Apoptosis is an evolutionarily conserved, extremely regulated procedure of programmed, active, cell decease, morphologically and biochemically different from mortification, and is of import in normal development and physiological homeostasis of multicellular beings [ 1, 2 and 3 ] . Cells deceasing by programmed cell death maintain membrane unity until late in the procedure but display several morphological and biochemical changes, including chromatin condensation, atomic cleavage, internucleosomal DNA atomization, cytoplasmatic vacuolization, cell shrinking and membrane blebbing with casting of apoptotic organic structures [ 4 and 5 ] . Since deceasing apoptotic cells and apoptotic organic structures are quickly phagocytosed by neighbouring cells, chiefly macrophages, before escape of the cellular contents, this cell decease procedure does non normally result in an inflammatory response [ 5 and 6 ] . In contrast, mortification is an inadvertent signifier of cell decease, ensuing from physically or chemically induced membrane harm and consequences in conceited cells that leak their cytoplasmatic contents. This escape normally induces an inflammatory response [ 5 ] .
Many bacterial pathogens are able to pull strings for their ain benefit the host apoptotic programme [ 7 ] . Some obligate intracellular pathogens, such as Brucella suis, Rickettsia rickettsii and Chlamydiae, can exercise an anti-apoptotic consequence upon host cells, so that these remain as a site for their growing and generation [ 8, 9 and 10 ] . Pathogen-induced activation of the host cell-death tract may, in bend, serve to extinguish cardinal immune cells, hedging host defences that otherwise would move to restrict infection [ 11 ] . Initiation of host cell programmed cell death by bacterium has been suggested as an of import factor in the pathogenesis of some infections [ 7 and 11 ] , since riddance of immune cells can ease invasion and spreading of the infective agent.
Photobacterium damselae subsp. piscicida ( antecedently Pasteurella piscicida ) is the causative agent of fish hemorrhagic septicemia, a serious bacterial disease impacting several economically of import Marine fish species such as Ocyurus chrysurus, gilthead seabream, striped doodly-squat and sea bass [ 12 and 13 ] . Different surveies have identified several factors/mechanisms of import in the virulency of Ph. damselae subsp. piscicida ( see [ 14 ] ) . Despite these surveies, information on the pathogenesis of hemorrhagic septicemia in sea bass is still scarce and the interaction of the bacteriums with the host scavenger cells is non sufficiently understood.
In this survey, we investigated the interaction of Ph. damselae subsp. piscicida with sea bass phagocytes in vitro and in fish by experimentation infected by the intraperitoneal ( i.p. ) path. Here, we report on the happening of programmed cell death of sea bass macrophages and neutrophils as a consequence of the host-pathogen interaction in vivo and discourse the possible function of the Ph. damselae-induced programmed cell death in the pathogenesis of fish hemorrhagic septicemia.
2. Materials and methods
2.1. Experimental fish
Sexually immature sea bass were purchased from a commercial fish farm. Fish weighing 16.5±2.9 g were used for the finding of the virulency of Ph. damselae subsp. piscicida isolates. Fish weighing 113.6±23.9 g were used for all the other experiments because they allowed the aggregation of a larger figure of peritoneal cells. The animate beings were maintained in recirculating aerated saltwater, at 18-19 & A ; deg ; C. The fish used in experiments with unrecorded bacteriums were antecedently acclimatized to 23-24 & A ; deg ; C. Water quality was maintained with mechanical and biological filtration and fish were fed ad libitum on commercial pellets. Merely healthy fish, as indicated by their activity and exterior visual aspect, were used in the experiments.
The beginning and virulency of the Ph. damselae subsp. piscicida strains used in this survey are listed in Table 1. Strains DI 21, B51 and EPOY 8803-II were provided by Professor Alicia E. Toranzo ( Departamento de Microbiolog & A ; iacute ; a Y Parasitolog & A ; iacute ; a, Facultad de Biologia, University of Santiago de Compostela, Spain ) , strains MT1415, PP3, MT1375, MT1588 and MT1594 were provided by Dr Andrew C. Barnes ( Marine Laboratory, Aberdeen, UK ) and strain PTAVSA95 was supplied by Dr Nuno M.S. Department of State Santos ( Institute for Molecular and Cell Biology, University of Porto, Portugal ) . The strain ATCC 29690 was obtained from the American Type Culture Collection, USA. The virulency of each isolate was determined by shooting groups of 12-16 sea bass ( average weight 16.5±2.9 g ) with different doses of bacteriums. Virulence was defined harmonizing to old surveies [ 15 ] . All the virulent strains used in this work killed 100 % of the fish when 107CFU were injected i.p. In contrast, less than 50 % mortality was recorded when 108CFU of the non-virulent strains were injected.
