systemic macrophage and neutrophil destruction by secondary necrosis induced by a bacterial exotoxin...

Systemic macrophage and neutrophil destruction bysecondary necrosis induced by a bacterial exotoxin ina Gram-negative septicaemia

Ana do Vale,1 Carolina Costa-Ramos,1

Alexandra Silva,1 Daniela S. P. Silva,1

Fátima Gärtner,2 Nuno M. S. dos Santos1 andManuel T. Silva1*1IBMC-Instituto de Biologia Molecular e Celular,Universidade do Porto, Portugal.2ICBAS-Instituto de Ciências Biomédicas de AbelSalazar, Universidade do Porto, Portugal.

Summary

Bacterial modulation of phagocyte cell death is anemerging theme in pathogenesis. Here we describethe systemic destruction of macrophages and neutro-phils by the Gram-negative Photobacterium damselaessp. piscicida (Phdp) in fish pasteurellosis, a deadlysystemic infection. Following experimental inocula-tion, Phdp spreads by bacteraemia and colonizesthe organs, producing a septicaemic infection, andsecretes the apoptogenic exotoxin AIP56 which issystemically disseminated. In experimental andnatural pasteurellosis, destruction of macrophagesand neutrophils by secondary necrosis followingcaspase-3-associated apoptosis was seen predomi-nantly in the spleen, head kidney and gut laminapropria. Identical phagocyte destruction occurredafter injection of rAIP56, but not of heat-inactivatedrAIP56, or AIP56-negative Phdp strains, indicatingthat AIP56 is responsible for phagocyte destructionoccurring in pasteurellosis. Active caspase-3 andactive neutrophil elastase are present in the blood inadvanced infection, indicating that phagocyte lysis bysecondary necrosis is accompanied by release oftissue-damaging molecules. The AIP56-induced lysisof phagocytes represents a very efficient, self-amplifying etiopathogenic mechanism, because itresults in two effects that operate in concert againstthe host, namely, evasion of the pathogen from acrucial defence mechanism through the destructionof both professional phagocytes, and release of

tissue-damaging molecules. The induction by a bac-terial exotoxin of in vivo systemic lysis of both pro-fessional phagocytes by secondary necrosis, nowdescribed for the first time, may represent an over-looked etiopathogenic mechanism operating in otherinfections of vertebrates.

Introduction

Bacteria producing extracellular infections are preferen-tially located in body fluids and other extracellular spaceswhere they multiply. One crucial, first line, antibacterialhost defence against extracellular pathogens is phagocy-tosis by macrophages and neutrophils (Nahm et al.,1999), especially in acute, rapid infections, where anti-bodies cannot be produced in time to be protective. Onemechanism used by successful extracellular parasites toevade phagocytosis is the production of molecules thatinactivate or destroy host phagocytes (Weinrauch andZychlinsky, 1999; Narayanan et al., 2002; DeLeo, 2004).Many of these molecules do so by triggering the endog-enous programmed cell death machinery of these leuco-cytes leading to their elimination by apoptosis (Weinrauchand Zychlinsky, 1999; Narayanan et al., 2002; DeLeo,2004). This anti-phagocytic mechanism can be processedthrough direct contact between the pathogen and the hostcell target and transfer of effector molecules by secretionsystems (Weinrauch and Zychlinsky, 1999), or at a dis-tance by exotoxins with cytotoxicity for leucocytes(Narayanan et al., 2002).

One of the main bacterial diseases affecting aquacul-ture is pasteurellosis, the infection caused by theextracellular pathogen Photobacterium damselae ssp.piscicida (Phdp). This Gram-negative is highly pathogenicfor wild and cultured marine fish, affecting more than 20fish species worldwide, including sea bass, sole and seabream (Magariños et al., 1992; 1996; Thune et al., 1993;Romalde, 2002). In the acute form, pasteurellosis is asystemic and deadly infection with a rapid course andvery high mortalities (Hawke et al., 1987; Nelson et al.,1989).

We have shown that, in experimental sea bass perito-nitis following intraperitoneal (i.p.) inoculation of Phdp,this pathogen induces selective apoptotic destruction of

Received 27 July, 2006; revised 28 September, 2006; accepted 29September, 2006. *For correspondence. E-mail [email protected];Tel. (+351) 22 6074900; Fax (+351) 22 6099157.

Cellular Microbiology (2007) 9(4), 988–1003 doi:10.1111/j.1462-5822.2006.00846.xFirst published online 6 December 2006

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

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peritoneal macrophages and neutrophils (do Vale et al.,2003). This destruction is mediated by AIP56, an apopto-genic, plasmid-encoded protein exotoxin secreted bygrowing virulent Phdp (do Vale et al., 2005). Apoptosis-associated cytotoxic alterations of peritoneal phagocytessimilar to those seen after the i.p. inoculation of live viru-lent Phdp cells occurred when purified rAIP56 was i.p.inoculated in sea bass or when isolated peritoneal seabass leucocytes were exposed ex vivo to the purifiedrecombinant toxin (do Vale et al., 2003; 2005). The aip56gene is absent from non-virulent Phdp strains, andpassive immunization of sea bass with anti-AIP56 anti-bodies provides significant protection against Phdp chal-lenge (do Vale et al., 2005), indicating that AIP56 exotoxinis a key virulence factor of Phdp. Studies aiming at theelucidation of the pathogenesis of pasteurellosis are jus-tified because they may lead to the development of muchneeded preventive or curative interventions and willextend the knowledge of mechanisms involved inbacteria-induced apoptosis of host immune cells. There-fore, we considered of interest to analyse the role ofAIP56 in acute natural pasteurellosis and in experimentalsystemic Phdp infection as a model of the natural disease.Here we show that the AIP56-dependent apoptoticdestruction of macrophages and neutrophils operatessystemically in acute natural pasteurellosis of sea bream,sole and sea bass and in experimental septicaemic infec-tions of sea bass. By inducing systemic lysis of macroph-ages and neutrophils, the exotoxin not only disarms thehost phagocytic defence mechanism thus allowing themassive extracellular multiplication of Phdp leading to asepticaemic infection, but also causes the release of cyto-toxic, tissue-damaging molecules from phagocytes.

Results

Fish with acute natural pasteurellosis are infected withPhdp bearing the aip56 gene

Sea bream, sea bass and sole affected by acute naturalpasteurellosis used in the present study were in anadvanced phase of the infection. The fish were notfeeding and quickly became moribund with typical signsas lethargy and loss of equilibrium. No obvious externalalterations were apparent except skin discoloration andpresence of scattered petechiae in the abdominal area.Microbiological analyses of spleens and kidneys showedpositive cultures for Phdp. Presence of Phdp in the organsof affected fish was also confirmed by immunocytochem-istry, as described below. All Phdp strains isolated fromthose fish were found to have the aip56 gene (not shown),in accordance with our previous observation that all viru-lent Phdp strains from our collection contained that gene(do Vale et al., 2005).

Fish with advanced acute natural or experimentalpasteurellosis have extensive apoptotic destruction ofmacrophages and neutrophils and massive Phdpaccumulations

The organization of the immune system in teleost fish issimilar to that in mammals except that there is no bonemarrow, lymph nodes and Peyer’s patches (Press andEvensen, 1999; Scapigliati et al., 2002; Rombout et al.,2005). Fish head kidney is equivalent to mammalian bonemarrow in terms of haematopoietic activity (Press andEvensen, 1999; Scapigliati et al., 2002; Rombout et al.,2005). Cells of the fish immune system are mostly locatedin the head kidney, spleen, gut and thymus (Press andEvensen, 1999; Scapigliati et al., 2002; Rombout et al.,2005).

