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IMMUNOLOGIE VAN DE KARPER
GER RIJKERS
tekeningen: Wim Valen
tikwerk
tV\ :
IT-M
: Anke Hoeksma
INHOUD
bladzijde
VOORWOORD
3
CELLET
IMMUNOLOGIE
antigenen en antistoffen
8
humorale en cellulaire immuniteit
11
organen
12
immunologische reactie
13
TECHNIEKEN
rozet test
15
piaque test
15
schübtransplantie
16
IMMUNOLOGIE VAN DE KARPER
18
VOORWOORD
Proefschriften worden in het algemeen nauwelijks
gelezen. In de meeste gevallen beperkt men zich
tot de laatste stelling, het dankwoord en de
levensloop. Hiervoor zijn 3 (goede) redenen aan
te wijzen:
1) de tekst is meestal in het Engels gesteld,
2) het onderwerp is erg gespecialiseerd en zeker
voor een leek nauwelijks te begrijpen,
3) wetenschappelijke teksten zijn saai.
In dit gedeelte van het proefschrift wilde ik
vertellen wat ik gedurende 3 jaar met "die karpers"
gedaan heb. De tekst is in het nederlands, verder
heb ik geprobeerd ook het 2e en 3e argument te
ondervangen.
Ik hoop dat U na het lezen van dit gedeelte met
mij de volgende stelling kunt onderschrijven:
Immunologie is niet moeilijk.
ALGEMEEN
Om zinvol te kunnen praten over immunologie is het noodzakelijk om
eerst iets te vertellen over cellen, DMA, RNA, aminozuren, eiwitten
etc. Iedereen heeft deze termen vast wel eens ergens gehoord, maar de
betekenis en onderlinge samenhang zal niet iedereen duidelijk zijn.
CELLEN
Een cel kunnen we omschrijven als de kleinste eenheid binnen een
organisme die nog tot alle basisfuncties in staat is. Als basisfunctie
beschouwen we het in leven blijven en het in staat zijn om zich te
delen. Omdat definities nooit zo erg duidelijk zijn (definitie van een
stoel?) volgen hier enkele voorbeelden.
Zoals een huis is opgebouwd uit stenen, een rekenmachientje uit
chips, zo zijn dieren en planten opgebouwd uit cellen. Hogere dieren
en planten zijn opgebouwd uit zeer veel cellen (de mens b.v. uit
2 à 300.000.000.000.000 c e l l e n ) . Uit de definitie van een cel volgt
dat er ook organismen kunnen voorkomen bestaande uit één enkele cel.
Voorbeelden hiervan zijn bacteriën en het pantoffeldiertje. Het pantoffeldiertje heeft het vermogen tot bewegen, voedsel- en zuurstofopname verenigd in die ene cel. De cellen van hogere organismen zijn
gespecialiseerd, dat wil zeggen elke cel heeft z'n eigen functie.
Specialisatie van cellen bij dieren
cel
rode bloedcel
darmcel
spiercel
functie
transport van zuurstof
opname van voedsel
beweging
Een cel moet aan een aantal vereisten voldoen om in leven te kunnen
blijven en om zijn speciale functie uit te kunnen oefenen. Deze vereisten zullen we bespreken aan de hand van een voorbeeld: de plasma
cel. De functie van een plasmacel is het produceren van antilichamen
of antistoffen (daar komen we later nog uitvoerig op t e r u g ) . Binnen
een cel kunnen we verschillende onderafdelingen onderscheiden: een informatiecentrum (de k e r n ) , energiecentrales (mitochondriën), fabrieken
(ribosomen), afvalverwerkingsbedrijven ( l y s o s o m e n ) , inpakafdelingen
(Golgi-apparaat) en verder nog een infrastructuur (endoplasmaties
reticulurn). Zie fig. 1.
Fig. 1: De onderdelen van een cel.
Om te voorkomen dat deze processen in de soep lopen (de afvalverwerking gaat het informatiecentrum opruimen) zijn al deze onderafdelingen
van elkaar gescheiden door een tussenwandje (membraan). De cel als
geheel is ook omgeven door een wand, de celmembraan. Deze celmembraan
zorgt er niet alleen voor dat het hele zaakje bij elkaar gehouden
w o r d t , maar ook dat grondstoffen (voor de fabrieken en de energiecentrale) naar binnen of naar buiten kunnen.
Een organisme ontstaat door versmelting van een eicel met een zaadcel. Door deling van deze ene cel ontstaan dan 2 cellen, die zich weer
delen, enz. Dat dit snel kan aantikken is bekend uit de parabel van de
graankorrel en het schaakbord (fig. 2 ) .Wanneer we links bovenaan met
1 graankorrel beginnen, op het 2e vakje 2 korrels, op het 3e vakje 4,
e n z . , bereiken we vrij snel de wereld graan productie. In fig. 2 zien
we ook waar we uitkomen voor een volwassen mens als we links boven op
het schaakbord beginnen met een eicel en een zaadcel.
Bij deling wordt de erfelijke informatie (opgeslagen in de kern)
keurig verdeeld over de twee cellen. Hierbij gaat geen informatie
verloren, want voordat een cel zich deelt, wordt eerst alle informatie
gecopieerd zodat de twee nieuwe cellen weer de oorspronkelijke hoeveelheid bezitten. Het gevolg hiervan is dat elke cel dus alle
erfelijke informatie bezit.
t k g GRAAN
JAARUJKSEWERELDGRAANPRODUCTIE
BABV. 1DAG
10TON URAAN
Fig. 2:De graankorrel ophet schaakbord.
De erfelijke informatie in de kern is gecodeerd. We kunnen dit vergelijken meteen computer, waar de informatie opeenmagneetband (DNA)
gecodeerd ligt. Alsereen bepaalde opdracht moet worden uitgevoerd,
b.v. de produktie vananti1ichamen, danwordt in de kern het benodigde
stukje informatie overgeschreven op "boodschapper RNA".Deze boodschapper brengt de opdracht (in code) over naar de ribosomen, die zich
buiten de kern verspreid in decel bevinden. Er zijn verschillende
-DNA:
«»»SÄ?
BOODSCHAPPER^
CELMEMBRAAAI
RIBOSOMEN
KERN
/
/
GRANULAIR ENDOPLASMATIES
RETICULUM
/
^
Wv
©
9 J
60LÖI APPARAAT
Fig. 3: Aanmaak van eiwitten.
(py\
>
J ANTILICHAMEN
soorten ribosomen. Zo zijn er losse ribosomen die eiwitten maken
voor eigen gebruik, zoals b.v. eiwitten die nodig zijn voor de vervanging van versleten onderdelen. Ribosomen die eiwitten maken die
bestemd zijn voor de export (zoals b.v. anti1ichamen) liggen op het
endoplasmaties reticulum zodat de eiwitten meteen naar de inpakafdelingen getransporteerd kunnen worden. Endoplasmaties reticulum met
ribosomen erop noemt men granulair endoplasmatisch reticulum ( G E R ) ;
kant-en-klare eiwitten worden door het Golgi-apparaat ingepakt in
blaasjes. Door middel van deze blaasjes worden de eiwitten uitgescheiden (fig. 3 ) .
Eiwitten zijn opgebouwd uit aminozuren. Er zijn 20 verschillende
aminozuren. De eigenschappen van een eiwit worden bepaald door het
a a n t a l , de samenstelling en de volgorde van de aminozuren. We kunnen
eiwitten vergelijken met w o o r d e n . Woorden zijn opgebouwd uit letters.
Er zijn 26 verschillende letters. De betekenis van een woord wordt
bepaald door het a a n t a l , de samenstelling en de volgorde van de letters.
IMMUNOLOGIE
Alle gewervelde dieren zijn uitgerust met een bewakingssysteem
(immuunsysteem), dat er voor zorgt dat binnengedrongen vreemde stoffen of cellen onschadelijk gemaakt worden. Deze vreemde zaken kunnen
bijvoorbeeld bacteriën zijn of virussen of eiwitten. Een immuunsysteem moet om die ongewenste bezoekers (die we antigenen noemen) te
herkennen, onderscheid kunnen maken tussen "vreemd" en "eigen".
Onder eigen verstaan we de cellen, eiwitten, hormonen enz. waaruit het
lichaam is opgebouwd. Dit vermogen om onderscheid te kunnen maken is
erg belangrijk omdat het natuurlijk niet de bedoeling van het immuunsysteem is om het eigen lichaam af te breken.
Een voorbeeld uit de praktijk kan veel van de immunologie duidelijk
maken. Als je een griepje oploopt kun je een paar dagen goed ziek
zijn. Het griepvirus is dan je lichaam binnengedrongen en is zich daar
naar hartelust aan het vermenigvuldigen. Na enige tijd ben je echter
weer genezen. Wat er zich in de tussentijd heeft afgespeeld is het
volgende: het immuunsysteem heeft het virus als vreemd herkend en het
daarna onschadelijk gemaakt.
Antigenen en antistoffen
Vreemde stoffen roepen een bepaalde reactie op wanneer ze bij mens
of dier worden ingespoten. De vreemde stoffen noemen we met een verzamelnaam antigenen. Men heeft gevonden dat voor het oproepen van een
immunologische reactie niet een compleet eiwit of een hele cel nodig
is. Een groepje van 6-7 aminozuren is al voldoende. Zo'n eenheid
noemen we antigene déterminant.
Als reactie op het inspuiten van een antigeen worden antistoffen
geproduceerd. Deze antistoffen binden specifiek met het antigeen: dat
wil zeggen ze reageren alléén met het antigeen waarmee het dier is ingespoten en nergens anders mee. Specificiteit is één van de belangrijkste kenmerken van een immunologische reactie.
A-griep virus: wél reactie
A-griep virus
antistoffen
Hongkong griep virus: geen reactie
Antistoffen, die ook wel immunoglobulines worden genoemd, komen voor
in het bloed, maar ook in de rest van het lichaam. Bij mensen komen
verschillende soorten immunoglobulines voor. De belangrijkste zijn
IgG en IgM (Ig = Immunoglobuline, G en M geeft de klassea a n ) .
In figuur 4 staan IgG en IgM schematisch getekend. Het gearceerde
gedeelte van het immunoglobuline bindt (specifiek) aan het antigeen.
We zien dat IgG 2 bindingsplaatsen heeft, IgM zelfs 10.
zoogdier
IgG
IgM
0 #
specifiekebinding
o
l f
Klontering
Fig. 4: Antistoffen
We zien dat immunoglobulines zich kunnen binden aan 1ichaamsvreemde
stoffen. Dit alleen is echter niet voldoende om b.v. een ziekteverwekkende bacterie onschadelijk te maken. Dit onschadelijk maken kan
op twee manieren gebeuren: de eerste manier is door macrofagen (fig.5)
Deze holle bolle Gijzen zijn grote cellen die graag rommel opruimen.
Ze doen dat niet specifiek; alles wat vreemd is wordt opgepeuzeld
(macrofaag betekent letterlijk veelvraat),
Fig. 5: Macrofaag
Omdat immunoglobulines meerdere bindingsplaatsen hebben, zijn ze in
staat om antigenen aan elkaar te doen klonteren. Deze samengeklonterde
antigenen zijn dan extra lekker voor macrofagen. Een tweede manier
waarop antigenen onschadelijk gemaakt worden nadat antilichamen gebonden zijn is de volgende.
In het bloed bevindt zich een hele reeks van stoffen die we met een
verzamelnaam het complementsysteem noemen. Wanneer een antilichaam
gebonden is aan een antigeen (fig. 6) verandert er iets aan het antilichaam waardoor het eerste deel van het complementsysteem (Cl) gebonden wordt. Cl activeert dan C 2 , C3 activeert C4 enz. totdat het
laatste deel C9 geactiveerd wordt. C9 is een stof die de membraan van
de vreemde cel doet oplossen, waardoor de vreemde cel gedood wordt.
Fig. 6: Werking van complement.
De belangrijkste kenmerken van het immuunsysteem zijn de specificiteit
en de vorming van (immunologisch) geheugen.
