A holistic view of the dynamisms of teleost IgM: a case study of Streptococcus iniae vaccinated rainbow trout (Oncorhynchus mykiss)

To date, little is known about how trout IgM, the primary antibody of fish, varies in titer, specificity, disulfide cross-linking, and affinity following immunization with a pathogen. Work using defined antigens has demonstrated that the disulfide cross-linking structure of IgM becomes increasingly more polymerized during an immune response, coinciding with an increase in affinity, but it is unknown if this has relevance to aquatic pathogens. Understanding how IgM varies following vaccination with an aquatic pathogen is of considerable importance as effector functions allocated to multiple antibody isotypes in mammals are essentially relegated to this single molecule. To gain insights into the dynamism of IgM, rainbow trout were immunized with Streptococcus iniae and individual serum titers, their specificity and affinity to S. iniae, and the disulfide cross-linking pattern of both total-serum and specific Ig were analyzed over a period of 37 weeks. We found that in vaccinated animals titer increased by a factor of ≈100 from starting levels, affinity increased 10-fold, and diversity of S. iniae proteins recognized by trout antibody increased at least 5-fold. Most intriguing, though less cross-linked IgM predominated early in response, by week 5, the fully tetramerized antibody comprised 50% of total specific protein. We propose that this is a mechanism to optimize efficacy of carrying out effector functions and recognizing a wide array of epitopes with higher affinity.     

A Yersinia pestis guaBA mutant is attenuated in virulence and provides protection against plague in a mouse model of infection

There is a need to develop effective countermeasures for Yersinia pestis, the etiologic agent of plague and a potential bioterrorism agent. Salmonella and Shigella spp. deleted in the guaBA genes involved in guanine biosynthesis have been shown to be attenuated in vivo. In this study, we sought to determine whether deletion of the guaBA operon would render Y. pestis auxotrophic for guanine and avirulent; such a strain could serve as a live attenuated plague vaccine candidate. A Y. pestis guaBA mutant was generated by specific deletion of a segment of the guaBA operon, producing a guanine auxotroph that was highly attenuated in a mouse model of Y. pestis infection. Furthermore, mice vaccinated with a single dose of 7 × 10⁴ CFU via the intravenous route were fully protected against subsequent lethal challenge with the Y. pestis parental strain. These findings identify guaBA as a target for deletion to generate a live attenuated plague vaccine.     

The Yersinia pseudotuberculosis YplA phospholipase differs in its activity,regulation and secretion from that of the Yersinia enterocolitica YplA

Analysis of the Yersinia pseudotuberculosis and Yersinia pestis genomes indicates that both species carry an identical copy of a gene that is predicted to encode a protein which shares 80% similarity to the Yersinia enterocolitica YplA, a secreted phospholipase that has been shown to contribute to virulence. In contrast to well tolerated production of the Y. enterocolitica YplA in Escherichia coli, Y. pseudotuberculosis YplA expression was found to be toxic; however, cell viability could be restored if the Y. pseudotuberculosis YplA was expressed in the presence of its accessory protein YplB. In vitro, Y. pseudotuberculosis YplB was shown to reduce the activity of its cognate phospholipase in a dose-dependent manner. To determine whether the Y. pseudotuberculosis and Y. enterocolitica YplAs were secreted and regulated in a similar manner, secretion and promoter activity assays were performed. Unlike the situation apparent in Y. enterocolitica, expression of the Y. pseudotuberculosis yplA gene did not appear to be controlled by the flagellar regulon, nor did the phospholipase appear to be efficiently exported through the flagellar apparatus. These results indicate that the Yersinia YplAs vary in many of their attributes despite their high degree of amino acid homology.     

Evaluation of a Yersinia pestis mutant impaired in a thermoregulated type VI-like secretion system in flea,macrophage and murine models

Type VI secretion systems (T6SSs) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499–YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26 °C in comparison to 37 °C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499–YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 h post-infection for bacterial cells pre-grown at 26 °C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499–YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37 °C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain.     

Long-term liquid culture of haematopoietic precursor cells from the head kidney and spleen of the rainbow trout (Oncorhynchus mykiss)

Cells from the spleen and head kidney of rainbow trout (Oncorhynchus mykiss) were cultured in vitro. The cells were held in culture for more than three months. Spleen cells lived only a few days if cultured between October and May, but long-term cultures were possible in late spring and summer. This variation is assumed to be due to seasonal variations in the content of precursor cells. An adherent cell layer (stroma) was found, which was occupied by other cells on the surface or in niches of the stromal network. Various cell types could be identified by different cytochemical methods and electron microscopy: macrophages, blast cells, fibroblastoid and epitheloid cells, adipocytes and multinucleated cells. About 20% of stromal cells were found to be capable of phagocytosis. Living cells were found in the supernatant of cultures: granulocytes, lymphocytes, blast cells, multinucleated cells. The admixture of phytohaemagglutinin (PHA) or horse serum (HS) was necessary for development of the stroma-like network. It is supposed, that PHA promotes the release of growth factors, and that HS could also produce the same effects. HS also causes strong growth of stromal structures and therefore the release of growth factors by the stroma.     