The histopathology of advanced pasteurellosis wassimilar in sea bream, sole and sea bass with the naturaldisease and in sea bass with experimental infectioninduced by the i.p. inoculation of Phdp virulent strains.High-power microscopical analyses of the areas of celldestruction present in haematoxylin-eosin (HE)-stainedsections showed apoptotic bodies and cells with frag-mented and/or condensed nuclei together with lysing cellsand cell debris (Fig. 1B and D). It is of note that sea bassneutrophils, contrary to mammalian neutrophils, have anon-lobulated nucleus, similar to that of macrophages (doVale et al., 2002) (see Fig. 2I), allowing a direct associa-tion of nuclear fragmentation to apoptosis. TUNEL-positive cells (Fig. 2A) and cells positive for activecaspase-3 (Fig. 2B) were present in the areas of celldestruction. Colocalization of areas of cell destruction withareas of TUNEL positivity and of active caspase-3 posi-tivity was seen when consecutive sections were analysed(Figs 1C and 2A and B). These results identify the celldeath process as apoptotic. Besides the foci of apoptosis,apoptosing cells were also seen scattered in the splenicand head kidney parenchymas (Fig. 2C and D), in theperipheral blood (not shown), in blood of the spleen, liverand head kidney vasculature (Fig. 2E) and in gut laminapropria (Fig. 2F).

Our previous results showed a selective apoptogenicactivity of AIP56 against sea bass peritoneal macroph-ages and neutrophils (do Vale et al., 2003; 2005). That theapoptosing cells seen in the organs with advanced Phdpinfections were macrophages and neutrophils was dem-onstrated using the specific staining of macrophages foralpha-naphtyl acetate esterase (ANAE) activity and ofneutrophils for myeloperoxidase activity (Fig. 2G–J). Inaccordance with the caspase-3-associated apoptoticnature of the cell destruction observed in affected fish,caspase-3 activity was detected by fluorimetry in lysatesof spleens and kidneys of fish with advanced natural(Fig. 3) or experimental (not shown) pasteurellosis.

Phagocyte apoptosis in a Gram-negative septicaemia 989

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 988–1003

Histologically the liver of affected fish was much lessaffected by pasteurellosis, with the only abnormality beingthe presence in a few fish of periportal leucocyte infiltra-tions with abundant apoptosing cells (not shown). Nohistological alterations were seen in the tubules of thecaudal kidney.

The here described destruction of fish phagocytes wasmost extensive in the spleen, head kidney and gut andinfrequent in the liver, a distribution that parallels AIP56activity against macrophages (do Vale et al., 2003; 2005)and localization of macrophage populations in teleosts(Abelli et al., 1997; Dalmo et al., 1997; Press andEvensen, 1999; Petrie-Hanson and Ainsworth, 2000;Scapigliati et al., 2002; Rombout et al., 2005). Accord-ingly, no increase in the basal level of apoptosing lympho-cytes was seen in the thymus of fish with advancedpasteurellosis (not shown). Besides the above localiza-tions, apoptosing phagocytes were seen in all territorieswith resident macrophages, or accessible to phagocytemigration, that is regions rich in connective tissue (Elliset al., 1976; Ainsworth, 1992) including gills, skin andheart (not shown).

Accumulations of bacteria (Fig. 2D), identified as Phdpby immunocytochemistry (Fig. 4C and D), appearedalmost exclusively as extracellular bacilli, frequently inmicrocolonies, without abscess formation, and were par-ticularly abundant in the spleen and head kidney. Phdpwas also present in gut lamina propria, liver sinusoids andblood (Fig. 4D). This pattern of distribution of Phdp issuggestive of bacteraemia and septicaemia.

Identical histopathological alterations were observed insea bass with advanced infection following i.p. inoculationwith all Phdp virulent, AIP56-positive strains tested. His-topathological alterations similar to those described in fishinoculated i.p. with virulent Phdp were present in sea bassexperimentally infected by the water route, the route ofinfection in the natural disease (not shown). This indicatesthat, after the establishment of a systemic infection follow-ing the introduction of the pathogen, the progress of thedisease is similar regardless of the route of infection,validating the systemic infection that follows the i.p. inocu-lation of Phdp as a model of acute natural pasteurellosis.

Systemic pasteurellosis is a septicaemia withexotoxaemia

As the progress of natural pasteurellosis is not accessibleto a detailed kinetic investigation because the timing ofthe beginning of the infection is not known, to study thetemporal development of the disease under the microbio-logical and histopathological points of view we inducedexperimental Phdp infections in sea bass using the i.p.route of infection.

Phdp bacilli injected i.p. were extensively phagocytosedduring the first 1 h post inoculation by peritoneal macroph-ages and recruited neutrophils (Fig. 4A), but at 6–12 hmany bacteria were extracellular (Fig. 4B) due to the apo-ptotic destruction of infected phagocytes (do Vale et al.,2003; 2005). When Phdp inocula producing systemicinfections were used, i.p. infection was followed by bacter-

Fig. 1. Histopathology of advanced naturalpasteurellosis in sections stained with HE.A. Spleen from a sea bass with advancednatural pasteurellosis showing one focus ofcell destruction (objective 5¥).B. High-power view of the periphery of thefocus in A, showing apoptosing cells (arrows)with fragmented and/or condensed nuclei andapoptotic bodies (arrowheads) and cell debris(*) in areas of cell destruction. Objective100¥.C. Head kidney of a sole with advancednatural pasteurellosis with a focus of celldestruction. Objective 5¥.D. Higher magnification of the focus in Cshowing apoptosing cells (arrows), apoptoticbodies (arrowheads), cell debris (*) andscattered bacilli. Objective 100¥.

990 A. do Vale et al.


Fig. 2. Apoptosing macrophages and neutrophils are present in spleen, head kidney, blood and gut lamina propria in advanced naturalpasteurellosis.A. Section of sole head kidney stained with the TUNEL technique for internucleosomal DNA degradation. TUNEL-positive cells (red) arenumerous in the focus of cell destruction shown in Fig. 1C. Objective 5¥.B. Immunostaining for active caspase-3 of the focus of cell destruction in A showing numerous active caspase-3-positive cells (brownish red).Objective 5¥. Stainings for A and B and Fig. 1C were done on consecutive sections.C. Low-power view of a section of the spleen of a sea bass. Section immunostained for active caspase-3 (brownish red). Abundant cellspositive for active caspase-3 are present in foci or scattered throughout the splenic parenchyma. Objective 10¥.D. Section of the spleen of a sole immunostained for active caspase-3. Numerous cells positive for active caspase-3 and with fragmentedand/or condensed nuclei, and apoptotic bodies are mixed with scattered bacilli or close to bacterial microcolonies (*). Objective 40¥.E. Apoptosing cells with fragmented and condensed nuclei and positive for active caspase-3 (brownish red) are present in the blood of avessel in the head kidney of a sea bass. Objective 100¥.F. Section of intestine of sea bass. Numerous apoptosing cells with fragmented nuclei and condensed chromatin and stained for activecaspase-3 (brownish red) are present in the lamina propria. Objective 20¥.G. A normal sea bass macrophage specifically labelled by detection of ANAE, showing a kidney-shaped nucleus and ANAE staining (red) ofthe cytoplasm. Preparation counterstained with haematoxylin. Objective 100¥.H. Apoptosing macrophage in the spleen from a sea bass with terminal pasteurellosis, showing cell shrinkage, a fragmented nucleus withcondensed chromatin and ANAE staining (red) of the cytoplasm. Preparation counterstained with haematoxylin. Objective 100¥.I. Normal sea bass neutrophil specifically labelled by detection of myeloperoxidase, showing a spherical, non-lobulated nucleus as is typical ofsea bass neutrophils (do Vale et al., 2002) and cytoplasmic granules stained for myeloperoxidase (brown). Preparation counterstained withHemacolor. Objective 100¥.J. Apoptosing neutrophil in the spleen from a sea bass with terminal pasteurellosis, showing cell shrinkage, nuclear fragmentation and chromatincondensation, and myeloperoxidase-positive (brown) cytoplasmic granules. Preparation counterstained with Hemacolor. Objective 100¥.



aemia and organ colonization as shown by histology(Fig. 4C and D) and by the assessment of the temporalprogression of bacterial counts in blood and spleen(Fig. 5). By 48 h, when some fish were already moribund,very high numbers of bacteria were counted in spleensand blood, in accordance with the presence of largenumbers of extracellular bacilli seen by histology inadvanced infections. The results of the bacterial countstogether with histopathological observations indicate theoccurrence of bacteraemia and septicaemia with spread-ing of Phdp throughout the infected host and, in theadvanced phase of the disease, massive extracellularmultiplication of Phdp, mainly in the spleen and headkidney.