Table 1. Beginning and virulency of the Ph. damselae subsp. piscicida isolates used in the present survey
Bacterias were routinely cultured at 22 & A ; deg ; C in tryptic soy stock ( TSB ) or tryptic soy agar ( TSA ) ( both from Difco Laboratories, Detroit, MI, USA ) supplemented with NaCl to a concluding concentration of 1 % ( w/v ) ( TSB-1 and TSA-1, severally ) and were stored frozen at ?70 & A ; deg ; C in TSB-1 supplemented with 15 % ( v/v ) glycerin.
To fix the inoculant for injection into the peritoneal pits of fish, the stocked bacteriums were cultured for 48 H at 22 & A ; deg ; C on TSA-1 and so inoculated into TSB-1 and cultured overnight at the same temperature, with shaking ( 100 revolutions per minute ) . Exponentially turning bacteriums were collected by centrifugation at 3000-g for 30 min, washed one time and resuspended in phosphate buffered saline ( PBS ) at the indicated concentrations. After anesthetization with 0.03 % ( v/v ) ethene ethanediol monophenyl quintessence ( Merck, Darmstadt, Germany ) , groups of sea bass were injected i.p. with 100 ?l of the bacterial suspensions. Plating consecutive dilutions of the suspensions onto TSA-1 home bases and numbering the figure of CFU following incubation at 22 & A ; deg ; C confirmed bacterial concentrations of the inoculant. In some experiments, UV-killed bacterial cells were used. For UV-treatment, exponentially turning bacteriums collected as described antecedently and resuspended in PBS were treated with UV-light for 1 h. The non-viability of the bacterium was confirmed by vaccination of TSA-1 home bases.
2.3. Bacterial civilization supernatants
For fixing civilization supernatants, bacterial strains were cultured nightlong in TSB-1 at 22 & A ; deg ; C with agitating ( 100 revolutions per minute ) and sub-cultured at a dilution of 1:100 in the same conditions, until the in-between exponential stage ( OD of 0.650 at 600 nanometer ) . The bacteriums were removed by centrifugation ( 3000-g, 30 min, 4 & A ; deg ; C ) and the civilization supernatants were collected, filtered through a 0.22 ?m-pore-size filter ( Schleicher and Shuell, Dassel, Germany ) and concentrated about 100-fold utilizing a Vivaflow 200 concentrator ( Sartorius AG, Goettingen, Germany ) . Concentrated supernatants were dialysed against 20 mM Tris-HCl ( pH 8.0 ) and were so aliquoted and stored at ?70 & A ; deg ; C until usage. Protein concentrations were determined utilizing a Micro BCA Protein Assay Reagent Kit ( Pierce, Rockford, IL, USA ) . The asepsis of the civilization filtrates was confirmed by the absence of settlements after plating on TSA-1 home bases. Concentrated supernatants were diluted in PBS to the indicated concentrations prior to the i.p. injection of 100 ?l per fish. In some experiments, bacterial civilization supernatants diluted in PBS were boiled for 10 min before proving.
2.4. Collection of peritoneal leucocytes
The peritoneal cells were collected from undisturbed peritoneal pits ( control ) or at the indicated times after injection of bacteriums or civilization supernatants, by a process described in item elsewhere [ 16 ] . Briefly, after being killed by an overexposure to ethylene glycol monophenyl quintessence ( Merck, Darmstadt, Germany ) at a concentration of 0.06 % ( v/v ) , the animate beings were exsanguinated by cutting the ventral aorta, and the abdominal side of the fish was so cleaned with ethyl alcohol. PBS with osmotic strength adjusted to 355 mOsm and supplemented with 20 U Lipo-Hepin ml?1and 5 % ( w/v ) glucose was injected into the peritoneal pit ( 5 milliliter per fish ) . PBS incorporating the peritoneal cells was so collected and placed on ice until processed, as follows.
2.5. Light microscopy
Entire peritoneal cell counts were performed with a hemocytometer and cytospin readyings were made utilizing a Shandon Cytospin 2 setup. The cytospins were fixed with formol-ethanol ( 10 % of 37 % methanal in absolute ethyl alcohol ) for 45 s and stained with Wright ‘s discoloration ( Haemacolor, Merck, Darmstadt, Germany ) . Detection of peroxidase activity to label neutrophils [ 17 ] was done by the Antonow ‘s technique [ 18 ] . The macrophages and neutrophils in the peritoneal exudations were differentially counted and the per centum of both cell types established after numbering a lower limit of 300 cells per slide. Additionally, where indicated, the per centums of apoptotic macrophages and neutrophils were determined.