Tegen griep kun je je in laten enten. Je krijgt dan een injectie met
gedood griepvirus. Omdat het virus dood is kan het zich niet meer ver-
10
menigvuldigen en kun je van zo'n injectie niet ziek worden. Het
immuunsysteem herkent het griepvirus echter wél als vreemd en reageert
daarop. Er worden antistoffen gevormd, maar er wordt ook immunologisch
geheugen opgewekt. Na een griepspuit ben je niet meer vatbaar. Dit
betekent echter niet dat je dan niet meer met het virus besmet kunt
raken. Het virus dringt nog wel het lichaam binnen, maar het immuunsysteem reageert zo snel en zo doeltreffend dat het virus al onschadelijk gemaakt is voordat het je ziek heeft kunnen maken. Je bent dan
immuun voor de griep. Het immunologisch geheugen, dat daarbij gevormd
w o r d t , houdt lang aan. De bekende DTKP prikken maken je gedurende de
rest van je leven immuun voor difterie, tetanus, kinkhoest en polio.
Toch biedt een griepspuit over het algemeen maar bescherming voor één
jaar; het volgend jaar kun je toch weer de griep krijgen. Dit wordt
niet veroorzaakt door een kortdurend immunologisch geheugen maar heeft
alles te maken met de specificiteit van het immuunsysteem. Een griepepidemie krijgt - net als orkanen - een naam: de A-griep, de Hongkonggriep enz. Men gebruikt die verschillende namen omdat het telkens weer
een ander virus betreft. Alhoewel die verschillende virussen wel allemaal griep veroorzaken, verschillen ze onderling zó sterk dat het immuunsysteem zich niet meer herinnert daarmee ooit in aanraking te zijn
geweest. Een A-griep spuit biedt dan ook geen bescherming tegen een
infectie met het Hongkong-griep virus.
Humorale en cellulaire immuniteit
In de immunologische reacties die we tot nu toe besproken hebben
wordt de vreemde stof onschadelijk gemaakt met behulp van anti1ichamen.
Men noemt deze vorm van verdediging humoral immuniteit. Humoraal betekent vloeibaar. Een dergelijke naam is gekozen omdat deze vorm van
immuniteit overgebracht kan worden door een dier met serum van een
immuun dier in te spuiten. De cellen die betrokken blijken te zijn bij
humorale reacties heten B-lymfocyten.
Een andere vorm van immuniteit is de zogenaamde cellulaire immuniteit. Voorbeelden uit de praktijk zijn de afstoting van een getransplanteerd orgaan en de Mantoux-reactie (krasjes bij TBC-controle ) . Bij
een cellulaire immunologische reactie worden geen antilichamen gemaakt,
de vreemde cel wordt onschadelijk gemaakt door direct contact met een
lymfocyt (fig. 7 ) .De lymfocyten betrokken bij cellulaire immuniteit
noemt men T-lymfocyten. De twee kenmerken van een immunologische
reactie specificiteit en geheugen gelden voor zowel humorale als
cellulaire reacties.
11
Fig. 7.
Organen
Belangrijke immuunorganen bijde mens zijn milt, thymus (zwezerik),
beenmerg, lymfeklieren en in mindere mate tonsillen (amandelen)en
appendix (blinde darm). T-lymfocyten komen uit de thymus en B-lymfocyten worden aangemaakt inhetbeenmerg. Vissen bezitten geen beenmerg
of lymfeklieren. De belangrijkste organen bijeenvis zijn thymus,
milt, kopnier en nier.(fig.8 ) .
A
B C D
A thymus
B kopnier
C milt
D nier
Fig. 8: Immuunorganen bijde karper
De thymus bestaat vrijwel uitsluitend uit lymfocyten, in milt, kopnier
en nier komen naast lymfocyten ook veel rode bloedcellen en granulo-
12
cyten voor. Behalve in de immuunorganen komen lymfocyten ook voor in
het bloed waar ze behoren tot de witte bloedlichaampjes.
Immunologische reactie
Wat gebeurt er nu precies als een vreemde stof het lichaam is binnengedrongen. We hebben al besproken dat antilichamen specifiek een
vreemde stof kunnen binden. Lymfocyten hebben antilichamen aan hun
celoppervlak. Door middel van deze oppervlakte-anti1ichamen zijn ze
in staat om vreemde stoffen te herkennen. Deze herkenning is specifiek,
d.w.z. een lymfocyt herkent maar één bepaalde vreemde stof en een
vreemde stof wordt maar door één lymfocyt (of een groepje identieke
lymfocyten) als vreemd herkend. In figuur 9 is deze specifieke herkenning vergeleken met een aangerande maagd (lymfocyt) die met haar
hand (oppervlakte-anti1ichaam) uit een hele reeks van verdachten haar
aanvaller (vreemde cel) herkent aan de vorm van zijn oren (antigene
determin a n t ) .
Fig. 9: Specifieke herkenning
Nadat de herkenning heeft plaats gevonden (tussen vreemde stof A en
lymfocyt a) gaat de desbetreffende lymfocyt zich delen. Op die manier
ontstaan er veel lymfocyten die allemaal specifiek zijn voor de vreemde stof A. Daarna veranderen (differentiëren met een mooi woord) de
13
lymfocyten in plasmacellen. Plasmacellen produceren veel anti1ichamen
(specifiek gericht tegen vreemde stof A) en deze antilichamen komen
onder andere in het bloed terecht. De manier waarop antilichamen een
vreemde stof herkennen en onschadelijk maken hebben we reeds besproken.
Nadat herkenning heeft plaatsgevonden veranderen gelukkig niet alle
lymfocyten in een plasmacel. Een gedeelte wordt namelijk geheugencel.
Deze geheugencellen zorgen ervoor dat als er weer een infectie met
vreemde stof A optreedt het hele proces van herkenning tot en met
antilichaamproductie veel sneller en heftiger verloopt.
Als voorbeeld dient een grafiek va.n het aantal plasmacellen in de kopnier van een karper tijdens een eerste en een herhaalde reactie (fig.
10).
dagennainjectie
Fig. 10: Een eerste (A) en herhaalde (A) immunologische reactie in de
kopnier van een karper ingespoten met schape rode bloedcellen.
14
TECHNIEKEN
In dit hoofdstukje worden enkele technieken besproken die gebruikt
zijn bij het onderzoek van het immuunsysteem van de karper.
Rozet test
Met behulp van de rozet test is het mogelijk om het aantal lymfocyten gericht tegen een bepaalde vreemde stof te bepalen. We maken
daarbij gebruik van het feit dat de oppervlakte-anti1ichamen van een
lymfocyt een vreemde stof kunnen binden.
Van een karper die ingespoten is met konijne rode bloedcellen
(KRBC) verwijderen we de milt. De milt wordt fijngeknipt en door een
fijnmazig gaasje gewreven zodat we een oplossing krijgen bestaande uit
losse miltcellen. Aan deze celsuspensie voegen we KRBC toe en vervolgens zetten we dit mengseltje een tijdje in de koelkast. Als er in de
milt lymfocyten aanwezig waren specifiek gericht tegen KRBC, dan binden de oppervlakte-anti1ichamen van de lymfocyt aan de KRBC. Op die
manier ontstaat er een krans van KRBC rondom zo'n lymfocyt: een rozet
(fig. 1 1 ) . Door op verschillende dagen na inspuiten het aantal rozetten te tellen kun je een idee krijgen hoe de lymfocyten reageren op
deze 1ichaamsvreemde stof.
Fig. 11: Rozet vormende cel.
PIague test
Bij de plaque test bepalen we het aantal plasmacellen dat antilichamen uitscheidt tegen een bepaalde vreemde stof of cel. Als vreemde cel gebruiken we nu niet KRBC maar schape rode bloedcellen ( S R B C ) .
Ook hier maken we weer een celsuspensie en mengen deze met SRBC.
Bovendien wordt complement aan het mengsel toegevoegd. Dit mengseltje
laten we tussen twee microscoop glaasjes lopen. De SRBC en de karpercellen zijn zodanig verdund dat 1) tussen de glaasjes een monolayer
15
ontstaat, d.w.z. de cellen liggen tegen elkaar aanineenenkele laag;
2) er veel meer SRBC zijn dan karpercellen. Alserinhet mengsel
plasmacellen aanwezig waren dan gaan deze cellen tussen de glaasjes
rustig door met de taak waar zemeebezig w a r e n , namelijk het maken
en uitscheiden vanantilichamen. De antilichamen binden zich aande
SRBC, dieimmers in overmaat aanwezig zijn, vervolgens wordt het complement geactiveerd ende SRBC in de buurt vande plasmacel wordengedood. Opdiemanier ontstaat er rondom de plasmacel eengatdatna
verloop van tijd methetblote oogkanworden waargenomen. Doorhet
aantal gaten (plaques) te tellen komen we teweten hoeveel plasmacellen er aanwezig waren ineen bepaald orgaan. Foto's van plaques
staan in appendix IenII.
Schubtransplantatie
Een methode omde cellulaire immuniteit te testen is door schubben
te transplanteren en vervolgens te bekijken hoe lang hetduurt voordat
zo'n schub afgestotenis.
Bij de karper liggen de schubben als dakpannen over elkaar heen.
Het is mogelijk ombijeenviseen schub te verwijderen en daarvoor
een schub vaneenandere visinde plaats te zetten. In figuur 12
staat aangegeven hoewe datongeveer gedaan hebben.
CONTROLE
VREEMDE SCHUBBEN
Fig. 12:Schema voor schubtransplantatie.
Fig. 13:Schub vaneenvis.Pigmentcellen zijn aangegeven meteen pijl
16
Een getransplanteerde schub wordt herkend als 1ichaamsvreemd en het
immuunsysteen neemt daarom maatregelen: de pigmentcellen, die in een
normale schub rond zijn (fig. 1 3 ) ,vertakken zich. Wat later krijgt de
getransplanteerde schub een meikwit-achtige schijn over zich. Dit
wordt veroorzaakt door de lymfocyten die op de vreemde schub afkomen
en deze afbreken. Na een aantal dagen verdwijnen de lymfocyten weer
en blijft er alleen nog maar een doorschijnend stukje bot over. Op
dit moment beschouwen we de schub als afgesoten.
17
IMMUNOLOGIE VAN DE KARPER
In het voorafgaande is de karper al af en toe ter sprake gekomen in
verband met het immuunsysteem. In appendix I van dit proefschrift
staan de resultaten vermeld van onderzoek verricht aan een tropisch
visje Barbus aonchonius
(prachtbarbeel in het nederlands). Aandacht
werd besteed aan de ontwikkeling van het humorale en cellulaire immuunsysteem. Humorale immuniteit werd onderzocht door bij dieren van verschillende leeftijden te kijken naar het aantal plaque vormende cellen dat in de milt ontstaat na inspuiten met SRBC. Cellulaire immuniteit werd onderzocht met schubtransplantaties. De resultaten laten
zien dat dieren van 3 maanden al kunnen reageren op SRBC, maar dat het
daarna nog wel 6 maanden duurt voordat dit vermogen maximaal is. De
cellulaire immuniteit ontwikkelt zich sneller: dieren van 6 maanden
oud stoten vreemde schubben even snel af als 9 maanden oude dieren.
In appendix II staat hoe we de plaque test bij de karper uitgevoerd
hebben. Het grootste probleem was het vinden van een geschikte complement bron. Het bleek dat karper-complement niet het meest geschikt
was. Veel beter was kopvoorn, sneep, barbeel en brasem. Omdat de kopvoorn, sneep en barbeel in Nederland erg zeldzaam of zelfs beschermd
zijn, hebben we besloten de optimale omstandigheden uit te werken voor
brasem-complement. Brasems zijn er gelukkig genoeg, er is zelfs een
meer naar genoemd.
In appendix III is gekeken naar de reactie van het immuunsysteem
van de karper op SRBC bij verschillende temperaturen. Eerst hebben we
gekeken in welke organen antilichaam producerende cellen voorkomen. In
de kopnier en nier zit ongeveer 90% van het totaal aantal "plaque
vormende cellen" (PFC). Slechts 5% zit in de milt en verder komen er
nog geringe aantallen PFC voor in het bloed, het hart en de thymus.
In figuur 10 staat weergegeven hoe het aantal PFC in de kopnier verloopt bij 24 C. Het hoogste aantal PFC wordt bereikt op dag 9 na inspuiten. Als we de karpers bij een lagere temperatuur houden, dan duurt
het langer voordat de piek van de PFC reactie bereikt wordt.