AIP56 protein was detected by Western blotting in theblood of 15 out of 19 fish infected for 40 h with Phdp strainPP3 and showing histopathological signs of advancedinfection, but in none at 18 h post infection or in thecontrols (Fig. 6). Using a semi-quantitative estimation ofthe amount of circulating AIP56 based on the intensity ofthe AIP56 bands in the blots, more toxin was detected inmoribund fish as compared with fish infected for 40 h butwithout signs of terminal disease (Fig. 6).

These results show that the septicaemia of advancedacute pasteurellosis is accompanied by AIP56 toxaemia.

Systemic apoptotic phagocyte destruction in acutepasteurellosis is due to Phdp AIP56 exotoxin

Bacteria, and apoptosing cells identified by fragmentedand/or condensed nuclei, and apoptotic bodies, werelabelled by the anti-AIP56 antibody as shown in Fig. 7 forspleen and blood in fish with histopathological signs ofadvanced pasteurellosis. Areas of AIP56 positivity colo-calized with areas of accumulation of apoptosing cellsdefined by fragmented and/or condensed nuclei anddetection of active caspase-3 (Fig. 7C and D).

Sea bass injected i.p. with rAIP56 showed dose-dependent mortalities. Moribund fish had phagocyte apo-ptotic destruction in spleen, head kidney and gut identicalto that in fish inoculated with virulent Phdp; these alter-ations were not present in sea bass injected i.p. withheat-inactivated rAIP56 (not shown).

These observations indicate that AIP56 exotoxin isresponsible for the systemic phagocyte destructiondescribed above in fish with natural or experimental sys-temic pasteurellosis.

Macrophages and neutrophils infiltrate Phdp-infectedorgans and are later depleted

Bacterial invasion of organs by bacteraemia triggers alocal inflammatory response in the invaded tissues withrecruitment of macrophages and neutrophils (Sebbaneet al., 2005). The kinetics of neutrophil and macrophagerecruitment into the spleens of infected fish was assessedin sea bass inoculated i.p. with Phdp strain PP3 (Fig. 8Aand B). The counts of splenic macrophages and neutro-phils increased at 24–48 h post infection when bacteriawere already detectable in the spleen by culture. Thenumbers of phagocytes then decreased with minimalvalues in moribund fish. This late decrease was accom-panied by an increase in the number of apoptosingphagocytes and phagocyte apoptotic bodies (Fig. 8C andD) and correlates with the above described histopatho-logical observations showing apoptotic cell depletion.Although not assessed quantitatively, phagocyte infiltra-tion followed by apoptotic depletion was also observed inadvanced infections in head kidneys, gut lamina propriaand, in a few fish, in periportal areas of the liver tissue.

Temporal development of histopathology in systemicpasteurellosis

In an experiment with sea bass infected i.p. with4.5 ¥ 103 cfu of Phdp strain PP3, we looked at the histo-pathological alterations in several organs at 18 and 40 hpost inoculation to evaluate the temporal evolution ofthose alterations culminating in the scenario described infish with advanced/terminal disease.

Fig. 3. Caspase-3 activity is increased in lysates of spleen andhead kidney of sea bass with advanced natural pasteurellosis.Caspase-3 activity determined with the fluorescent substrateAc-DEVD-AMC, in lysates from spleens and head kidneys collectedfrom non-infected (control) sea bass or from sea bass withadvanced natural pasteurellosis is expressed as RFU per mg ofprotein in the lysates. Lysates incubated with the caspase-3inhibitor Ac-DEVD-CHO were used as specificity controls. SP,spleen; HK, head kidney; M, moribund fish.



First, we performed a blind, semi-quantitative assess-ment of histological parameters in sections of 19 spleensstained with HE (six control, non-infected fish, seven fishinfected for 18 h and six fish infected for 40 h). At 40 hpost inoculation all six fish were with advanced infectionand three were moribund. No AIP56 exotoxin wasdetected by Western blotting in the plasma at 0 or 18 hpost inoculation but at 40 h all fish analysed had circulat-ing AIP56 exotoxin.

Fig. 4. Phdp bacilli are phagocytosed early after infection, and become extracellular and accumulate massively as the infection progresses.A. Cytospin of peritoneal leucocytes from sea bass with experimental pasteurellosis 1 h after i. p. inoculation of 4 ¥ 108 cfu of Phdp strainMT1415, stained for myeloperoxidase and counterstained with Hemacolor. Numerous Phdp bacilli are seen within phagocytes, mainlymacrophages. Objective 100¥.B. Twelve hours after inoculation most bacilli are extracellular. Apoptosing neutrophils (with brown granules labelled for myeloperoxidaseactivity) with fragmented nuclei and condensed chromatin and apoptotic bodies are present. Objective 100¥.C. Section of the spleen from a sea bass with advanced natural pasteurellosis showing numerous microcolonies of extracellular Phdp stainedby immunocytochemistry (brownish red). Objective 5¥.D. Section of liver tissue from a sea bass with advanced experimental pasteurellosis 40 h after the i.p. inoculation of 4.5 ¥ 103 cfu of strainPP3. Extracellular Phdp bacilli revealed by immunocytochemistry (brownish red) are present in a portal vein and in sinusoids. Nohistopathological alterations are seen in the hepatocytes. Objective 40¥.

Fig. 5. Acute pasteurellosis is a septicaemia. Kinetic analysis ofPhdp colonization in blood (A) and spleen (B) of sea bass withexperimental pasteurellosis following the i.p. inoculation of1.8 ¥ 105 cfu of strain PP3. In this experiment moribund fish (M)started to occur at 48 h and cumulative mortality was 73%. Cfu,colony forming units; P.I., post infection.

Fig. 6. AIP56 exotoxin is present in the plasma of fish withadvanced pasteurellosis. The presence of AIP56 exotoxin inplasmas from control (non-infected) sea bass and from sea bassinfected i.p. with 4.5 ¥ 103 of Phdp strain PP3 was determined byWestern blotting using an anti-AIP56 rabbit serum (upper panel).The equal loadings of the lanes were confirmed by staining thenitrocellulose membrane with Ponceau S (lower panel) prior toimmunodetection of the exotoxin. The arrow in the left indicates theposition of the AIP56. In this experiment, 40 h post inoculation 15out of 19 fish were in advanced phase of infection and four fishwere moribund (M). Cumulative mortality was 95%. None of thefour control fish tested (three shown in the figure), and none of the13 fish tested at 18 h post infection (three shown) had detectableAIP56 in plasma. Of the 15 fish tested at 40 h, 10 had AIP56 in theplasma (four AIP56-positive and one AIP56-negative fish are shownin the figure).



Disorganization of the parenchyma architecture withcongestion and haemorrhage was present in all fish at 18and 40 h. Macrophage and neutrophil infiltration, alreadydetected in the kinetic experiment of Fig. 8, was evidentwith either a dispersed or focal pattern in all fish at 18 hbut without significant apoptotic alterations except in onesea bass that showed a few apoptosing phagocytes. At40 h those foci of macrophage and neutrophil infiltration,present in all fish showed numerous phagocytes withfragmented and/or condensed nuclei and apoptoticbodies, and necrotic areas with cell debris. These histo-pathological features resemble those described above foradvanced natural pasteurellosis (Fig. 1) and reflect thephagocyte depletion and the increase in apoptosingphagocytes and phagocyte apoptotic bodies detected inthe kinetic experiment of Fig. 8. The quantitative assess-ment of cells positive for active caspase-3 in spleen sec-tions showed a low number of apoptosing cells at 0 or18 h and a significant increase by 40 h in fish with circu-lating AIP56 but not in fish with undetectable plasmatictoxin (Fig. 9).