2.6. TUNEL staining
Deoxyribonucleic acid atomization in single cells was detected by terminal deoxynucleotidyltransferase-mediated dUTP nick terminal labeling ( TUNEL ) of DNA strand interruptions ( In Situ Cell Death Detection Kit, Fluorescein ; Roche Diagnostics, Mannheim, Germany ) . Since preliminary observations showed that the TUNEL staining could be done in cytospins processed for peroxidase sensing by the Antonow method, cytospin readyings fixed with formol-ethanol and stained for peroxidase, as described above, were subjected to the TUNEL reaction harmonizing to the maker ‘s instructions. The cytospin readyings were mounted with the anti-fading agent Vectashield ( Vector Laboratories, Peterborough, UK ) incorporating 4 ?g ml?1propidium iodide and were observed under a Zeiss Axioskop epifluorescence microscope equipped with an HBO-100 quicksilver lamp, filter set 40 ( BP360/51, BP485/17, BP560/18 ) from Zeiss, excitement filter BP450-490, beam splitter FT510 and emanation filter LP520. Apoptotic cells were identified by yellow-green fluorescence within the karyon, due to the fluorescein-dUTP incorporated at the 3?OH terminals of disconnected DNA. Images were acquired with a Spot 2 camera ( Diagnostics Instruments ) , entering individually for each microscopic field the images of peroxidase staining ( bright field ) and TUNEL ( fluorescence ) . Comparison between prints of bright field and fluorescence images of the same cells allowed the designation of four leukocyte classs: cells positive for both peroxidase and TUNEL, cells negative for both stainings and cells positive for lone peroxidase or TUNEL. TUNEL-positive cells with abundant peroxidase-positive cytoplasmatic granules were counted as apoptotic neutrophils. Apoptotic macrophages were identified as big mononuclear, peroxidase-negative, TUNEL-positive, leucocytes.
For sensing of programmed cell death in tissue subdivisions, sea bass head-kidneys were fixed in 10 % ( v/v ) buffered impersonal formol, embedded in paraffin and sectioned. Sections were so processed for TUNEL staining, following the maker ‘s instructions for staining of subdivisions.
2.7. Electron microscopy
The ultrastructure of peritoneal leucocytes was examined by transmittal negatron microscopy. Peritoneal exudations were fixed in 2.5 % ( v/v ) glutaraldehyde in cacodylate buffer ( 0.1 M, pH 7.2 ) and post-fixed in 1 % ( w/v ) Os tetroxide in the same buffer. The samples were so embedded in Epon rosin ( TAAB Laboratories Equipment Ltd, Berkshire, UK ) after desiccation in a ranked series of ethyl alcohol. Ultrathin subdivisions were cut with an RMC MT-7 microtome and contrasted with uranyl ethanoate and lead citrate as described [ 19 ] . Observations and micrographs were done under a Zeiss EM10 electron microscope.
2.8. Analysis of DNA atomization by gel cataphoresis
Endonuclease-mediated internucleosomal DNA atomization is a characteristic characteristic of programmed cell death [ 20 ] that can be detected by the happening of a ladder form in agarose gel cataphoresis of DNA extracted from apoptotic cells.
Peritoneal leukocytes ( 2-106 ) collected as described above were centrifuged at 350-g for 5 min at 4 & A ; deg ; C. The supernatants were discarded and the cell pellets were stored frozen at ?20 & A ; deg ; C until processed for extraction of DNA following standard processs [ 21 ] . The Deoxyribonucleic acid was incubated for 2 H at 37 & A ; deg ; C with 20 ?g ml?1RNAse, before being electrophoresed in a 1.5 % ( w/v ) agarose gel incorporating 0.5 ?g ml?1ethidium bromide to let visual image of DNA. A 100 base-pair DNA ladder marker ( Amersham Pharmacia Biotech Inc. , Piscataway, NJ, USA ) was ever included as a mention. Gels were photographed under UV light utilizing a Polaroid camera and the exposure were digitalized and adjusted for brightness and contrast utilizing Adobe Photoshop.