Vissen zijn koudbloedige dieren, ze hebben geen vaste lichaamstemperatuur zoals zoogdieren maar de temperatuur van hun lichaam wordt bepaald
door de omgevingstemperatuur. Bij lagere temperaturen verlopen alle
levensprocessen van een vis trager. Het is daarom niet verwonderlijk
dat ook de immuunreactie trager verloopt. Wat echter opviel was dat
de hoogte van de reactie (het maximale aantal antilichamen vormende
cellen) gelijk was bij alle temperaturen. Van uitstel komt in dit geval dus geen afstel. Een tweede interressant aspect van dit experiment
blijkt als je in een grafiek de temperatuur uitzet tegen de dag waarop
18
de piekreactie bereikt wordt (fig. 1 4 ) .We zien dat de grafiek geen
rechte lijn vormt maar dat er een "knik" in zit.
80
10.
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20 25 30
temperatuur("C)
Fig. 14: Verband tussen de temperatuur en de immunologische reactie.
Dat betekent dat er in de immunologische reactie (het hele proces van
herkenning van de vreemde stof tot aan de productie van anti1 i c h a m e n )
stappen zijn die verschillen in t e m p e r a t u u r g e v o e l i g h e i d .
In appendix IV staan experimenten beschreven die gedaan zijn om
iets te weten te komen over vorming van immunologisch geheugen. We
hebben al besproken dat als je een dier met een vreemde stof inspuit
er niet uitsluitend specifieke antilichamen gevormd worden maar ook
g e h e u g e n c e l l e n . De geheugencellen zorgen ervoor dat na een tweede injectie met dezelfde vreemde stof de reactie sneller en feller i s . De
mate van geheugenvorming kun je dus aflezen aan de hoogte van de herhaalde reactie.
Karpers zijn ingespoten met 3 verschillende hoeveelheden SRBC (1
m iljar d, 10 miljoen en 100.000 c e l l e n / d i e r ) en langs 2 verschillende
routes: in de spieren of in de b l o e d b a a n . De hoogste dosis SRBC geeft
de hoogste eerste reactie terwijl bij de laagste dosis nauwelijks een
reactie te meten valt. Als je echter na 1 maand de geheugenvorming
test door een tweede injectie toe te dienen dan blijkt dat de beste
reactie wordt gevonden in dieren die met de middelste dosis geimmuniseerd w o r d e n . Na 6 maanden en 1 jaar is de situatie nog extremer: de
hoogste reactie wordt bereikt in dieren geimmuniseerd met de laagste
d o s i s . Dat betekent dat de dosis die in een eerste reactie nauwelijks
iets doet, het beste is voor de geheugenvorming. Je kunt dit fenomeen
ook vertalen in termen van vaccineren (inenten) en bescherming tegen
een ziekte ( i m m u n i t e i t ) . Het idee heerst nog steeds dat een vaccin
19
een zo hoog mogelijke anti1ichaamproductie op moet wekken wil het bescherming bieden. Op grond van deze experimenten zou je het tegenovergestelde kunnen beweren: een vaccin biedt pas dan optimale bescherming
als het bij een eerste injectie geen antilichamen opwekt.
In de laatste twee hoofdstukken, appendix V en V I , wordt beschreven
wat voor effecten antibiotica op het immuunsysteem van de karper kunnen hebben.
Antibiotica zijn stoffen die de groei van bacteriën remmen. De bekendste zijn penicilline, streptomycine, chlooramphenicol, tetracycline. Antibiotica worden bij de mens gebruikt voor de bestrijding
van infectieziektes. In de veehouderij worden ze behalve voor de bestrijding van ziektes ook preventief (ter voorkoming van ziektes) gebruikt. Een bijkomende reden voor het gebruik is de groeibevorderende
werking op de dieren. Het werkingsmechanisme is niet voor alle antibiotica gelijk. Penicilline b.v. verstoort de vorming van een goede
celwand, waardoor de bacterie, als hij gaat groeien, uit elkaar klapt.
Dit kunnen we vergelijken met de gevolgen van een kapotte buitenband
van een fiets; de binnenband klapt pas als je hem oppompt. Tetracycline en chlooramphenicol remmen de eiwitfabriek van bacteriën.
Antibiotica remmen de groei van bacteriën. Alleen remming van de
groei is natuurlijk niet voldoende om een bacterie uit te schakelen,
immers zodra je stopt met de behandeling gaan de bacteriën weer rustig
verder met groei en deling. Daarom zal altijd het immuunsysteem nodig
zijn om de, door antibiotica in bedwang gehouden bacteriën, definitief
onschadelijk te maken.
Het antibioticum dat we gebruikt hebben in onze experimenten is Oxytetracycline ( o x y T C ) . De scheikundige formule staat in figuur 15.
Fig. 15: Oxytetracycline
Het antibioticum is op twee verschillende manieren toegediend aan de
karper.
20
1. door karpers te voeren met korrels waarin oxyTC meegemengd is.
Dit is ook de manier waarop antibiotica in de visteelt worden toegediend.
2. door karpers om de 3 dagen met een oxyTC-oplossing in te spuiten.
Bij de behandelde karpers hebben we gekeken of het immuunsysteem nog
goed werkte. Het bleek dat het bij k a r p e r s , die met oxyTC waren ingespoten, veel langer duurde voordat getransplanteerde schubben werden
afgestoten dan bij controle-dieren. Er waren zelfs schubben die helemaal niet meer werden afgestoten. Injecties met oyxTC remmen dus in
sterke mate de cellulaire immuunreacties. De humorale immuniteit wordt
sterk geremd door zowel injecties als voeren van oxyTC:
test
aantal rozet vormende
cellen in de milt
controle
oxyTC voer
oxyTC injectie
2,5
16
aantal anti1ichaam
producerende cellen:
- in de mi11
34
- in de kopnier
86
- in de middennier
55
2
15
15
Zowel het aantal rozet vormende cellen als het aantal antilichaam
producerende cellen is sterk verminderd tijdens een humorale respons
in met antibiotica behandelde dieren.
In latere experimenten hebben we dit effect op de humorale immuniteit verder onderzocht. Daarbij bleek dat vooral de eerste reactie
gevoelig voor antibiotica w a s , herhaalde reacties werden niet beïnvloed.
Uit deze experimenten kun je concluderen dat er erg voorzichtig
met antibiotica moet worden omgesprongen. Immers, wanneer je een ziek
dier, dat een bepaalde infectie heeft, met antibiotica gaat behandelen
dan gebeuren er twee dingen:
a. de bacterie wordt door het antibioticum in zijn groei geremd
b. het immuunsysteem van het dier wordt onderdrukt.
Als het dier tijdens de antibioticumkuur besmet wordt met een virus,
een schimmel of een bacterie die niet gevoelig is voor het antibioticum dan kan het immuunsysteem deze micro-organismen niet meer onschadelijk maken. Tenslotte is het de vraag wie zich na beëindiging van de
antibioticum behandeling het snelst herstelt: de bacterie of het immuunsysteem.
21
THE IMMUNE SYSTEMOFCYPRINID FISH
CENTRALELANDBOUWCATALOGUS
OOOO00021192
printed by P U D O C , Wageningen
cover design and i l l u s t r a t i o n s : W.J.A. Valen
AAN MIJN OUDERS
VOOR RIKY EN JORIS
Promotor: dr. J.F. Jongkind, hoogleraar in de celbiologie
Co-referent: dr. W.B. van Muiswinkel, wetenschappelijk hoofdmedewerker aan de Landbouwhogeschool
Y\V^ Ö2J01
©i^
GER T .
(^
RIJKERS
THE IMMUNE SYSTEM OF CYPRINID FISH
Proefschri f t
t e r v e r k r i j g i n g van de g r a a d van
doctor in de landbouwwetenschappen,
op gezag vande rector magnificus,
dr. H.C.vander Plas,
hoogleraar in de organische scheikunde,
in hetopenbaar te verdedigen
op woensdag 1oktober1980
des namiddags te vier uurin de aula
van de Landbouwhogeschool te Wageningen.
/sv - //f7&
I-
o3
h n Ó^o i
CONTENTS
9
ABBREVIATIONS
ALPHABETICAL LISTOF FISHSPECIES
11
GLOSSARY
13
OPENING REMARKS
14
GENERAL INTRODUCTION
Chapter 1.Cells andorgans
Chapter 2.Non-lymphoiddefence
Chapter 3. Immunoglobulins
Chapter 4.Humoral immunity
Chapter 5.Cellular immunity
Chapter6. Lymphocyte heterogeneity
Chapter 7.Factors affecting the
15
19
27
39
47
53
61
immune response
67
Epilogue
71
INTRODUCTIONTOTHE PAPERS
73
GENERAL DISCUSSION
77
SUMMARY
79
SAMENVATTING
81
DANKWOORD
82
CURRICULUMVIT.AE
83
REFERENCES
103
APPENDICES
•J ï r ? £ t i \
SEP. 1980
WH d20\.
c?Q
STELLINGEN
Denierbijlagerevertebraten isvergelijkbaarmethetbeenmergvan
zoogdieren.
Turpen,J.B. (1980)in:Development
andDifferentiationofVertebrate
Lymphocytes (J.D.Horton, Ed.).
Elsevier/NorthHolland
BiomedicalPress,pp.15-24.
ditproefschrift.
II
Antibiotica zijnimmuunsuppressief.
ditproefschrift
III
Eenpositievegemengdehaemagglutinatiereactievormtgeenbewijsvoor
demultispecificiteitvannatuurlijkeantilichamen.
Sigel,M.M.,Lee,J.C.,
McKinney,E.C.&Lopez,D.M.
(1978).Mar.Fish.Rev.,40,6-11.
IV
HetideevanGorczynskienSteeledatgemuteerdesomatische genenin
hetgenoomvangeslachtscellenkunnenworden ingebouwd isattractief
enverdientnaderonderzoek.
Gorczynski,R.M. SSteele,E.J.
(1980)1P.N.A.S.,77,2871-2875.
Hetbestuderenvanwetenschappelijke literatuurbuitenheteigenvakgebiedmetalsenigdoelomtoteenstellingtekomenisinstrijdmet
degeestvanhetpromotiereglement.
VI
Debeslissingoverhetaldanniettoekennenvanspreektijdopeen
internationaalcongreskanwordenvereenvoudigd doorbijdeaanmelding
nieteengeschrevenmaarmondelinge,doordeauteuropeencasettebandjeingesproken,samenvattingteverlangen.
VII
Indienfietserswerkelijk alsvolwaardigeweggebruikerswordenbeschouwd,
danbehorenvrijliggende fietspadentewordenvoorzienvanverlichting
enwegmarkering.
VIII
Deaandachtdieeen"laatste stelling"indemediakrijgtbewijstde
overschattingvandemaatschappelijkebetrokkenheid vanacademici.
IX
DeexportsymbolenFrauAntjeenHansjeBrinkerzijnteprefererenboven
hetduovanAgt/vanderKlauwalszijnderepresentatiefvoorde
Nederlandsebevolking.
ProefschriftvanG.T.Rijkers
TheImmuneSystemofCyprinidFish
Wageningen,1oktober1980.
APPENDIX PUBLICATION I
103
The immunesystemofcyprinidfish.Thedevelopmentofcellularand
humoralresponsivenessintherosybarb (Barbus oonohonius) .
G.T.RijkersandW.B.vanMuiswinkel (1977)
In:Developmental Immunobiology (Ed.byJ.B.SolomonandJ.D.Horton)
Elsevier/North HollandBiomedicalPress,Amsterdam
p.233-240.
APPENDIX PUBLICATION II
The haemolytic plaque assay in carp (Cyprinus
113
oarpio)
G.T. Rijkers, E.M.H Frederix-Wolters and W.B. van Muiswinkel (1980)
J . Immunol. Methods, 33, 79-86.
APPENDIX PUBLICATION III
123
The immune system of cyprinid f i s h . Kinetics and temperature
dependence of antibody producing c e l l s in carp (Cyprinus oarpio)
G.T. Rijkers, E.M.H. Frederix-Wolters and W.B. van Muiswinkel
Immunology (in p r e s s ) .