Phdp bacteria were visible at 18 h in HE-stained sec-tions only in one fish which also had apoptosing cells, butwere detectable in low numbers in all fish in sections

stained with the anti-Phdp antibody. This is in accordancewith the observations that, at this time, Phdp was alreadycolonizing the spleen (Fig. 5B) and phagocyte infiltrationof this organ was occurring (Fig. 8A and B). The numbersof bacilli increased dramatically at 40 h when five out ofsix fish had conspicuous bacillary microcolonies, in accor-dance with the kinetics of bacterial counts in the spleen(Fig. 5B).

Although not assessed quantitatively, histopathologicalalterations similar to those above described for spleenswere seen in head kidneys collected at 18 and 40 h postinoculation.

Lysis of phagocytes in advanced pasteurellosis is due tosecondary necrosis and releases active neutrophilelastase

Phagocytes dying by apoptosis in the lesions abovedescribed eventually progressed to secondary necrosis.Figure 10A shows the sequence of alterations resulting inlysis of apoptosing cells which includes cells under lysisand with nuclei with apoptotic characteristics namely, frag-mentation and/or chromatin condensation (Fig. 10A, III).Lysing cells with apoptotic nuclei are a hallmark of apop-

Fig. 7. AIP56 exotoxin is present in apoptosing cells and apoptotic bodies in advanced pasteurellosis.A. Spleen of a sea bass with advanced natural pasteurellosis labelled by immunocytochemistry for AIP56 exotoxin. Numerous apoptosing cellswith fragmented and/or condensed nuclei and apoptotic bodies are labelled for AIP56 (brownish red) within an area of cell destruction.Objective 60¥.B. Blood in a vessel of the spleen of a sea bass with advanced natural pasteurellosis labelled by immunocytochemistry for AIP56 exotoxin.Apoptosing cells with fragmented nuclei and/or condensed chromatin are immunostained (brownish red) for AIP56. Objective 100¥.C. A focus of cell destruction in the spleen of a sea bass with advanced natural pasteurellosis shows immunostaining for AIP56 (brownishred). Several Phdp microcolonies in the lower right side of the micrographs are labelled by the anti-AIP56 antibody. Objective 10¥.D. The focus of cell destruction in C contains numerous cells positive for active caspase-3 (brownish red). Phdp microcolonies are stained byhaematoxylin (blue). Objective 10¥. Micrographs in C and D are from consecutive sections.



totic secondary necrosis (Wyllie et al., 1980; Hebert et al.,1996).

Secondary necrosis of cells undergoing caspase-3-associated apoptosis in vivo, as is the case with sea bassphagocytes exposed to AIP56, is accompanied by therelease of active caspase-3, with an increase in circulat-ing active protease (Sun et al., 2001; Gujral et al., 2003).We measured the levels of active caspase-3 in the bloodof sea bass infected i.p. with Phdp strain PP3. The verylow levels of that protease in the blood of control (non-infected) fish and of fish infected for 18 h, increased sig-nificantly at 40 h post infection in fish with detectableplasma AIP56 but not in fish without detectable circulatingexotoxin (Fig. 10B).

It has been shown that neutrophils undergoing apoptoticsecondary necrosis release active elastase (Liu et al.,2003). We found increased levels of circulating activeneutrophil elastase in Phdp-infected fish (Fig. 10C). Theincrease was significant in fish at 40 h post infection andwith circulating AIP56, but not in fish without toxaemia. Anexcellent correlation was found between the blood levelsof caspase-3 (Fig. 10B) and elastase (Fig. 10C) in fishwith toxaemia and advanced or terminal disease (correla-tion coefficient = 0.803; n = 11) suggesting that neutrophilelastase was released by lysis of apoptosing neutrophilsrather than by degranulation of stimulated neutrophils.These results show that neutrophil elastase, a highlytissue-damaging enzyme (Kawabata et al., 2002), is sys-temically released in acute pasteurellosis.

The disintegration of phagocytes by lysis due to sec-ondary necrosis explains the extensive occurrence of celldebris in the foci of cell destruction, as documented inFig. 1B and D.

No mortality or significant phagocyte destruction occurin sea bass inoculated with AIP56-negative strains

In contrast to the effects of i.p. inoculation of AIP56-positive Phdp strains, the i.p. inoculation of the AIP56-

Fig. 8. Macrophages and neutrophils initially infiltrate the spleenand are depleted in terminal disease in acute pasteurellosis. Thenumber per organ of normal and apoptosing macrophages andmacrophagic apoptotic bodies (stained for ANAE) and of normaland apoptosing neutrophils and neutrophilic apoptotic bodies(stained for myeloperoxidase) were counted in the spleens of seabass inoculated with 6.2 ¥ 105 cfu of Phdp strain PP3. In thisexperiment moribund fish (M) started to occur at 48 h postinoculation and the cumulative mortality was 93%. P.I., postinfection.A. The number of normal macrophages peaked at 24 h and thendecreased to values below basal numbers in moribund fish.B. Normal neutrophils peaked at 48 h and decreased to valuesclose to basal numbers in moribund fish.C. Apoptosing macrophages and macrophagic apoptotic bodies aresignificantly increased at 60 h post infection (P = 0.04) and inmoribund fish (P = 0.04).D. Apoptosing neutrophils and neutrophilic apoptotic bodies aresignificantly increased at 60 h post infection (P = 0.001) and inmoribund fish (P < 0.001).

Fig. 9. Caspase-3-positive cells increase in the spleen as the Phdpinfection progresses. Number of cells positive for active caspase-3per microscopical field (100¥ objective) (number of fields persample = 15) in sections of the spleen of sea bass infected i.p. with4.5 ¥ 103 of AIP56-positive Phdp strain PP3 (black bars) or with1.1 ¥ 104 of AIP56-negative strain EPOY 8803-II (white bars) andimmunostained for active caspase-3. In the experiment with strainPP3 moribund fish (M) started to occur at 40 h post inoculation andthe cumulative mortality was 95%. With strain EPOY 8803-IImortality was 0%. Results for strain PP3 are plotted according topresence or absence of AIP56 exotoxin in plasma as assayed byWestern blotting. ND, not determined. Values in the two highestcolumns are statistically different (P < 0.01) when compared withthe control. P.I., post infection.



negative strains EPOY 8803-II and ATCC 29690 did notlead to progressive infection, with all fish recoveringwithout exterior signs of disease or mortality. Both AIP56-positive and -negative Phdp strains injected i.p. inducedan intense local inflammatory reaction with importantinflux of monocytes/macrophages and mainly neutrophils,but while AIP56-positive Phdp bacilli injected i.p. persistedin the peritoneal cavity inducing extensive apoptosis andnecrosis of peritoneal macrophages and neutrophils soonafter inoculation (Fig. 4B), AIP56-negative bacilli wererapidly eliminated and no significant phagocyte apoptosisor necrosis was observed (not shown). These results indi-cate that with the non-virulent strains, in contrast withvirulent strains, no infection was established and neutro-phil elimination did not involve secondary necrosis.Accordingly, no histopathological alterations comparable

to those described above for fish infected with AIP56-positive strains, namely extensive apoptosis and second-ary necrosis of phagocytes in the head kidney, spleen andgut lamina propria, were seen in fish injected with strainsEPOY 8803-II (Fig. 11A and B) or ATCC 29690. As adirect control for the kinetic experiment carried out withthe AIP56-positive strain PP3 described above, strainEPOY 8803-II was injected i.p. with a slightly higher dose(1.1 ¥ 105 cfu) in sea bass of about the same weight (7 g)as with the virulent strain. We performed in these fish ablind, semi-quantitative kinetic assessment of histologicalparameters in sections of 18 spleens stained with HE (sixnon-injected fish as controls, six fish at 18 h and six fish at40 h post inoculation). At 18 and 40 h post inoculationneither infiltration of phagocytes nor significant phagocyteapoptosis or necrosis, nor bacteria were seen in any of