2.9. In vitro macrophage checks
Monolayers of sea bass macrophages were obtained basically as described [ 22 ] . Briefly, the head-kidney was removed and pushed through a 100 ?m nylon mesh with L-15 medium ( Gibco, Paisley, UK ) incorporating 2 % foetal bovine serum ( FBS, Gibco, Paisley, UK ) , 1 % penicillin/streptomycin ( P/S, Gibco, Paisley, UK ) and 10 U/ml Lipo-Hepin. The cell suspension was layered onto a 31-45 % Percoll denseness gradient and, following centrifugation at 400-g for 30 min at 4 & A ; deg ; C, the set of cells layering above the 31-45 % interface was collected and washed with L-15 incorporating 0.1 % FBS. The cell suspension in L-15 medium incorporating 0.1 % FBS and 1 % P/S was adjusted to 107cells ml?1, and 1 milliliter was added to each well of a 24-well civilization home base ( Nunc, Denmark ) incorporating a Thermanox coverslip ( Nunc, Naperville, IL, USA ) . The home bases were incubated at 20 & A ; deg ; C and after 24 H, the non-adherent cells were removed by rinsing twice with L-15 medium and the staying monolayer was fed with 1 milliliters L-15 medium supplemented with 5 % FBS and 1 % P/S and maintained at 20 & A ; deg ; C for 2-3 yearss before usage. Adherent cells were & A ; gt ; 94 % of the monocyte/macrophage line of descent, harmonizing to morphological features observed after Wright staining ( Haemacolor, Merck, Darmstadt, Germany ) , and peroxidase sensing [ 17 and 18 ] . The apoptotic activity of deadly bacterial civilization supernatants was assessed by incubating macrophage monolayers with 0.1, 1 or 10 ?l ml?1of MT1415 concentrated supernatants prepared as described above and diluted in L-15 medium incorporating 0.1 % FBS and 1 % P/S. Mock-treated Wellss and Wellss treated with the concentrated non-virulent ATCC 29690 bacterial supernatant were used as controls. After 3, 6 and 24 H, coverslips were removed ( in triplicate ) , fixed with 10 % formol in absolute ethyl alcohol, stained with Wright Stain ( Haemacolor, Merck, Darmstadt, Germany ) and observed under the light microscope for the presence of cells with apoptotic morphology.
2.10. Ex vivo peritoneal exudation checks
For antique vivo checks, the sea bass peritoneal exudation cells were collected from control sea bass basically as above, with minor alterations. The aggregation of the peritoneal cells was performed in unfertile conditions utilizing L-15 medium supplemented with 10 % FBS, 1 % P/S and 30 U/ml Lipo-Hepin alternatively of PBS. The cell denseness was determined as above. The collected cell suspensions were placed into a 10 milliliter tubing and incubated at 22 & A ; deg ; C, with soft agitating. Exudate cell suspensions were infected with exponentially turning DI21 deadly bacterial cells at a bacterium: leukocyte ratio of 10:1 or 100:1 or treated with 10 ?l ml?1virulent ( DI21, B51 ) and non-virulent ( EPOY 8803-II ) Ph. damselae subsp. piscicida concentrated supernatants. After 6 H of incubation for the experiments with bacterial cells and after 6 or 22 H with bacterial supernatants, aliquots of the cell suspensions were collected and analyzed for the presence of apoptotic cells by light microscopic techniques as described earlier.
2.11. Reproducibility of consequences
For each experimental state of affairs, at least six fish were used. Where indicated, statistical analysis of the information was done utilizing the Student ‘s t-test. P & A ; lt ; 0.05 was considered important.
Preliminary observations showed abundant cells with a morphology suggestive of programmed cell death in samples of peritoneal exudations and head-kidney from sea bass deceasing after i.p. injection of deadly doses of Ph. damselae subsp. piscicida virulent strains. That such a morphology was so apoptotic was supported by the observation of abundant TUNEL-positive cells both in peritoneal exudations and in head-kidney. These observations prompted us to analyze in more item the apoptotic procedure utilizing the peritoneal theoretical account of infection, which allows a precise cytological analysis [ 16 ] .
3.1. Virulent Ph. damselae subsp. piscicida cells induce programmed cell death of sea bass peritoneal macrophages and neutrophils in vivo
Following the i.p. injection of 106-108CFU of the virulent strain MT1415, macrophages and neutrophils with apoptotic morphological changes were seen by conventional light microscopy, get downing at 6 h post-inoculation ( Fig. 1a ) . The apoptotic nature of these changes was confirmed by TUNEL staining ( Fig. 2a-c ) , electron microscopy ( Fig. 3 ( a ) , Fig. 3 ( B ) and Fig. 3 ( degree Celsius ) ) and DNA cataphoresis ( Fig. 4a, lanes 1-3 ) . The analysis of fluorescence and bright field images of the same cells in peritoneal samples processed for peroxidase sensing and TUNEL staining ( as documented below ; see Fig. 5a, B ) , showed that the apoptotic procedure preferentially affects macrophages and neutrophils, with merely a little per centum of apoptotic little mononuclear leucocytes ( platelets and lymph cells ) and no apoptotic eosinophilic farinaceous cells ( EGCs ) being present. The per centum of apoptotic cells was found to correlate positively with the figure of bacteriums used for infection ( informations non shown ) . At 6 H after the i.p. injection, apoptotic macrophages were much more abundant than apoptotic neutrophils, a difference that was attenuated at subsequent times. After i.p. injection of 107CFU of the virulent strain MT1415, most of the fish died within 48 H and some were already moribund at 18 h. Extensive programmed cell death of peritoneal macrophages and neutrophils occurred in all the studied moribund fish ( Fig. 2c ) . Electron microscopy of peritoneal samples collected from fish in advanced phases of infection, showed free and intracellular bacteriums, and extended lysis of the two types of phagocytic cells and of apoptotic organic structures ( Fig. 3 ( a ) , Fig. 3 ( B ) and Fig. 3 ( degree Celsius ) ) . Some of the lysing scavenger cells ( identified by the presence of intracellular bacteriums ) showed ultrastructural changes typical of programmed cell death ( Fig. 3 ( a ) , Fig. 3 ( B ) and Fig. 3 ( degree Celsius ) ) .