APPENDIX PUBLICATION IV
137
The immune system of cyprinid f i s h . The effect of antigen dose and
route of administration on the development of immunological memory
in carp (Cyprinus oarpio)
G.T. Rijkers, E.M.H. Frederix-Wolters and W.B. van Muiswinkel
In: Phylogeny of Immunological Memory (Ed. by M.J. Manning)
Elsevier/North-Holland Biomedical Press, Amsterdam
p . 93-102.
APPENDIX PUBLICATIONV
Theimmunesystemofcyprinid fish.Theimmunosuppressive effectof
theantibioticOxytetracyclineincarp (Cyprinus oarpio L.)
G.T.Rijkers,A.G.Teunissen,R.vanOosteromandW.B.van
Muiswinkel (1980)Aquaculture,19,177-189.
149
APPENDIXPUBLICATIONVI
Theimmunesystemofcyprinidfish.Oxytetracyclineandthe
regulationofhumoralimmunity incarp [Cypvinus
aarpio)
G.T.Rijkers,R.vanOosteromandW.B.vanMuiswinkel
(submittedforpublication).
165
ABBREVIATIONS
ABC
AcBSA
BALT
B cell
BGG
BSA
C1-C9
CD
CG
antigen binding c e l l ( s )
acetylated bovine serum albumin
bronchus associated lymphoid t i s s u e
bursa (equivalent) derived lymphocyte
bovine gamma globulin
bovine serum albumin
components of the complement system
c i r c u l a r dichroism
chicken globulin
conA
CRP
complement concentration bringing about 50°i lysis
of a standard dose indicator erythrocytes
concanavalin A
C-reactive protein
CVF
DNP
dinitrophenol
CH
50
DTH
EDTA
ERM
Fab
Fe
FHM
FSP
GALT
HA
H chain
HGG
HL
HMW
HRBC
HSA
Ig
IHN
IPN
J chain
K
f
KLH
K
o
L chain
LMW
cobra venom factor
delayed type h y p e r s e n s i t i v i t y
ethylenediaminetetraacetic acid
e n t e r i c redmouth disease
antigen binding fragment of immunoglobulin
c r y s t a l l i z a b l e fragment of immunoglobulin
f a t head minnow
fructosan specific protein
gut associated lymphoid tissue
haemagglutinating
heavy chain
human gamma globulin
haemolysing
high molecular weight
horse red blood cells
human serum albumin
immunoglobulin
infectious haemopoietic necrosis
infectious pancreatic necrosis
joining chain
functional association constant
keyhole limpet hemocyanin
i n t r i n s i c association constant
l i g h t chain
low molecular weight
LPS
MI
lipopolysaccharide
migration i n h i b i t i o n
MLR
mixed leukocyte reaction
MST
MW
NIP
median survival time
molecular weight
3-iodo-4-hydroxy-5-nitrophenyl a c e t i c acid
NNP
3,5-dinitro-4-hydroxy-phenyl a c e t i c acid
OSA
oxyTC
Pen
PFC
PHA
O-antigen of Salmonella abortus
Oxytetracycline
penicillin
antibody forming cells/plaque forming c e l l s
phytohaemagglutinin
PPD
PVP
PWM
RBC
purified protein derivate of tuberculin
polyvinylpyrrolidone
pokeweed mitogen
red blood c e l l s
RE c e l l s
RFC
S?n
SAQ
reticulo-endothelial cells
r o s e t t e forming c e l l s
sedimentation coefficient (in Svedberg units)
sum of squared differences
slg
SRBC
surface immunoglobulin
sheep red blood c e l l s
T cell
TNP
UV
VHS
thymus derived lymphocyte
trinitrophenol
ultra violet
virus haemorrhagic septicemia
WC
white c e l l s
10
ALPHABETICAL LIST OF FISH SPECIES
commonname
scientificname
arrowana
Osteoglossum bicirrhosum
Myxine glutinosa
Barbus barbus
Atlantichagfish
barbel
bigmouthbuffalo
black-spotbarb
bluegill
latiobus
ciprinellus
Barbus filamentosus
Lepomis macroohirus
bluegoUrami
Trichogaster
bluestripedgrunt
Haemulon sciurus
bowfin
Amia calva
Abramis brama
Lampetra reissneri
bream
brooklamprey
browntrout
carp
channelcatfish
chinooksalmon
chub
cod
cohosalmon
trichoptevus
Salmo trutta
Cyprinus carpio
latalurus
punctatus
Oncorhynohus tshawytsha
leuciscus oephalus
Gadus gadus
Oncorhynohus kisitoh
cunner
Carassius carassius
Tautogolabrus adspersus
dab
Limanda limanda
dace
dogfish
Leuciscus
leuciscus
Scyliorhinus
caniculus
eel
Anguilla anguilla (Litman,Kreutzmann)
eel
Anguilla chrysypa (Nardi)
Anguilla vulgaris (vonHagen)
Pimephales promelas
Platichthys
flesus
Lepisosteus platyrhincus (McKinney,Clem)
Lepisosteus osseus (Acton)
Carassius auratus
Epinephelus
itaria
Rhinobatus productus
Lebistes
reticulatus
Heterodontus
francisai
Crusiancarp
eel
fatheadminnow
flounder
gar
gar
goldfish
grouper
guitarfish
guppy
hornedshark
icefish
Notothenia
Japaneseeel
Anguilla
rossii
japonioa
11
commonname
scientificname
killifish
Fundulus
heteroclitus
lat janus synagris
Negaprion
brevirostris
Triakis
semifasoiata
Haemulon album
Tilapia mossambioa
Chondrostoma nasus
Gingly'mostoma cirratum
Eptatretus
stouttii
Polyodon spathula
Perca
fluviatilis
Esox luoius
Pleuroneotes
platessa
Lepomis gibbosus
Barbus
nigrofasaiatus
Salmo gairdneri
Ambloplites
rupestris
Barbus oonohonius
Anoplopoma fimbria
Salmo salar
Petromyzon marinus
Raja naevus
Mustelus oanis
Lutjanus griseus
Onoorhynohus nerka
Dasiatis amerioana
Barbus
lateristriga
Dasiatis centrouva
Centrarchidae sp.
Cymatogaster aggregata
Morone amerioana
Pseudopleuroneotes amerioanus
lane snapper
lemon shark
leopardshark
margate
Mozambique mouthbrooder
nase
nurse shark
Pacific hagfish
paddlefish
perch
pike
plaice
pumpkin seed
purple-headed barb
rainbow trout
rock bass
rosy barb
sable fish
salmon
sea lamprey
skate
smooth dogfish
snapper
sockeyesalmon
southernray
spannerbarb
stingray
sunfish
surfperch
whiteperch
winterflounder
12
GLOSSARY
allograft
anamnestic
graft derived from one animal and t r a n s planted to a genetically different animal of the same species.
reaction
the manifestation of immunological memory
whereby a second or subsequent exposure to an antigen leads
to a greater or more rapid reaction than the f i r s t .
anaphy Iaxis
a major type of immediate hypersensitivity
dependent on the formation of antigen-antibody complexes. The
reaction i s accompanied by pathological symptoms i n tissues
and organs due to the release of pharmacologically active
agents.
autograft
a graft in which the donor and r e c i p i e n t
are the same individual.
delayed type hypersensitivity
a s t a t e of increased r e a c t i v i t y to an
antigen, depending on previous s e n s i t i z a t i o n , giving r i s e to
a specific inflammatory reaction in the area where the antigen
i s localized.
migration
mixed
inhibition
leukocyte
the i n h i b i t i o n of the movement of cultured
macrophages by a factor released by s e n s i t i z e d lymphocytes.
reaction
t h e transformation of leukocytes into
b l a s t c e l l s in mixed cultures of leukocytes from normal
allogeneic individuals
xenograft
a graft between individuals of different
species.
13
OPENING REMARKS
There are several reasons for studying the immune system of f i s h :
1) From a phylogenetic point of view fish are i n t e r e s t i n g because they are the f i r s t
group of animals in which an immune system characterized by the presence of
immunoglobulins occurs.
2) The immune system of poikilotherms - including fish - is dependent upon the
environmental temperature offering the unique p o s s i b i l i t y to manipulate the
immune response by a mere variation in temperature.
3) When studying the ontogeny of the immune response oviparous fish are p a r t i c u l a r
i n t e r e s t i n g because the early stages and free swimming larvae are readily
accessible to experimental work.
4) A b e t t e r understanding of the immune system of fish may aid i n prophylaxis and
therapy of fish diseases in fish c u l t u r e .
In the General Introduction relevant data about fish immunology w i l l be
discussed. Since i t is not the i n t e n t i o n of t h i s thesis to review the whole f i e l d of
comparative immunology, data are r e s t r i c t e d to fishes although a comparison with
invertebrate defence mechanisms and mammalian immune systems w i l l be made when
needed.
During the l a s t 5-10 years the i n t e r e s t in fish immunology has accumulated in a
vast number of publications. However the data are d i f f i c u l t to unify: only in the
introduction of t h i s t h e s i s 69 species are mentioned. During evolution the different
classes of fish have diverged long before mammals were present. For t h i s reason i t
i s d i f f i c u l t , i f not impossible, to draw definite conclusions when comparing data of
species belonging to different c l a s s e s . In t h i s respect i t i s worthwhile to mention
the observation of Heuzeroth, Resch, Richter & Ambrosius (1973) who studied the
degree of s i m i l a r i t y of immunoglobulins. The differences between classes of fishes
were as large as the differences between other vertebrate orders (e.g. amphibians
and marnais). Even within one class of fish data are d i f f i c u l t to compare due to a
variety of immunization schedules and antigens used. Moreover, species may differ
in t h e i r p r e f e r e n t i a l temperature. In the Epilogue an attempt w i l l be made to give
a general and more personal idea about the immune system of bony fish.
In most experiments described in this thesis carp (Cyprinns oarpio L. 1758) were
used. This animal was chosen because i t i s an excellent experimental animal for
biological s t u d i e s . In addition i t i s worthwhile to mention t h a t t h i s species,Tilapia
and salmonids form the base for large scale fish culture in the world.
14
GENERAL INTRODUCTION
CHAPTERI
CELLSANDORGANS
Cells
Atthelightmicroscopicalaswellaselectron-microscopical levellymphocytes,
plasmacells,mononuclearphagocytesandgranulocytesoffishcloselyresemble their
mammaliancounterparts.
Lymphocytesaresmallroundcellswithalargenucleusandasmallrimof
basophilic cytoplasm (Weinreb,1963;Ferguson,1976b;Mattison&Fänge, 1977;
Kreuzmann,1977;Davina,Rijkers,Rombout,Timmermans&vanMuiswinkel, 1980).
Plasmacellspossessinganeccentricnucleuswithaprominentnucleolusanda
cytoplasmpackedwithroughendoplasmic reticulumhavebeenobservedinrainbow
trout (Chiller,Hodgins,Chambers&Weiser,1969;Etlinger,Hodgins&Chiller, 1978).
Monocytesandmacrophageshavebeendescribedinfishbutaconfusingnomenclatureisusedbymanyauthors (seereviewofEllis, 1977a).Thesameholdstrue
forgranulocyte identification.Adescriptionofthemorphologyandadiscussionon
thenomenclatureofthese cellsispublishedbyBarber&Westermann (1975, 1978),
Ellis (1977a)andDavinaetal. (1980).
©Lymphoid cellsaswellasmononuclearphagocytesandgranulocytesofmammals
andfisharequite comparablewhenmorphological criteriaareused.
Lymphoid organs
Themostprimitivevertebrates,thehagfish lackonmorphologicalgroundsa
definitethymusandspleen (Harboe,1963).Haematopoietic fociarepresentinthe
laminapropriaoftheentiregutlenght (Good,Finstad,Pollara&Gabrielsen,
1966).Thepronephros,which containsnephrostomes,istransformedpartly intolymphoidtissue (Gérard, 1954;Fänge, 1966).Inperipheralbloodofsealamprey,cells
wereobservedwhichresembledmammalian lymphocytes.Aprimitive spleenislocated
inaninvaginationofanteriorguttissue.Erythro-,thrombo-,granulo-andlymphopoiesis occursinthisorgan.Aprimitivebonemarrow,locatedinthefibrocartilaginousprevertebralarch,hasbeenobserved.Thebonemarrowcontains haematopoietic
tissue;proliferationoflymphoidcellswasobservedafterstimulationwithbovine
gammaglobulin (BGG).Adefinite thymusisabsentinlamprey.However,smallgroups
oflymphoidcellsintheepitheliumofthepharyngealgutterareconsideredasa
primitive thymus (Goodetal., 1966).