Fig. 10. Apoptosing cells in acute pasteurellosis lyse by secondary necrosis.A. High-power magnifications of cells from the focus of cell destruction in Fig. 1C and D showing progression through the several phases ofapoptotic secondary necrosis (Hebert et al., 1996). (I) Control cell with spherical single nucleus, with peripheral chromatin. (II) Shrunkapoptosing cell with dense cytoplasm, fragmented nucleus and condensed chromatin. (III) Cell under apoptotic secondary necrosis withapoptosing nucleus and lysing cytoplasm (note the low density of the cytoplasm in comparison with cell in panel II). (IV) Cell in advancedsecondary necrosis with a lysing cytoplasm and chromatolysis. (V) Cell remnant at the terminal phase of secondary necrosis. Objective 100¥.B and C. Levels of active caspase-3 and of neutrophil elastase were determined in the plasma of sea bass infected i.p. with 4.5 ¥ 103 of Phdpstrain PP3. In this experiment, 40 h post inoculation 15 out of 19 fish were in advanced phase of infection and four fish were moribund.Cumulative mortality was 95%. Results are expressed as RFU per ml of plasma and are plotted according to the presence or absence ofAIP56 exotoxin in plasma as assayed by Western blotting.B. Levels of active caspase-3 in plasmas of non-infected (control) and Phdp infected sea bass, determined as in Fig. 3. Incubation with thecaspase-3 inhibitor Ac-DEVD-CHO used as specificity control resulted in readings bellow 1 RFU ml-1 plasma (not shown). Basal levels ofplasmatic active caspase-3 seen in control fish and in fish without circulating AIP56 exotoxin are significantly increased (P < 0.001) in fish withadvanced disease and circulating exotoxin.C. Levels of plasmatic neutrophil elastase in the same fishes as in B, determined with the specific fluorescent substrateMeOSu-Ala-Ala-Pro-Val-AMC. Incubation with the neutrophil elastase inhibitor MeOSu-Ala-Ala-Pro-Val-CMK used as specificity control resultedin readings bellow 2 RFU ml-1 plasma (not shown). Basal levels of plasmatic active neutrophil elastase in control fish and in fish withoutcirculating AIP56 exotoxin are significantly increased (P < 0.01) in fish with advanced disease and circulating exotoxin. P.I., post infection.



the studied fish, the only histopathological alteration beingslight congestion. In contrast with the result obtained withstrain PP3 (Fig. 9), the quantitative assessment of cellspositive for active caspase-3 in the spleen sections of fishinjected with strain EPOY 8803-II showed very lownumbers of apoptosing cells similar to the control (Figs 9and 11B). Mortality in this experiment was 0%, contrastingwith 95% in the parallel experiment with strain PP3.

These results support the interpretation that apoptoticdestruction of sea bass phagocytes in acute pasteurello-sis is AIP56-mediated, representing a key pathogenicityfactor of AIP56-positive virulent Phdp that contributes tolethality of this infection.

Discussion

Our results show that the AIP56-dependent pathogenicitymechanism involving destruction of macrophages andneutrophils operates systemically in acute septicaemicpasteurellosis, including in the natural disease. This con-clusion is based on the following observations: (i) AIP56protein was found in the plasma and apoptosing phago-cytes in the organs of fish with advanced infection, indi-cating that the exotoxin is secreted in vivo by the infectingPhdp and is systemically distributed. (ii) The characteris-tics of the systemic phagocyte destructive process seen infish with advanced pasteurellosis are identical to thosepreviously seen in the process affecting peritoneal neu-trophils and macrophages in sea bass with experimentalPhdp peritonitis, injected i.p. with rAIP56 or in peritonealphagocytes exposed ex vivo to rAIP56 (do Vale et al.,2003; 2005). (iii) The amount of toxin in plasma correlateswith the severity of the disease and of the histopathologi-cal alterations. (iv) Most importantly, systemic phagocytedestruction similar to that seen in fish with advancednatural or experimental pasteurellosis was seen inspleens, head kidneys and gut of moribund sea bassfollowing the i.p. inoculation of rAIP56. As is the case of

all pathogenic bacteria, virulence of Phdp must bemultifactorial. However, several results indicate that theAIP56-dependent pathogenic mechanism is a key viru-lence factor in Phdp infections. First, the aip56 gene ispresent in all virulent Phdp strains studied so far (do Valeet al., 2005), being absent in non-virulent strains (do Valeet al., 2005). Second, the injection of rAIP56 leads todose-dependent lethality with phagocyte destruction iden-tical to that induced by Phdp infection. Third, passiveimmunization with anti-AIP56 antibodies provides signifi-cant protection against Phdp challenge (do Vale et al.,2005).

At the histopathological level, the AIP56-dependentmechanism of phagocyte destruction found in advancedpasteurellosis translates into depletion of phagocytesinstead of abscess formation, rarity of intraphagocyticbacteria and accumulation of large numbers of extracel-lular bacteria. This histopathological scenario is similar tothat recently described in a rat model of septicaemicplague (Sebbane et al., 2005).

The temporal progress of pasteurellosis analysed in thesea bass model of pasteurellosis induced by i.p. inocula-tion showed that, in the initial phases of the infection,virulent Phdp bacilli are extensively phagocytosed andbecome intracellular for several hours until lysis ofinfected macrophages and neutrophils due to the apopto-genic activity of AIP56 releases the bacteria. It has beenshown that Phdp is able to multiply within fish macroph-ages in vitro (Elkamel et al., 2003). However, it is notknown whether intramacrophage growth occurs in vivo inthe early phase of the infection before the occurrence ofapoptotic destruction of phagocytes, and this pointdeserves further studies. In the advanced phase withgeneralized bacterial colonization of the organs, intracel-lular Phdp were rarely seen but, instead, evidence forextensive extracellular multiplication was provided by ourobservation of the accumulation of large numbers ofextracellular bacteria with formation of numerous

Fig. 11. No significant apoptosis or necrosis occurs in sea bass organs after inoculation of AIP56-negative Phdp.A. HE-stained section of head kidney 40 h after i.p. inoculation of 1.1 ¥ 104 cfu of strain EPOY 8803-II. No apoptosis or necrosis is seen(compare with Fig. 1A and B). Objective 40¥.B. Section immunostained for the detection of active caspase-3 in spleen 40 h after i.p. inoculation of 1.1 ¥ 104 cfu of strain EPOY 8803-II.Very rare caspase-positive cells (red) are visible (compare with Fig. 2C and D). Objective 60¥.



microcolonies. This septicaemic spreading of the extra-cellular infection is accompanied by systemic dissemina-tion of AIP56 and by generalized apoptogenic activity ofthis exotoxin.