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Fig. 1. Light microscopic images of sea bass peritoneal exudations stained for the sensing of peroxidase and counterstained by the Wright technique. ( a ) Sample collected 6 H after the i.p. injection of 107CFU of the virulent strain MT1415 of Ph. damselae subsp. piscicida. Note peroxidase-positive and peroxidase-negative cells with changes typical of programmed cell death. ( B ) Sample collected 24 H after the i.p. injection of 107CFU of the non-virulent strain EPOY 8803-II. Note the presence of legion neutrophils ( identified by the peroxidase activity ) bespeaking the happening of an inflammatory reaction. No cell with apoptotic morphology is present.
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Fig. 2. Fluorescence microscopy of peritoneal exudations processed for the sensing of DNA atomization by TUNEL staining. ( a-c ) Samples collected from sea bass injected i.p. with 107CFU of the virulent strain MT1415 of Ph. damselae subsp. piscicida, demoing abundant TUNEL-positive cells. ( a ) After 6 H ; ( B ) after 24 H ; ( degree Celsius ) from a moribund fish, 18 H after injection. ( vitamin D ) Sample collected 48 H after the i.p. injection of 107CFU of the non-virulent strain EPOY 8803-II. Merely a really little figure of cells are TUNEL-positive.
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Fig. 3 ( a ) .
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Fig. 3 ( B ) .
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Fig. 3 ( degree Celsius ) . Fig. 3. Transmission electron microscopy of peritoneal exudations of sea bass collected after the i.p. injection of 107CFU of the virulent strain MT1415 of Ph. damselae subsp. pisicida. Sections contrasted with uranyl ethanoate and lead citrate. Bar=2 ?m. ( a ) Sample collected after 6 h. Note the leucocytes with apoptotic changes including cell shrinking, chromatin condensation, atomic atomization and cell blebbing. The macrophage on the upper right corner has intracellular apoptotic organic structures. ( B ) Sample collected after 12 h. Note the leucocytes in advanced programmed cell death with condensed chromatin. Arrows indicate bacteriums inside apoptotic scavenger cells. ( degree Celsius ) Sample collected after 12 h. Note the lytic changes in most leucocytes. Arrows indicate bacteriums inside an apoptotic scavenger cell.
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Fig. 4. Agarose gel cataphoresis of DNA extracted from peritoneal leucocytes of sea bass. ( a ) Deoxyribonucleic acid extracted from peritoneal leucocytes collected after the i.p. injection of 107CFU of Ph. damselae subsp. piscicida MT1415 ( lanes 1-3 ) , EPOY 8803-II ( lanes 4-6 ) and ATCC 29690 ( lanes 7-9 ) strains. Lanes 1, 4 and 7-after 6 H ; lanes 2, 5 and 8-after 24 H ; lanes 6 and 9-after 48 H ; lane 3-from a moribund fish 18 H after injection ; M-100 base-pair ladder marker. ( B ) Deoxyribonucleic acid extracted from peritoneal leucocytes collected after the i.p. injection of concentrated civilization supernatants from the Ph. damselae subsp. piscicida strains MT1415 ( lanes 1-6 ) and ATCC 29690 ( lanes 7-9 ) . Lanes 1 to 3-after injection of 100 ?l of PBS with 0.1 ?l concentrated civilization supernatant ( incorporating about 0.7 ?g of protein ) from the strain MT1415 ; lanes 4 to 6-after injection of 100 ?l PBS with 1 ?l of MT1415 concentrated civilization supernatant ( incorporating about 7 ?g of protein ) ; lanes 7 to 9-after injection of 1 ?l, in 100 ?l PBS, of ATCC 29690 concentrated civilization supernatants ( incorporating about 7 ?g of protein ) ; lanes 1, 4 and 7-after 6 H ; lanes 2, 5 and 8-after 12 H ; lanes 3, 6 and 9-after 24 H ; lane 10-DNA from peritoneal leucocytes of a control fish ; M-100 base-pair ladder marker.