Pronephros and spleen cells from rainbow trout (Oncorhynchus mykiss) in vitro: Growth factors produced under the influence of mitogens

Supernatants or conditioned media (CM) were produced by rainbow trout pronephros cells (PNC) and spleen cell cultures (1×10⁶/ml), by addition of 20 g/ml phythaemagglutinin (PHA), 5 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (PHA) (for 2h), PHA together with PMA, 10% horse serum (HS), or 100 g/ml concanavalin A (ConA) after a culture of 7 days. Only PNC were effective growth factor producers. The effect of different concentrations of CM (5%–28%) on cell number was tested after a cultivation period of 14 days. PNC at a concentration of 1×10⁶/ml were cultured with various concentrations of CM and the proliferation was tested by the XTT-test (testing the dehydrogenase activity of the cells by formation of a formazan) after 10 days. CM produced by cells with PHA, PHA and PMA and HS increased the proliferation in a concentration-dependent manner. CM produced with PMA alone was effective in the XTT-test. There was no synergistic or additive effect of PMA with PHA. CM produced with ConA had no effect, although in the XTT-test a strong proliferation of PNC in presence of 100 g/ml ConA was observed. In a semisolid culture with collagen, CM treatment resulted in prevention of cell death and an increase in cell size but did not induce proliferation. All CM obtained from spleen cells had no effect. Stimulation of spleen cells by CM could be seen only in the XTT-test. PHA and HS trigger the PNC to release growth factors in vitro which stimulate cell growth and/or prevent cell death.     

Leucocyte migration in rainbow trout (Oncorhynchus mykiss [Walbaum]): Optimization of migration conditions and responses to host and pathogen (Diphyllobothrium dendriticum [Nitzsch]) derived chemoattractants

A rainbow trout leucocyte-derived chemoattractant(s) was prepared and tested as a stimulant of leucocyte migration. It was used to optimize an in vitro leucocyte migration assay using a 48-well micro chemotaxis chamber. This assay has subsequently been used to test the chemoattractant activity of antigen extracts from the tegument of Diphyllobothrium dendriticum plerocercoids and conditioned medium obtained after in vitro maintenance of live plerocercoids. Leucocytes were found to have an increased directional motility (chemotactic response) to the host-derived chemoattractant(s) but a random increased motility (chemokinetic response) following stimulation/contact with parasite-derived antigens.     

The Longus type IV pilus of enterotoxigenic Escherichia coli (ETEC) mediates bacterial self-aggregation and protection from antimicrobial agents

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. ETEC pili and non-pili adherence factors designated colonization surface antigens (CSA) are believed to be important in the pathogenesis of diarrhea. Longus, a type IV pilus identified as the CSA₂₁, is expressed in up to one-third of ETEC strains, and share similarities to the toxin-coregulated pilus of Vibrio cholerae, and the bundle-forming pilus of enteropathogenic E. coli. To identify longus phenotype and possible function, a site-directed mutation of the lngA major subunit gene in the E9034A wild type ETEC strain was constructed. Lack of longus expression from the lngA mutant was demonstrated by immunoblot analysis and electron microscopy using specific anti-LngA antibody. Formation of self-aggregates by ETEC was shown to be dependent on longus expression as the lngA mutant or wild type grown under poor longus expression conditions was unable to express this phenotype. Longus-expressing ETEC were also associated with improved survival when exposed to antibacterial factors including lysozyme and antibiotics. This suggests that longus-mediated bacterial self-aggregates protect bacteria against antimicrobial environmental agents and may promote gut colonization.     

Association of cellular and molecular alterations in Leydig cells with apoptotic changes in germ cells from testes of Graomys griseoflavus × Graomys centralis male hybrids

Spermatogenesis is disrupted in Graomys griseoflavus × Graomys centralis male hybrids. This study was aimed to determine whether morphological alterations in Leydig cells from hybrids accompany the arrest of spermatogenesis and cell death of germ cells and whether apoptotic pathways are also involved in the response of these interstitial cells. We used three groups of 1-, 2- and 3-month-old male animals: (1) G. centralis, (2) G. griseoflavus and (3) hybrids obtained by crossing G. griseoflavus females with G. centralis males. Testicular ultrastructure was analyzed by transmission electron microscopy. TUNEL was studied using an in situ cell death detection kit and the expression of apoptotic molecules by immunohistochemistry. The data confirmed arrest of spermatogenesis and intense apoptotic processes of germ cells in hybrids. These animals also showed ultrastructural alterations in the Leydig cells. Fas, FasL and calbindin D_28k overexpression without an increase in DNA fragmentation was detected in the Leydig cells from hybrids. In conclusion, the sterility of Graomys hybrids occurs with ultrastructural changes in germ and Leydig cells. The enhancement of Fas and FasL is not associated with cell death in the Leydig cells. Probably the apoptosis in these interstitial cells is inhibited by the high expression of the antiapoptotic molecule calbindin D_28k.