The mechanism of Phdp-induced peritoneal phagocytedestruction was found to be apoptotic secondarynecrosis. In multicellular organisms, apoptosing cells aredoomed to final disintegration by necrosis which normallyoccurs within scavenging cells, usually macrophages(Savill and Haslett, 1995; Lauber et al., 2004), afterphagocytosis triggered by engulfment signals at thesurface of the apoptosing cells (Lauber et al., 2004). Con-sequently, these cells are removed before lysis, whichprevents the occurrence of an inflammatory response(Savill, 1997; Haslett, 1999; Serhan and Savill, 2005).When removal by scavenging cells does not occur, apo-ptosing cells continue the apoptotic death program andultimately disintegrate by secondary necrosis (Wyllieet al., 1980; Haslett, 1999). This cytolysis triggers aninflammatory response and induces tissue damage (Renand Savill, 1998; Li et al., 2003; Erjefalt, 2005). Underphysiological conditions, apoptosing cells are rarely seen,because the clearing process through phagocytosis byscavenger cells is very efficient (Wyllie et al., 1980;Haslett, 1999). Thus, accumulation of high numbers ofapoptosing cells in tissues, as seen in acute pasteurello-sis, implies a significant increase in the rate of apoptosisand/or a deficiency in their removal (Wyllie et al., 1980;Haslett, 1999; Tuder et al., 2003), which, in turn, leads tothe demise of the apoptosing cells by terminal secondarynecrosis (Wyllie et al., 1980; Haslett, 1999). In fact, insuf-ficient clearance of apoptosing cells in vivo has beendescribed in situations of massive apoptosis that over-whelms the available scavenging capacity (Ogasawaraet al., 1993; Haslett, 1999; Devitt et al., 2004) or when thiscapacity is directly impaired (Medan et al., 2002; Li et al.,2003; Devitt et al., 2004). In acute pasteurellosis thesetwo mechanisms operate because phagocyte apoptosis isextensive and affects the principal scavenger of apop-tosing cells, the macrophage.

The AIP56-dependent pathogenicity mechanism relieson the concomitant destruction of both professionalphagocytes by an exotoxin, first at the location of entryand later at the systemic level. Bacterial toxins with cyto-toxicity for leucocytes have been described (Narayananet al., 2002). However, these leucotoxins are chemicallydistinct from AIP56, and have not been implicated in sys-temic infections (Narayanan et al., 2002).

Destruction by apoptotic secondary necrosis of fishmacrophages and neutrophils by Phdp AIP56 exotoxinrepresents a subversion of the endogenous apoptoticprocess of phagocytes. In normal situations, apoptosis ofsenescent macrophages and neutrophils occurs at theright time and is beneficial for the host, representing an

example of a physiological quiescent process (Kerr et al.,1972) whereby the apoptosing cells are eliminated byscavenger cells before lysis, thus preventing inflammationand tissue damage (Savill et al., 1993; Savill, 1997;Haslett, 1999). However, it has become progressivelyevident that the intrinsic apoptotic machinery of hostimmune cells can be put at work by an invading pathogento its own advantage (Weinrauch and Zychlinsky, 1999;Gao and Abu Kwaik, 2000; DeLeo, 2004). When apopto-sis affects important partners in defence mechanisms, asphagocytes, it goes awry and turns beneficial to the infect-ing agent. Bacterial modulation of phagocyte cell death isan emerging theme in pathogenesis (Weinrauch andZychlinsky, 1999; Gao and Abu Kwaik, 2000; DeLeo,2004) but in vivo induction of systemic destruction of bothprofessional phagocytes by a bacterial exotoxin, asdescribed here, has not been reported in other instances,and investigation of the role of apoptotic secondary necro-sis of macrophages and neutrophils in the genesis ofinfectious pathology has been neglected. Moreover,bacteria-induced apoptosis of neutrophils in vivo has notin general been linked to pathogenicity but rather viewedas beneficial to the host (Zysk et al., 2000; Kobayashiet al., 2003; DeLeo, 2004) because, through a silent, anti-inflammatory, elimination of apoptosing neutrophils bymacrophages, it would help in the resolution of neutrophil-associated inflammation (Savill et al., 1993; Savill, 1997;Haslett, 1999). However, for a safe elimination of apop-tosing neutrophils to occur, a healthy population of scav-enging macrophages is necessary, which is not the casein pasteurellosis, making AIP56-induced neutrophil apop-totic destruction detrimental for the host.

The AIP56-dependent pathogenicity mechanism ap-pears decisive for the rapid course and high mortality ofexperimental and natural acute fish pasteurellosis. First,by affecting both macrophages and neutrophils, AIP56releases intraphagocytic bacteria phagocytosed in theinitial phase of the infection and leads to evasion of thepathogen from the crucial host phagocytic defences, twoeffects that promote survival and unrestricted extracellularmultiplication of Phdp. Destruction of macrophages andneutrophils would also affect cytokine production by theseleucocytes, further impairing the host immune response.Second, by using an exotoxin, the AIP56-dependent cyto-toxicity can operate at a distance without requiring contactbetween the pathogen and the target cells. Third, the hostprotective immune response against AIP56 toxin requiresantibody production, a response that cannot be mountedin the short time lapse of the disease. Finally, it is mostlikely that cytotoxic phagocyte molecules released byAIP56-induced phagocyte lysis participate in the genesisof infection-associated pathology that, adding to the neu-tralization of the host phagocytic defence mechanism,cooperates in the etiopathogenesis of the disease.



Indeed, there is an impressive body of evidence showingthat neutrophils are rich in many cytotoxic molecules,including proteases that, when released, catalyse thehydrolysis of matrix macromolecules and damage manytypes of cells, therefore having the potential to producetissue injury with important local and systemic conse-quences (Henson and Johnston, 1987; Weiss, 1989;Anderson et al., 1991; Savill and Haslett, 1995; Savill,1997; Owen and Campbell, 1999; Kawabata et al., 2002).Released neutrophil cytotoxic molecules, includingelastase, one of the most tissue destructive enzymesknown (Kawabata et al., 2002), has been implicated in thepathogenesis of critical situations including systemicinflammatory response syndrome, acute respiratory dis-tress syndrome and multiple organ failure (Jochum et al.,1994; Kawabata et al., 2002; Inoue et al., 2006). It is ofrelevance that we have found in sea bass with advancedpasteurellosis elevated blood concentrations of activeneutrophil elastase. This result indicates that the enzymewas systemically released, and able to overwhelm theendogenous protease inhibitors, as has been found whenit is released in great amounts (Owen and Campbell,1999; Kawabata et al., 2002). Macrophages also containtissue-damaging molecules including several proteases,although to a lesser degree as compared with neutrophils(Owen and Campbell, 1999; Kawabata et al., 2002),which are, as well, released by the macrophage lysisobserved in Phdp infection. It is most likely that releasedneutrophil elastase and other neutrophil and macrophagecytotoxic molecules contribute to the extensive tissuedestruction seen in advanced pasteurellosis, as has beenreported in several instances (Weiss, 1989; Jochum et al.,1994; Owen and Campbell, 1999; Kawabata et al., 2002;Dockrell and Whyte, 2006; Inoue et al., 2006), thus par-ticipating in the pathogenesis of the terminal phase ofacute pasteurellosis. The AIP56-induced lysis of mac-rophages and neutrophils is therefore a very efficient,self-amplifying etiopathogenic mechanism, because itresults in two effects that operate in concert against thehost, namely, evasion of the pathogen from a crucialdefence mechanism, and release of tissue-damagingmolecules.

The very high resistance of fish to endotoxic shock iswell documented (Iliev et al., 2005) and suggests thatAIP56 exotoxaemia rather than LPS endotoxaemia is thekey pathogenic factor in acute pasteurellosis, a uniquefeature for a Gram-negative septicaemia.

A general picture of the progress of acute pasteurellosiscan be deduced from the results of the kinetic experi-ments assessing Phdp colonization (Fig. 5), numbers ofnormal and apoptosing phagocytes in the spleen (Fig. 8)and histopathology. From the site of entry, where Phdpinitially multiplies, the developing infection spreads bybacteraemia to spleen, kidney, gut lamina propria, liver

and other territories, where the invading bacteria inducephagocyte infiltration. AIP56 exotoxin secreted by multi-plying Phdp is systemically distributed and inducesphagocyte lysis with release of cytotoxic, tissue-damagingmolecules both at the places of bacterial multiplicationand at a distance due to toxaemia and toxin diffusion.Death follows the generalized tissue bacterial colonizationand cell destruction.