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Fig. 5. Peritoneal exudate collected from a sea bass 6 H after the i.p. injection of 100 ?l of PBS with 0.1 ?l of concentrated civilization supernatant ( incorporating about 0.7 ?g of entire protein ) of the virulent strain MT1415. Cytospin processed for the coincident sensing of DNA atomization ( TUNEL-staining ) and peroxidase activity ( Antonow technique ) . ( a ) Fluorescence microscopy. ( B ) Bright field of the same microscopic field. Note, in ( a ) , the high proportion of TUNEL-positive cells. TUNEL and peroxidase-positive cells are marked with white stars. TUNEL-positive, peroxidase-negative cells are marked with black stars. The analysis of ( B ) shows that most of the TUNEL-positive cells are big mononucleate, peroxidase-negative cells, that is, macrophages. Some cells positive for both TUNEL and peroxidase, that is, apoptotic neutrophils, can besides be observed.
Besides strain MT1415, all the other virulent Ph. damselae subsp. piscicida strains listed in Table 1 induced programmed cell death of peritoneal macrophages and neutrophils when 106-107CFU were injected i.p.
No addition in the really low figure of apoptotic cells normally present in control peritoneal exudations was detected by TUNEL staining either at 6, 24, 48 H or 4 yearss after the i.p. injection of 107CFU of the non-virulent Ph. damselae subsp. piscicida strains ATCC 29690 and EPOY 8803-II ( Fig. 2d ) . Electron microscopy and DNA cataphoresis ( Fig. 4a, lanes 4-9 ) confirmed this observation. In the peritoneal pits of these fish, a transeunt inflammatory response with important neutrophil inflow was seen ( Fig. 1b ) .
3.2. In vivo programmed cell death of sea bass peritoneal macrophages and neutrophils is induced by Ph. damselae subsp. piscicida civilization supernatants but non by UV-killed bacteriums
In order to qualify the constituents of Ph. damselae subsp. piscicida involved in the initiation of programmed cell death of sea bass scavenger cells, we tested UV-killed bacterial cells and bacterial civilization supernatants for the ability to bring on macrophage and neutrophil programmed cell death in vivo. No addition over the control degrees of programmed cell death could be observed in peritoneal exudations collected 6 H after the i.p. injection of 108UV-killed deadly bacterial cells ( informations non shown ) . In contrast, extended programmed cell death of macrophages and neutrophils was detected, in a dose-dependent manner, in exudations collected 6, 12 and 24 H after i.p. injection of civilization supernatants of deadly bacterial civilizations ( strains MT1415, B51 and DI21 ) . The apoptotic procedure induced by the injection of 100 ?l PBS with 0.1 or 1 ?l concentrated supernatants, matching, severally, to about 0.7 or 7 ?g protein and to 10 or 100 ?l of the initial civilization supernatants, was characterized by TUNEL analysis ( Fig. 5a ) , electron microscopy and DNA gel cataphoresis ( Fig. 4b ; lanes 1-6 ) . As in the instance of the i.p. injection of unrecorded virulent Ph. damselae subsp. piscicida cells, the analysis of bright field and fluorescence images of the same cells in peritoneal exudations processed for TUNEL staining and peroxidase sensing ( Fig. 5a, B, severally ) showed that merely a little per centum of platelets and lymph cells and no EGCs were TUNEL-positive, bespeaking that the apoptotic procedure induced by the injection of deadly civilization supernatants affects preferentially macrophages and neutrophils. Supernatants from non-virulent bacterial civilizations, incorporating sums of entire protein similar to those of deadly supernatants, were non able to bring on programmed cell death of sea bass peritoneal scavenger cells, as shown by DNA cataphoresis ( Fig. 4b, lanes 7-9 ) and TUNEL staining ( non shown ) .
The apoptogenic activity of civilization supernatants from the virulent strains tested ( MT1415, B51 and DI21 ) was abolished by boiling for 10 min, as assessed by TUNEL staining and DNA cataphoresis of cells harvested 6 H after injection ( non shown ) .
A more elaborate survey on the development of the apoptotic procedure induced by 1 ?l of concentrated civilization supernatant of strain MT1415 was carried out. This survey showed that following the injection of the supernatant there is an initial uninterrupted addition of apoptotic macrophages accompanied by a uninterrupted lessening of non-apoptotic macrophages so that at 6 h post-injection, apoptotic macrophages represented 61.9±31.0 % of entire peritoneal macrophages ; this proportion increased to 86.1±36.1 % at 24 H, when 1.2±0.5-107apoptotic macrophages per peritoneal pit were present. As a effect of this procedure, the figure of non-apoptotic macrophages during the initial 24 H was ever below the entire figure at clip zero and below the figure in fish injected with the non-virulent supernatants.
