiation is limited, yet such information could suggest new methods of disease control. Phosphoinositide-3 kinase (PI3K) signalling pathways have a central involvement in many vital eukaryotic processes, such as regulation of cell growth, intracellular membrane trafficking and cell motility. Here we present evidence for the existence of functional PI3K intracellular signalling pathways in G. intestinalis.     

Maternal prenatal depressive symptoms predict infant NR3C1 1F and BDNF IV DNA methylation

Prenatal maternal psychological distress increases risk for adverse infant outcomes. However, the biological mechanisms underlying this association remain unclear. Prenatal stress can impact fetal epigenetic regulation that could underlie changes in infant stress responses. It has been suggested that maternal glucocorticoids may mediate this epigenetic effect. We examined this hypothesis by determining the impact of maternal cortisol and depressive symptoms during pregnancy on infant NR3C1 and BDNF DNA methylation. Fifty-seven pregnant women were recruited during the second or third trimester. Participants self-reported depressive symptoms and salivary cortisol samples were collected diurnally and in response to a stressor. Buccal swabs for DNA extraction and DNA methylation analysis were collected from each infant at 2 months of age, and mothers were assessed for postnatal depressive symptoms. Prenatal depressive symptoms significantly predicted increased NR3C1 1F DNA methylation in male infants (β = 2.147, P = 0.044). Prenatal depressive symptoms also significantly predicted decreased BDNF IV DNA methylation in both male and female infants (β = −3.244, P = 0.013). No measure of maternal cortisol during pregnancy predicted infant NR3C1 1F or BDNF promoter IV DNA methylation. Our findings highlight the susceptibility of males to changes in NR3C1 DNA methylation and present novel evidence for altered BDNF IV DNA methylation in response to maternal depression during pregnancy. The lack of association between maternal cortisol and infant DNA methylation suggests that effects of maternal depression may not be mediated directly by glucocorticoids. Future studies should consider other potential mediating mechanisms in the link between maternal mood and infant outcomes.