Most studies on induction of apoptosis of phagocyticcells by bacterial pathogens have been conducted inin vitro models of infection (DeLeo, 2004). These modelsoffer valuable advantages for dissection of mechanismsinvolved in the apoptotic destruction of immune cells butthey have, however, limitations when the full host/pathogen interactions operating in the natural diseasesare to be disclosed. In vivo studies directed at the searchof bacteria-induced simultaneous macrophage and neu-trophil destruction, and at the analysis of the role of thisdestruction in immune impairment and tissue injury, maywell reveal other septicaemic infections, in fish andmammals, where the agents use pathogenic mechanismssimilar to that here described in acute fish pasteurellosis.

In conclusion, the present results extend our previousobservations on AIP56 obtained with a model of localizedinfection, by showing that the apoptogenic antiphagocyticactivity of that exotoxin operates systemically in thenatural disease, which gives to that activity the characterof a relevant virulence mechanism. This mechanism rep-resents the first example of in vivo systemic destruction bysecondary necrosis of both professional phagocytes byan apoptogenic bacterial exotoxin with release of highlycytotoxic molecules.

Experimental procedures

Fish

Sea bass (Dicentrarchus labrax) were purchased from a com-mercial fish farm and were maintained in re-circulating aeratedseawater, at 19 � 1°C. Water quality was maintained withmechanical and biological filtration and UV disinfection and fishwere fed ad libitum on commercial pellets. Fish used for infectionexperiments were previously acclimatized to 23 � 1°C.

Sea bass, sea bream (Sparus aurata) and sole (Solea sene-galensis) with natural Phdp infection were obtained from two fishfarms during outbreaks of fish pasteurellosis. The disease wasidentified by isolation of Phdp from infected fish and identificationof the pathogen by conventional microbiological procedures.

Detection of aip56 gene in Phdp isolated from fish with naturalpasteurellosis was done as previously described (do Vale et al.,2003; 2005).

Phdp strains

Six virulent, AIP56-positive Phdp strains (PP3, MT1415,MT1375, MT1588, PTAVSA95 and B51) and two non-virulent,



AIP56-negative Phdp strains (EPOY 8803-II and ATCC 29690)were used. The, origin and virulence of each isolate and pres-ence or absence of the aip56 gene had previously beendescribed (do Vale et al., 2003; do Vale et al., 2005). Strains B51,and EPOY 8803-II were provided by Prof Alicia E. Toranzo(Departamento de Microbiología y Parasitología, Facultad deBiologia, University of Santiago de Compostela, Spain), strainsMT1415, PP3, MT1375 and MT1588 were provided by DrAndrew C. Barnes (Marine Laboratory, Aberdeen, UK). StrainPTAVSA95 is from our collection. Strain ATCC 29690 wasobtained from the American Type Culture Collection, USA.

Growth conditions and inocula preparation were carried out asdescribed (do Vale et al., 2003; 2005). Plating serial dilutions ofthe final bacterial suspensions onto TSA-1 plates and countingthe number of cfu following incubation at 22°C for 2 days con-firmed bacterial concentrations of the inocula.

Experimental Phdp infections

An experimental model reproducing acute natural pasteurellosiswas developed. Groups of sea bass with approximately the sameweight were injected i.p. with inocula of virulent Phdp adjustedaccording to the size of the fish and the virulence of the strain soto consistently produce systemic, lethal infections in most fish,with deaths occurring in 2–3 days. In the kinetic experiments, thePhdp strain PP3 was used with the inocula varying between4.5 ¥ 103 for fish weighing approximately 7 g and 6.2 ¥ 105 forfish of approximately 20 g. These inocula produced infectionswith mortalities from 73% to 95%. Fish were injected i.p with100 ml of Phdp suspensions after anaesthetization with 0.03%(v/v) ethylene glycol monophenyl ether (Merck, Darmstadt,Germany). At the post-infection times indicated in Results, fishwere euthanized after anaesthesia for sample collection. Fishshowing lethargy and loss of equilibrium were found to die withina very short time; therefore, these moribund fish were sampledregardless of the time post infection. In the experiments heredescribed, following i.p. inoculation of Phdp fish started tobecome moribund between 40 and 72 h post inoculation. Theinoculation of sea bass by the water route was done bycohabitation. A group of 30 fish was kept in a recirculatingaquarium system together with six groups of 30 fish injected i.p.with Phdp strain PP3. During the infection period, the disinfectionsystems were shut down. Cumulative mortalities in the i.p.injected groups ranged from 86% to 100%. Fish from thecohabitation-infected group started to die at day 8 post infectionand the cumulative mortality in that group was 27%.

As a control for the inoculation of the virulent, AIP56-positivePhdp strains, sea bass were injected i.p. with the non-virulent,AIP56-negative strains EPOY 8803-II and ATCC 29690. Ascontrol for the kinetic experiment with inoculation of sea bassweighing approximately 7 g with 4.5 ¥ 103 cfu of the virulentstrain PP3, sea bass of the same weight were injected i.p. with100 ml of a suspension prepared as described above of strainEPOY-8803-II, containing 1.1 ¥ 104 cfu.

Production and inoculation of rAIP56

Soluble His-tagged AIP56 (AIP56H+) protein was purified byaffinity chromatography from the soluble fraction of BL21Escherichia coli carrying the pETAIP56H+ plasmid induced at

17°C, as described (do Vale et al., 2005). The endotoxin contentof purified rAIP56 was determined by Cambrex Bio SciencesVerviers (Belgium) using the LAL kinetic chromogenic QCL®

assay. The sample was pretreated to avoid interference by dilu-tion in LAL reagent water, followed by heat inactivation at 75°Cfor 40 min, a further dilution with MgCl2 and the addition ofPyrosperse to a final concentration of 0.5%. The rAIP56 con-tained 298 000 EU ml-1 of endotoxin, corresponding approxi-mately to 29.8 mg ml-1 LPS. rAIP56 and known amounts of BSAwere separated in a 12% SDS-PAGE (Laemmli, 1970), the gelwas stained with Coomassie blue, digitalized, and the concen-tration of rAIP56 determined by densitometry using the QuantityOne® program (Bio-Rad) program. The concentration of rAIP56was found to be 16 mg ml-1.

Groups of sea bass were injected i.p. with 100 ml saline con-taining increasing doses of rAIP56, starting at 20 mg. As controls,two groups of sea bass were injected i.p. with the same amountof rAIP56 boiled for 30 min. Moribund fish were killed by anaes-thetic overdose and organs collected for histological analysis, asdescribed below.

Histological techniques

For conventional microscopy of paraffin-embedded samples,tissues were fixed with 10% buffered formalin. Sections weredeparaffinized, rehydrated and stained with HE. The internucleo-somal DNA fragmentation was detected by TUNEL staining ofDNA strand breaks with the In Situ Cell Death Detection Kit, AP(Roche Applied Sciences) following the manufacturer’s instruc-tions and using 3-amino-9-ethylcarbazole (AEC) as substrateand methyl green or haematoxylin as counterstain.

Antibodies for the specific labelling of sea bass macrophagesand neutrophils are not available. Therefore, we used enzymaticcytochemical methods to label those leucocytes. Sodiumfluoride-inhibitable ANAE activity had been shown to specificallylabel mammalian (Li et al., 1973; Ennist and Jones, 1983) andfish (Passantino et al., 2003) macrophages. ANAE activity wasdetected as previously described (Li et al., 1973; Ennist andJones, 1983). ANAE-positive cells show red cytoplasmic staining.Sea bass neutrophils are specifically labelled by staining formyeloperoxidase activity (do Vale et al., 2002). This staining wasdone as previously described (Afonso et al., 1998). Neutrophilicmyeloperoxidase staining appears as yellow-brown cytoplasmicgranules.