15
Fromtheevolutionarypointofview,theguitarfish (aprimitiveElasmobranch)
isthefirstrepresentativewhere athymus is found. Itisafully developed,
encapsulated lymphoidorganwithawellorganizedcortexandmedulla.Inthespleen
aredandwhitepulpareaisfound.Furthermore lymphoidtissuewas foundinthegut
andinthegonads.InthemoreadvancedElasmobranchs,theleopardsharkandnurse
shark,awelldeveloped thymusandspleenwerepresent.Abundantlymphoid tissue
wasalsofoundalongthegastrointestinaltractandinthegonads.Thekidneydid
notcontainlymphoidcells.Cells,identicaltomammalianplasmacells,were found
amongthelymphoidcellsinspleenandgonads (Goodet al., 1966).
TheChondrosteanpaddlefishhasawell-developedthymusorganizedintolobules.
Peripheralbloodcontains large,mediumandsmalllymphocytes.Thespleeniswell
developedandclearlydivided intoredandwhitepulp.Plasmacellswereobserved
inspleenandinhaematopoietictissueoverlyingtheheart.Theholosteanbowfin
hasathymuswith somedegreeoforganization intocortexandmedulla.Thespleen
isadiscreteorganwithtissuecomponentsdistributedintoredandwhitepulp
(Goodet al., 1966). Inholostean fishauniqueorganisfound,themeningeal
myeloidtissue,which isactively involvedinhaemopoiesis andthoughttobe
primitivebonemarrow (Scharrer,1944;McLeod,Sigel&Yunis, 1978).
Inteleost fish,nouniformityexists inthehistological appearanceof
lymphoidorgans.Forinstancethethymusofeel (vonHagen,1936)andMozambique
mouthbrooder (Sailendri&Muthukkaruppan, 1975)isclearlydividedintoacortex
andamedullawhereasthisdistinctioncanhardlybemade inthesalmonandrainbow
trout (Ellis,1977b;Grace&Manning, 1980).A spleenwith redandwhitepulpis
foundinMozambiquemouthbrooder (Sailendri &Muthukkaruppan, 1975),perch (Pontius
&Ambrosius,1972)andpike (M.G.Vos,pers. comm.). Inplaice thisdivisionisless
clear,theareaoccupiedbythewhitepulpbeingrelativelysmall (Ellis &deSousa,
1974),whereasincarp (Secombes&Manning,1980)andrainbowtrout,spleenismainly
redpulp (Anderson,1974;Grace&Manning, 1980).Thekidneyisanimportant lymphoid
organinteleosts.Theexcretion functionofpronephros iscompletely lostinadult
fishandtheorganshowsmainlyhaemopoieticandlymphoidcells (Ellis&deSousa
1974). Somesinuses inpronephrosaresurroundedbyendocrine cells (Secombes&
Manning, 1980). Lymphoidtissueofmesonephrosissituated inbetweennephrictubules. InMozambiquemouthbroodernoorganized lymphoidtissuewasobservedin
mesonephros (Sailendri&Muthukkaruppan, 1975). Furthermore individualorsmall
groupsoflymphoidcellsaresituated inbetweenintestinalepithelialcellsas
wellasinthelaminapropria (Bullock,1963;Pontius & Ambrosius,1972;Krementz
& Chapman,1975;Reiffel&Travill,1977;Zapata,1979a;Davinaetal., 1980).Teleost fish lackbonemarrow,bursaofFabricius andlymphnodes.
# I n cartilaginousandbony fishadiscretethymusandspleenarefound. Inbony
fishthekidneycontains lymphoidcells.Withtheexceptionofsealampreyand
Holosteans,fishlackbonemarrow.The cellularorganizationofthymusand
spleenisstronglyspeciesdependent.
16
function
of lymphoid organs
Afterinjectionofcarpwithasolubleantigen (humangammaglobulin (HGG)),
antigentrappingoccurredinspleen,pronephrosandtoalesserdegreeinmesonephros.
Usingacellularantigen (Aeromonas salmonieida),
mesonephrosplayedamajorpart
inantigentrapping.Thymusandliverwerenotinvolvedinthisproces (Secombes&
Manning, 1980).Antigenbindingcells (orrosetteformingcells)weredetectedin
spleenandpronephrosofrainbowtroutafterimmunizationwithsheepredbloodcells
(SRBC) (Chiller,Hodgins,Chambers&Weiser,1969).Unfortunatelymesonephroswas
not includedinthisstudy.Electronmicroscopyrevealedthatlymphocytes,blastlikecells,macrophagesandcellsresemblingeosinophilsboundtheantigen.Antigen
bindingcellshavealsobeenobservedinpronephrosandspleenofgoldfish,but
only lownumberswere foundinthymus (Ruben,Warr,Decker&Marchalonis, 1977;
Warr,DeLuca,Decker,Marchalonis&Ruben,1977). Antibodyproducingcellshave
beendemonstratedinspleenandpronephrosofbluegill (Smith,Potter&Merchant,
1967), rainbowtrout (Chiller,Hodgins&Weiser,1969;Anderson, 1978),perch,
(Pontius&Ambrosius, 1972)andMozambiquemouthbrooder (Sailendri&Muthukkaruppan,
1975). Furthermore antibodyformingcellsweredetectedinthemesonephrosof
rainbowtroutandgoldfish (Anderson,1978;Neale&Chavin,1971).Organcultures
ofsnapperandgrouperrevealedthatantibodysynthesisoccurrednotonlyinspleen
andpronephrosbutalsointhethymus (Ortiz-Muniz&Sigel, 1968,1971).InMozambiquemouthbrooderantibody formingcellswerepresentinthethymus (Sailendri,
1973).Inotherteleost speciesstudied,thymuscontainednoorvery lownumbers
ofantibody formingcells.Inliverofrainbowtroutandseveralshark species
antibody formingcellsweredetected (Chiller,Hodgins&Weiser,1969;Gitlin,
Perricelli&Gitlin, 1973).
Ontogenetic studiesinsalmon (Ellis,1977b)andcarp (Grace&Manning, 1980),
suggestedthatthespleenisnotvitalforimmunologicalmaturity since lymphocytes
ofthymusandkidneycarrysurfaceimmunoglobulinanddisplaymixed leucocytereactionsatatimewhenspleeninonlypresentinarudimentary form. Splenectomy
inkillifishdidnotaffectallograft rejection (Goss,1961).Insnapper,splenectomy
hadnoeffectontheantibodyresponseagainstbovineserumalbumin (BSA)(Ferren,
1967).Ontheotherhand,thespleenofbluegouramiissupposedtobeamajor
lymphoidorgan (Yu,Sarot,Filazzola&Perlmutter, 1970).
Adult thymectomyinMozambiquemoutbrooderandsalmonhadnoeffectuponallograftrejection (Sailendri,1973;Botham,Grace&Manning, 1980).Thymectomyin
4monthsoldMozambiquemouthbrooderprolongedsurvivalofallograftsandthymectomy
in2monthsoldanimalstotallysuppressedtheanti-SRBCresponse (Sailendri, 1973).
Thepronephroshasbeenregardedasaphylogeneticanalogueofbonemarrow (Zapata,
1979b)and/orlymphnode (Ellis, 1977a).
# T h e thymusofbony fishmaybelookeduponasaprimarylymphoid organ important
forthecontinuousproductionoflymphoidcells.Kidney (pronephrosandmesonephros)canbeconsideredasastemcellcompartment,aprimarylymphoidorgan
17
andaperipheral lymphoidorgan. Itseems that thespleen doesnotplay an
important role intheimmune response ofmostbony fish.
18
CHAPTER2
NON-LYMPHOIDDEFENCEMECHANISMS
Non-lymphoid cellular
defence
Phagocytosisofforeignmaterialboth servesasadefencemechanisminitself
andasaninitial stepintheonsetofthespecific immuneresponse.Inmammals,
mononuclearphagocytesaswellascellsbelongingtothegranulocyteseriesposses
phagocyticcapacity.
Intheholosteangarstudies in vivo andin vitro showed thatmonocytesand
macrophageswerecapabletophagocytizebacteria,yeastandSRBC.These cellsdestroyedtheengulfedmicro-organisms,asrevealedbyelectronmicroscopy. Granulocytes
appearedtobeinert in vivo andin vitro uptakeofparticles (McKinney,Smith,
Haines&Sigel, 1977).Inplaice,monocytesandthrombocytesweretheonlyphagocytic
celltypesinperipheralblood (Ellis,1976;Ferguson,1976b). Macrophagesfoundin
pronephros,mesonephros,spleen,thymus,heart,mesentaryandperitonealfluid,
consistedofthreetypes:1)freeroundedcellsresemblingmonocytes2)reticuloendothelial (RE)cells liningblood sinusesand3)melano-macrophages.Afterintraperitoneal injectionofcarbonparticles,phagocytosiswasperformedpredominantlyby
theellipsoidsofthespleenandthenetworkofREcellsthroughouthaemapoietic
tissueofpronephrosandmesonephros (Ellis,Munroe&Roberts,1976). Macrophages
liningtheatrialendocardiumwerealsoinvolvedinphagocytosis (Ferguson, 1975).
Followingphagocytosis somemacrophagesinpro-andmesonephrosandspleenformed
aggregateswithmelano-macrophages (Ellisetal., 1976,M.G.Vos,pers. comm.).
Amazingly,granulocyteswerenotphagocyticinplaice (Ellis,1976).
Incontrasttothesituationingarandplaice,neutrophilicgranulocytesin
rainbow troutandgoldfishhavephagocyticproperties (Finn&Nielsen,1971a,
1971b;Watson,Shechmeister&Jackson,1963;Weinreb&Weinreb,1969). Eosinophilic
granulocytesofgoldfishphagocytizebacteria (Watsonetal., 1963;Weinreb&Weinreb,
1969). Similarobservationshavebeenmadeinthecunner (Mackmull&Michels, 1932),
carp (Pliszka,1939)andguppy (Jakowska&Nigrelli,1953).
Thrombocyteshavebeendescribedtobephagocytic (Yokoyama,1960;Fänge, 1968;
Ferguson,1976b). Itremainstobedemonstratedforthesecellsiftheuptakeof
foreignmaterialisfollowedbyintracellulardigestion.
Thereareanumberofpublicationsdealingwithnaturalorexperimentalinfectionsinfish.Anextensivediscussionofthesereportsfallsbeyondthescopeof
thisthesis.Inthiscontextitisonlyworthwhiletomentionthatphagocyticcells
are involvedininflammatoryreactions evokedbyviruses (Finn,1970),bacteria
Post, 1963)andparasitic infections (Joy&Jones,1973).
Insomestudiestheclearanceofcarbonorantigenicmaterialfrombloodwas
usedasanindicationforthephagocytizing capacityoftheanimal.Followingintravenousorintramuscular injectionofT 2 bacteriophage intolemonshark,phageparticleswerecompletelyeliminatedfromthecirculationbyday4-5(Sigel,Acton,
19
Evans,Russell,Wells,Painter&Lucas, 1968). Inbrowntroutandcarp,keptatoptimal temperaturesMS2bacteriophageiscleared fromthebloodstreamwithin7days.In
theicefish,keptat2°Cthisprocess takes42-56days (O'Neill, 1980).Theeffectof
temperatureonnonlymphoid defencewillbediscussedinChapter7.
• Phagocytosisinfishisaccomplishedbymononuclear phagocytes.Insome species
granulocytesarealso involvedinthisprocess.
Non-lymphoid humoral defence
Inhighervertebratesanumberofcomponentshavebeendescribedwithapotent
anti-bacterial and/oranti-viral activitywhichfunctioninanon-specificway.
Although somecomponentsacttotally independentoftheimmune systemothersare
activatedbyantibody (e.g.complement).