On the other manus, the figure of apoptotic neutrophils besides increased continuously during the initial 24 H, but at a slower gait as compared to the macrophages. At 6 H post-injection merely 18.7±12.4 % of the peritoneal neutrophils were apoptotic, and apoptotic macrophages were 10.8±6.5 times more abundant than apoptotic neutrophils ( P=0.004 ) , but at 24 h 0.9±0.4-107neutrophils were apoptotic, which represents 66.8±34.2 % of entire neutrophils.
The farther analysis of the development of the procedure showed that in the peritoneal exudations of fish that were moribund as a effect of the injection of 1 ?l of the concentrated virulent supernatant, most of the macrophages and neutrophils were apoptotic and lysing. In contrast, the lasting fish showed a progressive control of the apoptotic procedure, about no apoptotic scavenger cells being present after 4 yearss post-injection. In these fish, 15 yearss after the injection the Numberss of peritoneal macrophages and neutrophils were diminishing and nearing the values of the controls.
3.3. Ph. damselae subsp. piscicida do non bring on programmed cell death of sea bass peritoneal scavenger cells in vitro
The figure of apoptotic macrophages in in vitro head-kidney macrophage civilizations or of apoptotic neutrophils and macrophages in peritoneal exudations incubated antique vivo, treated with deadly supernatants or virulent bacteriums, under the described conditions, was low and similar to that in untreated samples or in samples treated with avirulent supernatants ( informations non shown ) . In the ex vivo experiments with Ph. damselae subsp. piscicida, phagocytosis of the bacteriums by macrophages and neutrophils was seen.
Bacterial pathogens have developed different schemes to last inside the host and to get the better of its natural defences, and therefore do disease. One of these schemes is the use of mechanisms that quickly and expeditiously kill host cells involved in antimicrobic defence. In the instance of bacteriums that proliferate extracellularly, devastation of host scavenger cells by the occupying micro-organism will strip the septic host of the important defence mechanism represented by phagocytosis. In many beings, including fish, the phagocytic system consists of two types of specialised leucocytes. One is the fixed, long-living, resident macrophage or macrophage-like cell, present in all compartments of the organic structure, which, hence, plays a really of import function in the initial phases of infection, as it is the first scavenger cell to meet occupying micro-organisms. The other professional scavenger cell is the neutrophil, a leucocyte armed with powerful antimicrobic molecules that is quickly recruited to the infective focal point from modesty pools in the hematopoietic variety meats and blood and that, accordingly, besides is of import in the phagocytic defence mechanism.
Zychlinsky et Al. [ 23 ] were the first to demo that macrophages can undergo programmed cell death in vitro as a consequence of infection. Since so, several writers have reported on the triggering of programmed cell death of host immune cells by several bacterial species and proposed this as a infective mechanism ( see reappraisals in [ 7 and 11 ] ) . Almost all of the reported cases of microbial-induced apoptotic decease of host scavenger cells are in vitro surveies with individual cell populations. Most concern macrophages [ 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34 ] , a few refer to neutrophils [ 31, 35 and 36 ] but none describes the coincident devastation of macrophages and neutrophils.
The present paper describes the happening of extended programmed cell death in the peritoneal pit and in the head-kidney of sea bass by experimentation infected with Ph. damselae subsp. piscicida. Further analysis of the apoptotic procedure, utilizing the peritoneal theoretical account, showed for the first clip the happening of extended programmed cell death of neutrophils and macrophages during an infection by a microbic pathogen. We found that virulent, but non non-virulent, isolates of Ph. damselae subsp. piscicida induce, in by experimentation infected sea bass, programmed cell death of macrophages and neutrophils. The happening of such a procedure was detected by visible radiation and negatron microscopic observation of peritoneal neutrophils and macrophages with morphological characteristics of apoptotic cells, by specific in situ sensing of DNA atomization in peritoneal cells by TUNEL staining, and by the happening of a characteristic ladder form after agarose gel cataphoresis of DNA extracted from peritoneal leucocytes.
Our present consequences do non place the bacterial factor ( s ) responsible for the initiation of sea bass macrophage and neutrophil programmed cell death by Ph. damselae subsp. piscicida. Since UV-killed virulent bacterial cells do non trip the apoptotic procedure, secreted bacterial merchandises emerge as the likely campaigners for the apoptogenic factor. In support of this reading is the observation that extended programmed cell death of macrophages and neutrophils was observed in peritoneal exudations after injection of virulent, but non of non-virulent, bacterial civilization supernatants. Since the apoptogenic activity of civilization supernatants was abolished by heat-treatment, our informations are consistent with the secernment of one ( or several ) protein ( s ) by virulent isolates of Ph. damselae subsp. piscicida, with apoptogenic activity towards macrophages and neutrophils in by experimentation infected sea bass.