Immunocytochemistry

To become proteolytically active, procaspase-3 has to be pro-cessed by cleavage of its precursor (Boatright and Salvesen,2003). For detection of processed (activated) caspase-3 a 1:500dilution of an affinity-purified rabbit Anti-active human caspase-3(catalogue n° AF835, R&D Systems) raised against the peptideCRGTELDCGIETD of human caspase-3 was used. This antibodydetects the processed large subunit of activated caspase-3, butdoes not detect, at the used dilution, the precursor form. TheAnti-active caspase-3 antibody recognizes processed sea basscaspase-3 because of the presence of the peptide CRGTDLDPGIETD in sea bass caspase-3 and because this caspase isprocessed in a similar way as the human caspase-3 (Reis et al.,2007). For detection of AIP56, an AIP56-specific rabbit serum (do



Vale et al., 2005) was used at a dilution of 1:6000 and for detec-tion of Phdp a Phdp-specific rat serum (see below) was used at adilution of 1:500. Sections were deparaffinized and rehydratedand the endogenous peroxidase activity inactivated by incubationwith 3% hydrogen peroxide in methanol. For detection of AIP56,sections were heated in a microwave oven for 25 min at 720 W in0.1 M citrate buffer, pH 6.0 for antigen retrieval before beingsubjected to endogenous peroxidase inactivation. Incubationswith the Anti-active caspase-3 and anti-Phdp antibodies weredone at room temperature for 2 h. Incubations with the anti-AIP56antibody were performed overnight at 4°C. Detection of primaryantibodies was done using the Histostain®-Plus Bulk Kit (ZymedLaboratories), following the manufacturer’s instructions and usingAEC as substrate. Sections were counterstained with haematoxy-lin. As negative controls of immunostaining techniques, sectionsfrom non-infected fish subjected to the complete stainings andsections not exposed to the primary antibodies were used.

Enumeration of macrophages and neutrophils in thespleen

Sea bass spleens were homogenized in PBS adjusted to355 mOsm and supplemented with 5% glucose and 30 units ml-1

of heparin by squeezing through 70 mm Nylon cell strainers (BDFalcon). Absolute cell counts were performed in a haemocytom-eter using the Natt-Herrick solution as diluent (Natt and Herrick,1952). Differential macrophage and neutrophil counts were per-formed as described (Traver et al., 2003) except that splenocytesmears were stained for ANAE activity (Ennist and Jones, 1983)and for myeloperoxidase activity (Afonso et al., 1998) to specifi-cally label macrophages and neutrophils respectively.

Detection of AIP56 in blood

Blood was collected from the caudal sinus using a heparinizedsyringe and immediately centrifuged at 2000 g for 5 min at 4°C.Plasma was aliquoted and stored at -80°C until used. For detec-tion of AIP56, 1 ml of aliquots was subjected to SDS-PAGE(Laemmli, 1970) and subsequently blotted onto nitrocellulosemembranes (Schleicher and Shuell), according to manufacturer’sinstructions. Immunodetection of AIP56 was performed with a1:15 000 dilution of an anti-AIP56 rabbit antiserum (do Vale et al.,2005). Rabbit IgGs on Western blots were detected with adonkey anti-rabbit IgG horseradish peroxidase-linked secondaryantibody (The Binding Site) using the SuperSignal West Durachemiluminescence kit (Pierce biotechnology).

Production and characterization of anti-Phdp antibody

Phdp strains PP3 and MT1415 were grown as described above,washed, resuspended in saline and killed by exposure to an UVlamp for 2 h. Immunization of two Wistar rats was performedaccording to established procedures (Harlow and Lane, 1998),using UV killed bacterial cells (mixture 1:1 of cells from strainsPP3 and MT1415) as antigen. Each rat received three injections.The rats were bled after the third injection and the sera tested fortiter by slide agglutination, and pooled. The specificity of thepooled serum was tested by Western blotting. Briefly, total pro-teins from several bacteria pathogenic for fish were subjected to

12% SDS-PAGE and blotted onto a nitrocellulose membrane(Schleicher and Shuell). Western blotting was performed asdescribed above, using a 1:6000 dilution of the pooled rat serumas primary antibody and a 1:4000 dilution of a Goat anti-rat IgGhorseradish peroxidase-linked species-specific whole antibody(Amersham Biosciences) as secondary antibody. Tested bacteriaincluded Tenacibaculum maritimum KI 74.1, Streptococcusparauberis RM 222.1, Photobacterium damselae ssp. damselaeTW 397, Aeromonas salmonicida ssp. salmonicida EW 22.1 andRIM 44.1, Vibrio anguillarum RM 253.1 and PC 557.01 andPhotobacterium damselae ssp. piscicida PP3. All the bacterialstrains, except PP3, were provided by Prof Alicia Toranzo (Depar-tamento de Microbiología y Parasitología, Facultad de Biologia,University of Santiago de Compostela, Spain). The anti-Phdpantibody was found to recognize several protein bands from thetwo subspecies of Photobacterium damselae but no staining wasobserved in lanes loaded with total proteins from the other bac-terial species tested.

Detection of caspase-3 activity in plasma and tissues

Caspase-3 activity was determined using a fluorimetric caspase-3assay kit (Sigma-Aldrich) as described (do Vale et al., 2003;2005). Plasma was obtained as described above. Soluble extractswere prepared from homogenates of spleens and kidneys byincubation for 20 min on ice with the kit’s lysis buffer. Results wereexpressed as relative fluorescence units (RFU) per microlitre ofplasma, or per microgram of protein in organ extracts determinedusing the Micro BCA protein assay kit (Pierce biotechnology).

Detection of neutrophil elastase activity in plasma

It has been shown that Phdp does not possess elastase activity(Magariños et al., 1992). Preliminary experiments had shown thatsea bass neutrophils, like mammalian neutrophils, containelastase activity that degrades the specific substrate MeOSu-Ala-Ala-Pro-Val-AMC. Proteolytically active neutrophil elastasewas detected in plasma obtained as described above, by fluo-rimetry using the specific neutrophil elastase substrate MeOSu-Ala-Ala-Pro-Val-AMC (Sigma) (Pelus et al., 2004). Briefly, 5 ml ofaliquots of plasma was dispensed in duplicate in microtiter plates.To control the non-specific hydrolysis of the substrate, 4 ml of a10 mM solution of the specific neutrophil elastase inhibitorMeOSu-Ala-Ala-Pro-Val-CMK (Sigma Aldrich) was added to oneof the duplicates (Pelus et al., 2004). Plates were incubated for5 min at room temperature and 200 ml of reaction mixture (50 mMTris-HCl pH 7.5, 150 mM NaCl, 0.05% Triton X-100, 0.01% BSA,50 mM MeOSu-Ala-Ala-Pro-Val-AMC) were added to each well.The kinetics of substrate cleavage was measured in a SpectraMax Gemini XS fluorimeter (Molecular Devices) at an excitationof 380 nm and emission of 460 nm. Results were expressed asRFU per microlitre of plasma.

Statistics

Statistical analysis of the data was performed using the Student’st-test and P < 0.05 was considered significant.

Acknowledgements

The authors are grateful to Tony Ellis for helpful discussions andsupport, to Alberto Villena for help with leucocyte cytochemistry,



to Jorge Pedrosa, Gil Castro and Diana Nascimento for criticalreading of the manuscript and to Alexandra Rema, Célia Lopesand Fátima Faria for assistance with histology. This work wassupported by FCT project POCI/MAR/56111/2004 funded byPOCI 2010, cofunded by FEDER. Ana do Vale and CarolinaCosta- Ramos were supported by FCT Grants SFRH/BPD/11538/2002 and SFRH/BPD/20881/2004 respectively.

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systemic macrophage and neutrophil destruction by secondary necrosis induced by a bacterial exotoxin...

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