Thefollowing componentspresentinhighervertebrates havebeendescribedfor
fish.Theirbiological significanceandtheirrelationtothespecificdefence system,especiallyunder circumstancesweretheimmune systemfunctionspoorly(low
temperatures),arelargelyunknownatpresent.
a) complement
Complementisagroupofserumcomponents involvedinbothspecificandnonspecificdefence.Inmammalsthecomplement systemconsistsofaseriesofatleast
18proteins (including C1-C9)whichcanbeactivated intwoways.Intheclassical
pathwaycomplementisactivatedbycontactwiththealteredFcpartofanantibody
molecule afterbindingwithanantigen.Inthealternativepathway activationis
accomplishedbycontactwithbacterial cellwallpolysaccharides. Complement activity
hasbeendemonstrated inserafromallvertebrate classes (Gewürz,Eugster,Muschel,
Finstad&Good,1965;Gewürz,Finstad,Muschel&Good,1966;Legier,Evans&Dupree,
1966; Legier,Evans&Dupree,1967;Gigli&Austin,1971;Ross&Jensen, 1973). Fish
serainwhich complement activityhasbeendemonstratedareshowninTABLE1.
Basicpropertiesofmammaliancomplement (thermolability,requirementofCa ,
Mg )aresharedbyfishcomplement.However,thetemperature rangeoverwhich complement remains activeisfargreaterforfishes:at4Cperchandcarp complement
retainsitshaemolytic activity (Pontius&Ambrosius, 1972;ownobservations).
Forheat-inactivationoffishcomplement lowertemperaturesarerequired thanto
inactivatemammalian complement (TABLE 1).
Asinotherpoikilothermscomplementisinmost casesnotexchangable
betweenunrelated fish species.SRBC,sensitizedbyrainbowtrout antibodies,were
haemolyzedonlyincombinationwith isologous serum (rainbouwtrout)orwith serum
fromclosely related speciesascohosalmon,sockeye salmonandchinook salmon
(Chiller,Hodgins&Weiser, 1966).ForSRBC,sensitizedwithbluegillantibodies,
blue gill,pumpkin seedandwhiteperchserumareeffective complement sources
(Smith,Potter&Merchant, 1967). Rosybarbandrainbow trout serumaresuitable
complement sourcesforSRBCsensitizedwithrosybarb antibodies (Rijkers&
20
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21
VanMuiswinkel,1977). InthehaemolyticplaqueassayusingSRBCand immunecarp
cells,seraofcarp,bream,barbel,chub andnasewere capableofbringingabout
haemolysis (Rijkers,Frederix-WoIters&VanMuiswinkel,1980a). Itappearsthat
isologousserumorserumfromcloselyrelated speciesiseffectiveascomplement
source inthehaemolyticplaqueassay.However,intheplaqueassayofrosybarb,
serumfrompurple-headedbarb,black-spotbarbandspannerbarbdonotdisplaycomplementactivity (Rijkers&VanMuiswinkel,1977). Intheplaqueassayofcarp,serum
ofgoldfishisineffective (Rijkersetal.,1980a). Itisthereforeunlikelythat
functional interchangabilityofcomplementcanbeusedasataxonomicalcriterium.
Fishcomplement ispresentnotonly inserum. Inrainbowtroutaheatlabile
anti-Vibrio
activitywasobserved inmucus,presumablycomplement+antibody (Harrell,
Etlinger &Hodgins, 1976).
Onlyfewdataexistonisolatedcomponentsoffishcomplement.Ross&Jensen
(1973b)haveisolatedandpurified thefirstcomponent (C1n)ofthecomplementsystem
ofnurse shark.C1nwas incompatiblewithrabbit immunoglobulinbutformedanintermediatecomplexwithsheeperythrocytessensitizedwithnursesharkantibody.These
intermediatescouldbespecifically lysedwithguineapigserumdevoidof C1.Onthe
otherhand,wholeguineapigcomplementdidnotreactwithSRBCsensitizedwithshark
antibody. Itwasconcluded thatthefirstcomponentofsharkcomplementcaninitiate
thecascadereactionofthemammaliancomplement systemprovidedthat itisallowed
toreactwithanimmunecomplexcontainingacompatibleantibody (Ross&Jensen,
1973a). InadditionGigli&Austen (1971)havedemonstrated thepresenceofC9in
nursesharkserumwhichreactedwiththecellular intermediateEAC 14235678prepared
withguineapigserum.
Cobravenomfactor (CVF)depletestheterminalcomplementcomponentsbyactivatingthecomplement sequenceatC3.ThecomplementactivatingeffectofCVFrequires
thecooperationofthealternativepathway system.Thisalternativecomplementactivitywaspresentinhagfishserumand inhemolymphofhorseshoecrab (Limulus
polyphemus)
andinsipunculidworm (Golfingea sp.) butabsentin lamprey,nurseshark,paddlefish
andcarpsera (Day,Gewürz,Johannsen,Finstad &Good, 1970). Classicalcomplement
activitywasnotobserved inhagfish,lamprey,horseshoe,crabandsipunculidworm.
Thesefindingssuggestthattheterminalcomponentsofthecomplementsystem,which
canbeactivatedwithoutantibody,appeared earlyduringevolution.A complement
system,which requires antibody-mediated activationisonly foundinthevertebrates
were immunoglobulinhasbeendemonstrated.
b) Lysozyme
Lysozyme,anenzymwithbacteriolyticproperties,ispresent inserum,mucus
andphagocyticcellsofmanyfishspecies (Luk'yanenko,196S;Fletcher &Grant,1968;
Fletcher &White,1973b;Ourth,1980). Themolecularweight (15x 10)issimilarto
mammalianlysozyme.Thedifferentelectroporeticmobilityreflectsdifferencesin
aminoacidcomposition (Fletcher&White, 1973b).
22
Variationsinlysozyme activitybetween individualmemberswithinonespecies
areconsiderable.Ingeneral,serum lysozyme activityincarnivorous fishes likepike
andperchishigher thaninomnivorous species likecarp (Luk'yanenko, 1965). After
immunizationofcarpwith Aeromonas punctata
thehighest lysozomeactivity coincides
withtheserumantibodypeak (Vladimirov, 1972).
Using immunohistochemicaltechniques,lysozymeactivity couldbedemonstrated
inmonocytesandneutrophils (Murray&Fletcher, 1976). These celltypesprobably
contributetotheserum lysozyme activity sincethenumberofmonocytesandneutrophils
increases concommitantlywith serum lysozome levelsafter intravenous injectionof
latexbeads (Fletcher&White, 1973b).
[ a) Interferon
Interferonisasubstanceproduced duringviral infectionsinordertoincrease
cellresistancetodifferent typesofviruses.Theantiviral actionofinterferonis
species specific,i.e.mouse interferondoesnotpreventvirusreplication inhuman
cell lines.Infish,interferonproductionhasbeendemonstrated both in vivo and in
vitro.
Virus haemorrhagic septicemia (VHS)isadisease causedbyarhabdovirus
(Egtved virus). Inrainbowtroutthedisease occursmainlyatwater temperaturesof
6-12C anddisappears spontaneouslyattemperatures over 14-15C.Following injectionofrainbow troutkeptat15CwithEgtvedvirus,serumwascollected after3,9
and 14days.Theantiviral activityofthese seratested in vitro wasmaximalonday
3.Thiswasshownbyprotectionofrainbow trout gonad cells against challengewith
Egtvedvirus,butalsotoinfectioushaemopoieticnecrosis (IHN)virusorinfectious
pancreaticnecrosis (IPN)virus.Thisbroad antiviral activityisspecies specific
sincefatheadminnow (FHM)cellsarenotprotected aftertreatmentwiththis serum
againstthevirus challenge.Thearguments fortheinterferonnatureoftheserum
factorwere enforcedsinceitappearedtobenon-sedimentable,non-dialysable,heat
andlowpHstable,trypsin labileandRNase resistant (deKinkelin&Dorson, 1973).
Rainbow troutandbrown troutinjectedwith rhabdovirus strain 23.75synthesize interferonwithmaximumtitres reachedwithin2days.Theinterferonsynthesispreceedsthe
neutralizing activityduetospecific antibody (deKinkelin,Baudouy&LeBerre, 1977).
AttemptstoinfectFHMcellswithreovirus type2werenotsuccesful.FHMcells
exposedtothisvirusdeveloped resistanceandproducedanantiviral substance which
appearedtobeinterferon (Oie&Loh, 1969). GFA,avirus-like component,iscapable
of inducing interferon synthesisinacell lineofthebluestriped grunt (Beasly,
Sigel&Clem, 1966).
d) C-reaotive protein
C-reactive protein (CRP)isaproteinwhich appearsinmammalian serumduring
theacutephaseofinfectionwithmicro-organisms.CRPbindstophosphoryl choline
residueswhicharepresentincellivallglycopeptidesofvariousbacteria,fungiand
23
parasites.CRPcancause agglutination,precipitationandcomplement activation
(Siegel,Rent&Gewürz,1974;Pepys,Balz,Mussalam&Doenhoff, 1980).
A serumcomponentwithCRPpropertieshasbeendescribedforplaice, dab, cod,
flounderanddogfish (Baldo&Fletcher, 1973;Pepys,Dash,Fletcher,Richardson,Munn
& Feinstein, 1978).CRPprecipitated aqueous extracts frombacteria,fungiand
nematodes.Theprecipitation reactioncouldbeinhibitedbyphosphoryl choline.
Immunoelectrophoresis revealedaprecipitation lineinthecu-region,excludingthe
possibilityofanimmunoglobulinnaturebecause antibodyactivitywasrestrictedto
the ß-regionintheirexperiments (Baldo&Fletcher, 1973).AccordingtoPepysetal.
(1978)CRPofplaice,flounderanddogfish closelyresemblehumanCRPinmolecular
3
weight (135-160x10)andE.M.morphology (twopentameric discs).
Incontrasttomammals,CRPinplaiceisnotanacutephaseproteinbuta
normal serumconstituentwhichmayprovidetheanimalwithapermanent defence line
against invadingmicro-organisms.
e) Natural
hemagglutinins
Inrepresentatives fromallclassesoffish,naturalagglutininshavebeen
detected. Theiractivityisusually directed againstxenogeneicerythrocytes,more
precisely towards carbohydratemoietiesonsurfacemembranes.
Thisnatural agglutinins sharethefollowingpropertieswhich distinguish
themfrom immunoglobulins:
1) theyarecomposedofidentical subunits,sonodistinctionbetweenheavyand
lightchainscanbemade.
2) nointerchaindisulfidebridgesoccur,themoleculeislinkedbynoncovalent
bonds.
3) uponelectrophoresisnoheterogeneity inaminoacid compositionofthesubunits
isdisplayed.
Aproteinwithagglutinating activitytohumanerythrocyte "0"antigenshasbeen
demonstrated inlamprey serum (Litman,Frommel,Finstad,Howell,Pollara&Good,
1970b).Themoleculewhich consistedof4non-covalentlinkedsubunits (M.W.90.000)
dissociated spontaneously. Inaddition,thecirculardichroic (CD)spectraandelectrophoreticmobility differedmarkedly fromIg.
Innon-immune lamprey seruma48Sproteinwasobservedwith agglutinating
activitytohorseerythrocytes (Marchalonis&Edelman, 1968b).Themoleculeiscomposedoflowmolecularweightsubunits linkedbynoncovalentbonds.Ca requirement
andaminoacid compositionoftheproteinresembled natural invertebrate hemagglutinin
illustratingtheevolutionary positionofAgnathabetween invertebratesand
vertebrates.
Innormaleelserumanagglutininisfoundwhichreacts specificallywith human
O-erythrocytes (Springer&Desai,1970).Theagglutinin appearedtobeaproteinwith
amolecularweightof123.000 (7.2S ) ,butwithout significant amountsofcarbohydrate
characteristic forimmunoglobulin (Desai&Springer, 1972).Themolecule consistsof
24
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25
3 subunits,eachsubunitcomposedof4identicalpolypeptide chains,joinedbydisulfidebridges.CDspectraofthismolecule differsignificantly from thoseofhuman
7Simmunoglobulin (Jirgensons,Springer &Desai, 1970J.