The per centum of macrophages undergoing programmed cell death early after the oncoming of the procedure was found to be greater than that of neutrophils. This consequence most likely reflects the predomination of macrophages ( 5.7±3.4-107per fish ) over neutrophils ( 0.69±0.37-107per fish ) in the resting peritoneal pit when the supernatant was injected. Furthermore, the dynamicss of the apoptotic procedure impacting neutrophils was significantly affected by the inflammatory response to the i.p. injection of the supernatants. It is widely documented that the early response to a peritoneal aggression is characterized by a quick and extended inflow of neutrophils, while the macrophage response is more delayed [ 17 and 18 ] . It follows, hence, that the high Numberss of incoming neutrophils during the initial 24 H of the procedure significantly increase the counts of non-apoptotic neutrophils. In contrast, a similar state of affairs does non happen with macrophages.
The consequences sing the apoptotic procedure that follows the i.p. injection of deadly doses of Ph. damselae subsp. piscicida cells or civilization supernatants show that both peritoneal macrophages and neutrophils are extensively affected, the bulk of macrophages and neutrophils being apoptotic 24 H after injection and making values around 107per peritoneal pit for both cell types. Furthermore, it is relevant that in those state of affairss the figure of non-apoptotic macrophages was ever below the figure in the resting peritoneal pit. On the other manus, negatron microscopy showed that at the late phases of this procedure, extended lysis of macrophages, neutrophils and apoptotic organic structures occurs. One relevant effect of the procedure is the terrible depletion of functional scavenger cells with the failure of the antibacterial phagocytic defences. Virulent Ph. damselae subsp. piscicida cells, although able to occupy sea bass epithelial cells, multiply extracellularly in the septic fish [ 37 ] . We have late shown that peritoneal neutrophils and macrophages of sea bass extensively phagocytose i.p. injected Ph. damselae subsp. piscicida [ 17 ] . On the other manus, it has been demonstrated that this fish pathogen is killed by sea bass macrophages in vitro, superoxide anions being implicated in the violent death procedure [ 38 ] . The susceptibleness of these bacteriums to the macrophage killing activity is supported by the consequences of Barnes et Al. [ 39 ] demoing that this pathogen is non good equipped for the protection against onslaught by the reactive O species produced by macrophages and neutrophils [ 39 ] . It is, hence, probably that extended devastation of sea bass scavenger cells by the apoptogenic activity of virulent Ph. damselae subsp. piscicida would be an of import factor in the pathogenesis of sea bass hemorrhagic septicemia by forestalling phagocytosis and violent death of the pathogen and by let go ofing bacteriums already inside the scavenger cells.
The coincident devastation of sea bass neutrophils and macrophages by Ph. damselae subsp. piscicida has another effect advantageous to the pathogen. Indeed, macrophages, besides holding an efficient antibacterial phagocytic activity, besides are the scavenger cells with the critical function of extinguishing the potentially cytotoxic, moribund, apoptotic neutrophils and neutrophilic apoptotic organic structures in infectious/inflammatory focal point [ 40 ] . Therefore, the inefficient clearance by macrophages of deceasing apoptotic neutrophils and neutrophil apoptotic organic structures consequences in the advancement towards secondary mortification [ 5 and 41 ] and eventual lysis of the apoptotic, moribund neutrophils and apoptotic organic structures exposing host tissues to the extremely cytotoxic neutrophilic contents [ 40, 42 and 43 ] .
The present observation that no apoptogenic activity towards sea bass scavenger cells of virulent Ph. damselae subsp. piscicida cells or civilization supernatants was seen in vitro suggests an indirect consequence of the apoptogenic factor and may good explicate how the bacteria is so infective in vivo but is expeditiously killed by macrophages in vitro [ 38 ] .
Several surveies have been carried out taking at the designation of the factors/mechanisms involved in the virulency of Ph. damselae subsp. piscicida. However, understanding of the pathogenesis of hemorrhagic septicemia in sea bass is still limited and the aspects of the interaction of the bacteriums with the host scavenger cells are far from clear. The soon reported initiation of coincident macrophage and neutrophil programmed cell death by Ph. damselae subsp. piscicida in sea bass, with eventual lysis of high Numberss of the two scavenger cells appears as a novel and really powerful infective scheme of this fish pathogen. Examination of fish agony from natural Ph. damselae subsp. piscicida infection will find if initiation of programmed cell death is of import for the pathogenesis of natural hemorrhagic septicemia.