Inrainbowtroutnaturalhemagglutinins againstrabbit,mouseandhumanerythrocyteswerepresent.Whereas induced antibodieswerealwayselutedwith the first high
molecularweightpeak fromaSephadexG-200column,naturalhemagglutininswere
eluted inthefirstand thirdproteinpeak. Induced antibodieswereprecipitable by
221Na ? S0,andwere ß-globulinsinImmunoelectrophoresis,naturalhemagglutininswere
nonprecipitable by 221Na^SO.andwere faster than0-globulins (Hodgins,Weiser &
Ridgway, 1967).Moreover,sucha lowmolecularweightagglutinin similar tothe
natural agglutininivasnot inducedafterexposure tovariousantigens (Hodgins,
Wendung,Braaten&Weiser, 1973).
Innurse shark serumaproteinwasfoundwhich specificallyprecipitates fructosans.Thisprotein (FSP)was antigenicallyunrelated toshark immunoglobulin
(Harisdangkul,Kabat,McDonough &Sigel, 1972a).
Themolecularweightwas 280,500 (10.6S)anditconsisted of4noncovalent linked
subunits.Nocarbohydratewasdetected (Harisdangkul,Kabat,McDonough &Sigel,
1972b). CD spectraofpurified FSPfromnurse sharkresembledmore closely tophytohemagglutininorconcanavalinA thantovertebrate immunoglobulin (Pflunm,WangS
Edelmann, 1971).
InTABLE 2somecharacteristics ofagglutinins fromplant,invertebrates and
vertebrateoriginaregiven.Natural occuringhemagglutinins ininvertebrates are
largemolecularweightproteinswithanelectrophoreticmobilitysimilartothatof
immunoglobulins (Marchalonis, 1977). Inthemajority ofinvertebrate species,hemagglutininspossessanopsonicactivity,indicating arole inthedefencesystem (Acton
&Weinheimer, 1974).Thebiological significance ofagglutinins ofplantoriginis
notclear.
# A numberofhumoral defence factorshavebeenobserved infish:complement,
lysozyme,interferonandC-reactive protein. Ingeneral theyresemble their
mammaliancounterparts infunctional andphysiochemicalcharacteristics.Furthermore,non-antibody hemagglutinins arepresent infish serumwhich resemble
invertebrate agglutinins.
26
CHAPTER3
IMMUNOGLOBULINS
AccordingtotheWorldHealthOrganization immunoglobulins (Ig's)arethose
proteinsofanimaloriginwithknownantibodyactivityandcertainproteins related
toantibodybychemical structure.Inmanfiveclassesofserum Ig'sarefound,as
illustratedinTABLE3.Allthese immunoglobulin classesareserologically related
because theysharethesame lightchains.Twoantigenically different types,of
lightchains,termed K or X,arefoundinallclasses.Theheavychainsdefinethe
immunoglobulinclasses.Heavy chains,whichdifferinavarietyofproperties, includingmolecularweight (M.W.)andaminoacid sequence,havebeengiventhedesignations:u, y ,a, 5andE .Themolecular formulaofIgM (L?u7)5 indicates that5
sub-units,eachcomposedof2lightand2heavychains,arelinked together.
TABLE 3
Characteristics ofimmunoglobulinclasses inman
Class
S„
IgM
18-20
IgG
6.7
IgA
7-11
IgD
6.2-6.8
IgE
8.2
20
3
Molecularweight (xlO)
Native
H-chain L-chain
Carbohydrate
content(%)
Formula
a 2 u 2 )5*
950
70
22.5
150
53
22.5
2
150-400
64
22.5
6-10
180
60
22.5
12.7
L
262
190
72.5
22.5
11.7
L
2E2
9-12
L2Y2
a2c*2)n
S„ n =sedimentationcoefficient;H-chain=heavy chain;L-chain =lightchain; L
canbeXor K; ncanbe 1,2orhigher.
DatafromMarchalonis (1977).
Twomajorpropertiesbywhichproteinscanberesolvedareelectric chargeand
molecularweight.Upon zoneelectrophoresisinstarchblocksatpH8.6mammalian
immunoglobulinsmigratetothecathodeandwereoriginally termed Y~gl°bulins.From
early studiesitwasconcluded thatfish lacked immunoglobulins becausey-globulins
werenotdetectedinserum (Engle,Woods,Paulsen&Pert,1958;Good&Papermaster,
1961;Clem&Sigel,1963;Post, 1966). This conclusionturnedouttobeincorrect
sincecarpandplaice immunoglobulinforinstancemigrateasß-globulins (Ambrosius,
1966;Baldo&Fletcher, 1973).Ontheotherhand,proteinswhichmigrateasy globulinsdonotnecessaryrepresent immunoglobulins:transferrinandC-reactive
proteinmigratealsoasy-globulins (seealsoChapter 2). Thereforereportsinwhich
thepresenceorabsenceofimmunoglobulinisbaseduponelectrophoresis aloneshould
be interpretedwithgreatcare.
27
Immunoglobulin of Agnatha
Onbaseofthefailureofdetectingcirculatingantibodyafterimmunization
withBSAandtheabsenceofserumglobulinuponImmunoelectrophoresis,Good&Papermaster (1961)concludedthatPacifichagfishlackanimmuneresponse.Whenusing
otherantigens (heat-killed Brucella
abortus,
typhoid-paratyphoidvaccine,hemo-
cyanin,bacteriophageT,)withorwithoutcompleteFreund'sadjuvant,noagglutinating,complement-fixing,neutralizingorantigenbindingantibodies couldbe
detected (Papermaster,Condie&Good,1962;Good&Papermaster,1964).Thecausefor
thisfailureinelicitingantibodyresponsesprobablyliesinpooranimalhusbandry
(lowwatertemperatures,starvation)especiallysincelaterstudieshaveshownthat
hagfisharecapableofmountingahumoral immuneresponse.Afterimmunizationof
hagfishwithkeyholelimpethemocyanin (KLH)theproductionofspecificantibodies
couldbedemonstratedbypassivehaemagglutinationandimmunodiffusioninagargels
(Thoenes&Hildemann,1969).Fractionationofimmuneserumshowedtheantibodiesto
bepresentinthemacroglobulinfraction (28S).A smallercomponent (7S)whichlacked
antibodyactivitywasantigenically identicalwiththe28Scomponent.The28ScomponentwasthoughttobeIgM (possiblyadimerof19Smolecules)whilethe7ScomponentrepresentedmonomericIgM (Thoenes&Hildemann, 1969).
NaturaloccurringagglutininsforSRBCwereheatlabilesincehagfishserum
heatedat56Cfor30minlostallactivity.Antibodies inducedafterrepeated immunizationwithSRBCwereheatstable (Linthicium&Hildemann,1970).
MorerecentlyRaison,Hull&Hildemann (1978a)succeeded inraisingspecific
antibodiesagainstgroupAstreptococcal carbohydrate inhagfish.Gelfiltrationand
sodiumdodecylsulfate/polyacrylamidegelelectrophoresisundernon-reducingconditions indicatedthattheintact immunoglobulinhadamolecularweightofapproximately 160,000.Theelectrophoreticmobilityofheavychainswasidenticalwiththat
ofmurine v chains.Thetertiarystructureofhagfish immunoglobulinwasverylabile
(completedissociation in0.005M 2-mercaptoethanol)suggestingtheabsenceof
disulfidebondingofthepolypeptidechains (Raison,Hull&Hildemann, 1978b).The
hexosecontentofhagfishimmunoglobulin is3.41 (W/W) (Hildemann,cited inLitman,
1977).
Uponrepeatedimmunizationthesealampreyiscapableofproducingneutralizing
antibodiestobacteriophage f2.Theantibodyactivityislocalized in6.6S and14S
fractionsofimmunelampreyserum.The6.6S and 14Sfractionswereantigenically
identical.Theyieldof14Smaterialwasinsufficientforfurthercharacterization.
The6.6Smoleculecontainedheavy (H)andlight (L)chainswithamolecularweightof
70,000and22,000respectively.LampreyHchainsresembledmurineychainsinmolecular
weightandelectrophoreticmobility.HandLchainswerenotcovalentlylinkedand
dissociated spontaneously (Marchalonis&Edelman,1968b).Thereforethemolecular
weightoftheintactimmunoglobulin (180,000)couldonlybeestimated (Marchalonis&
Cone, 1973).
28
Stimulation ofsea lampreywithhemocyaninorBSAdidnot evokeproductionof
circulating antibody,whilehighdoses Brucella induced anantibodyresponse (Pollara,
Finstad &Good, 1966). Theantibodies (9S)hadamolecular weightof 150,000-200,000
and showedupon Immunoelectrophoresis anaglobulinarc.Theproteinwith y mobility
lacked antibodyactivity (Finstad &Good, 1964;Pollara,Finstad,Good &Bridges,
1966).When lampreywas immunizedwithhuman"0"erythrocytes,antibody production
wasdemonstrated (Pollara,Litman,Finstad,Howell &Good, 1970). These antibodies
were associatedwith the9Sserum fraction.Uponelectrophoresis antibody activity
resided intheanodalmigrating fractionwith achargedensitycharacteristic foran
a globulinrather thanayglobulin (Good,Finstad &Litman, 1972).A molecular
weightof320,000wasobtained for thepurified immunoglobulin (Litman,Howell,
Finstad,Good &Pollara, 1969). Thenativemolecule spontaneously dissociates into
antigenically identical subunits ofapproximately 150,000and 75,000M.W.After
reductionandalkylationno counterparts ofheavy andlightchainswereobtained but
subunitswithaM.W. of90,000. Itwas suggested thatthenatural formof lamprey
immunoglobulin isrepresentedbyatetramercomprised of4equivalent subunits held
togetherbynoncovalentbonds (Litman,Frommel,Finstad,Howell,Pollara &Good,
1970b).
• Studies onimmunoglobulins inAgnatharesultinconflicting conclusionsregardingstructural conformations.According toMarchalonis lamprey immunoglobulin
iscomprised ofheavy and light chains (Marchalonis &Edelman, 1968b)while
Litmansuggests thatthemolecule contains identical subunits (Litmanet al.,
1970b). Thisdisagreement canbeexplained byconsidering thenatureof the
antigensused for immunization.The immunoglobulinsofMarchalonis &Edelman
(1968b)were inducedbyviralproteinantigenswhile Litmanetal. (1970b)may
havecharacterized an inducible classofantibody tocell surface carbohydrate.
Naturalagglutinins toxenogeneic erythrocytes havebeendescribed for lower
vertebrates (seealsoChapter 2).Thenatural occurring agglutinin forSRBCinhagfishwasalreadymentioned (Linthicium&Hildemann, 1970). Serafromthearctic
lamprey contained natural agglutininswhichreactedwithvariousvertebrateerythrocytes.Antibodies induced afterrepeated immunizationwithSRBCwereheat stableanddisplayed ahighdegreeofspecificity forSRBC,whereasnatural agglutinins lost
theiractivityafterheating serumat46°Cfor30min. (Fujii,Nakagawa &Murakawa,
1979b). Normal sealamprey serumhasbeenshowntocontainnatural agglutinating
activity forSRBC (Gewürz,Finstad,Muschel &Good, 1966). Thisactivitywasblocked
byEDTAandwasnot stimulated by immunization.Marchalonis &Edelman (1968b)described
a natural agglutinating activity towardshorseerythrocytes insealampreywhich
resided inthe48Sserumfraction.
©Natural agglutinins canbefunctional distinguished frominducibleantibodiesby
theirheat stability.Aphysiochemicalcomparison reveals thatnatural agglutininsresemblemore invertebrate agglutinins thanvertebrate immunoglobulins.
29
(TABLE 2).ThelampreyantibodydescribedbyLitmanetal. (1970b)mayrepresent
aninducibleformofnaturalagglutinins.Asfarastheirimmunecapacityis
concernedAgnathamightrepresentanintermediatebetweeninvertebratesand
vertebrates,possessingdefencesystemcharacteristicsofbothgroups.
Immunoglobulin of
Chondvichthyes
After immunizationofleopard sharkwithbacteriophageT 2 antibodyactivityis
presentinthe17Smacroglobulinserumfraction.Lateantiseracontained antibody
activityinthe7Sfractionalso (Suran,Tarail&Papermaster, 1967).Theimmunoglobulinfractionscouldbepurified fromshark serumbygelfiltrationandion
exchangechromatography (Clem&Small,1967).The7Sand17Simmunoglobulins appeared
tobeidenticalinimmunodiffusionusingrabbitantisharkserum (Suranetal., 1967).
Afterextensive reductionandalkylationfollowedbygelfiltrationin5MguanidineHC1both immunoglobulinscouldbeseparatedinHandL-chains.Hchainsof7Sand19S
wereindistinguishablebyfingerprintsoftrypticdigests,disc electrophoretic
patterns,antigenicpropertiesandmolecularweight (77,000).TheMWofsharkH
chainsiscomparabletomammalianuchain.BysimilarcriteriaLchainswereidenticaltoeachotherbutdifferent fromHchains.HandLchains accountforabout75
and 25%respectivelyofthemassoftheimmunoglobulinmolecule.BasedupontheM.W.
ofthenative immunoglobulinmolecule,thepolypeptidechainsandthemassratiosof
thesechainsshark7Simmunoglobuliniscomposedof2Hand2Lchains.The17Smolecule
consistsofapentamerofdisulfide-linked subunits,eachsubunitbeingcomposedof
211and2Lchains (Clem&Leslie,1966). PropertiesofotherChondrichthyesarelisted
inTABLE4.Allthesespeciespossesspentamericaswellasmonomericimmunoglobulin.
Thepentamericnatureofthe17Simmunoglobulinhasbeenconfirmedbyelectronmicroscopy.Dogfish17Simmunoglobulinshowedafivebranchedcyclicsymetryanddimensions
similartomammalianIgM (Feinstein&Munn, 1969).
Interrelationshipbetween7Sand17Smolecules
-Newbornnursesharkhaveessentialnoserum immunoglobulin.Duringfurtherdevelopmentthesynthesisof17SIgpreceeds7SIgsynthesis (Fidler,Clem&small, 1969).
-The18Santibodyto Salmonella
typhirrwœiwn 0antigenis150-200timesmoreefficient
inagglutinationreactionsthan7Santibody (Schulkind,Robins&Clem, 1969).
-Antibodyactivityduringthefirst 10-12monthsafter immunizationwith
Salmonella
typhimvœium wasrestrictedto17Simmunoglobulin.Notuntil1yearafterimmunizationantibodyactivitywasobservedin7Simmunoglobulin (Clem&Leslie, 1966).
-Administrationofradiolabeled7Sand17Simmunoglobulinhasdemonstrated that7S
IgMisneitheraprecursernoradegradationproductof17SIgM.
Thehalflifeoflemonsharkimmunoglobulinwas4-5days.Furthermoreitwasdemonstratedthat 17SIgMispredominantly intravascularwhereas7SIgMisdistributed
bothextravascularlyandintravascularly (Clem,Klapper&Small,1969;Small,
Klapper&Clem, 1970).
30
OnbaseofthesedataClem&Leslie (1969)concludedthatthetwomolecular formsof
shark IgMfunctionallymimicmammalian IgMandIgGmolecules.
An interesting exceptiontothepentamericIgMfoundinmost Chondrichthyes
istheimmunoglobulinmoleculeofstingray.HandLchainsaresimilartomammalianu
and lightchainsinmolecularweightandamino acidcompositions,butthemolecular
TABLE4 Physicochemicalpropertiesofcartilaginousandbony fish immunoglobulin
d a a s andspecies
Molecular weight (x!0 )
Native
H-chain
L-chain
Carbohydrate
Formula
content 7.
CHONDRICHTHYES
horned shark
19
7
900
180
69
69
23
23
9.3
11.3
a2u2)5
cam's)
17
7
982
198
72
73
20
21
8.7
7.6
«•2U2>5
breuiroatria)
19
7
850
160
71
71
23
23
3.7
3.5
(HetepodontitB
smooth dogfish
fvaneisci)
(Muetelua
(Negaprion
lenra sherk
leopard shark
scmfaaciata)
17
7
77
77
(Gir.nlino3iorr,a cirvat:*!",)
19
7
70
70
22
22
72
22
70
23
(Triakia
nurse sha?:k
2P2
a2p2)5
L
2U2
1
1
2,3
2,3
4
4
5,6
5,6
3.5
3.6
s t i n g ray
{Daeictie
rentrcura)
II
southern ray
(Dasiatia
amencona)
17
(Polyeder.
upat''!uLi)
14
662
58
21
6.8
(Polyodcn
spathula)
59
870
180
75
75
24
24
9.0
8.1
70
24
10.7
52
24
9.1
360
L
9.3
V2>5
L W
2 2
7
7
a2u2)2
8
a
9
2u2>5
CH0NDR0STEAN5
padaiefish
a2*2>4
a2u2>4
L2P2
10
II
II
HOLÛ5ÎEAKS
bovfin
(Amia calva)
14
600-900
6
gar
(Lepiaoateua
platyrkimua)
(LepiBOBteuB
oeaeus)
14
650
70
22
14
610
58
23
(L
(L
4.9
2 y4
L2P2
2"2 ) 4
a2p2)2
12,13
12,13
14,15
16,17,18
TELEOSTS
carp
goldfish
catfish
grouper
rainbow trout
brown trout
coho salmon
margate
plaice
pike
(CyprinuB
earrio)
15
720
71
24
(CyprinuB
oai^pio)
14
608
77
24
anratuß)
16
76
23
punctatuB)
14
610
70
ztaria)
16
6
900
120
nairdneri)
16
(Caraaaiue
(IctaluruB
(PpinspheluB
(Salmo
<L2*2>4
6.8
a2x2>4
23
5.5
a2u2)4
70
40
22
22
3.5
I.I
70
22
19
670
kiautah)
17
756
75
26
(Haermlon album)
15
7
900
180
70
70
22
22
70
22
8.8
60
23
9.2
(Pleuronectee
(Eeox
plateaBa)
12
luaiua)
15
638-651
16,24
25,26
25,26
27
28
17
(OnoorhynohuB
<^2>4
* ab'2
a2P2)4
trutta)
(Salmo
19,20
21,22,23
L
< 2"2>*
29
30
30
31
<V2>4
32,33
S__ •sedimentation coefficient, H-chain-heavy chain; L-chain•light chain. 1)Frommel, Litman, Finstad &Good (1971);
27Marchalonis 4Edelman (1965); 3)MarchalonisSEdelman (1966); 4)Clem«Small (1967); 5)Suran, Tarail t Papermaster
(1967);6)Litman, Frommel, Chartrand, Finstad «Good (197le); 7)Clem, DeBoutaud 5Sigel (1967); 8)Marchalonis «Schonfeld (1970);9)Johnston, Acton,Weinheimer, Niedermeier, Evans, Shelton S Bennett (1971); 10)Acton, Heinheimer, Dupree,
Russell,Wolcott, Evans, Schrohenloher S Bennett (1971d); II)Pollara, Suran, Finstad I Good (1968); 12)Litman, Frommel,
Finstad SGood ( I 9 7 U ) ; 13)Litman, Frommel, Finstad S Good (1971b); 14)Bradshav, Clem SSigel (1969); 15) Bradshaw,
ClemaSigel (1971); 16)Acton, Weinheimer, Hall,Niedermeier, Shelton 5Bennett (1971b); 17)Acton, Evans, Weinheimer,
Dupree1Bennett (1970); 18)Acton,Weinheimer, Dupree, Evans «Bennett (1971c); 19) Marchalonis (1971); 20)Shelton«
Smith (1970); 21)Ambrosius, Richter Ä König (1967); 22)Richter, Frenzel, Hädge, Kopperschlager Ä Ambrosius (1973);
23)Andreas,Richter, Hädge&Ambrosius (1975); 24)Hall,Evans,Dupree, Acton, Weinheimer &Bennett (1973); 25)Clem&
Small (lr,70); 26)Clem (1971); 27)Dorson (1972); 28)IngramSAlexander (1979); 29)Cisar SFryer (1974); 30)Clem S
Leslie (1969);3 D Fletcher t Grant (1969); 32)Clerx (1978); 33)Clerx, Castel,BolSGervig (1980).
31
«
cN
tn
s
+
c+
O
o
>,
• *
Ö
•H
o;
S
m
m
r-»
(T.
—"
Tl
C
.* «
o
-O
C ^-s
m u~t
£d
o in
o r-»
O ON
» to
c B
^i eu
eu B
ao
t-i H
m
33
^j
* aj
en ,c
3 O
tO I-H
>
- to
B J=
i-H
.0
a. oo
o o
•4
3
H
34
S
«H
,-)
highnegative charge.Using this isolationtechnique,Jchainanalogueshavebeen
demonstratedinnurseshark (Klapper&Clem, 1972;McCumber&Clem, 1976), leopard
shark (Klaus,Halpern,Koshland&Goodman,1971;Weinheimer,Mestecky&Acton, 1971)
andchannelcatfish (Weinheimeretal., 1971;Mestecky,Kulhavy,Schrohenloher,
Tomana&Wright,1975a,b ) .Thephysiochemical characteristicsoffishJchain
arecomparedwithhumanJchaininTABLE5.Jchainisanevolutionary conservative
protein sincerabbitantiseratohumanJchainalsoreactwithdogfishandleopard
sharkJchain (Koshland, 1975).
J chainsprobablyplayanimportantroleintheassemblyofIgMandIgAmonomers
intopolymers (Kownatzki,1973).Jchainshowever,arenotalwaysessentialforpolymerassemblyasillustratedbytheapparentabsenceofJchaininpolymericIgMof
gar (Mesteckyetal., 1975),paddlefish (Weinheimeretal., 1971),pike (Clerx, 1978)
andcarp (Zikan,1974).Moreover,mildlyreducedhighmolecularweight immunoglobulin
ofcarp,which lacks Jchain,canreassociate in vitvo intonativemolecules (Richter
&Ambrosius,1978).
%Inbony fishpentamericortetramericimmunoglobulinisfound.Physicochemical
characteristicsoffishimmunoglobulin closelyresemblemammalianIgM.
Specificity
and affinity
Goldfishanti-BSAantibodies showedthesamedegreeofspecificitytowardsthe
antigenasrabbitanti-BSAantibodies (Everhardt&Shefner, 1966).
Studiesonantigen-combining sitesrequirewelldefined antigenicdeterminants.
Dinitrophenol (DNP)isasuitablemoleculeforthispurpose.Inmammalstheaffinity
ofantibodies increasesduringtheimmuneresponse.ThisphenomenoniscalledmaturationandholdstrueforIgGbutforIgMthisisquestionable (discussedinFiebig,
Hörnig,Scherbaum&Ambrosius, 1979).
NursesharksimmunizedwithDNPsubstitutedKLHproducebothHWMandLMWantiDNPantibodies (Voss,Russell&Sigel, 1969). GiantgrouperrespondtoDNPbythe
productionofHWMandLMWantibodies (Clem&Small,1968,1970). Inboth experiments
antibodieswithahighaffinity (K = 1 0 - 5 x 1 0 M )andlowaffinity
4-1
°
(K = 1 0 M )wereproduced.Anincreaseinaffinitywasneverobserved.
AntibodiesproducedbycarpimmunizedwithDNPconjugatedtohumanserumalbumin (DNP-HSA),possessed4measurablecombining sitespermolecule.Theintrinsic
affinity(K)tothemonovalenthaptenDNP-L-lysinevariedfrom1 0 M -2.5x10M .
Duringtheinitialphaseoftheimmuneresponsetheaffinitydoubledbutoverthefollowing25monthsperioditremained constant (Fiebig&Ambrosius,1975,1977).The
functionalaffinity (KJ oftheanti-DNPantibodiesforthemultivalenthaptenDNP-T.
3 7
bacteriphagewas 10-10 fold greaterthantheK (Gruhn,Fiebig&Ambrosius, 1977).
K f values increased considerably aftersecondary immunizationwith DNP-HSA,upto
10 1 2 M
(Fiebig,Gruhn&Ambrosius,1977). Carpimmunizedwith thymus independentDNP
conjugates (DNP-Ficoll)synthesizeantibodieswithK similartoantibodies elicited
bythethymusdependentDNP-HSA (Fiebig,Gruhn,Scherbaum&Ambrosius, 1979).
35
1
2
K fhoweverisonly4x10 -4x10 higherthantheircorrespondingK values.A
possibleexplanationforthisobservationistheinvolvementofT-likecellsinthe
differentiationofdifferentB-like cellclones (Fiebig,Scherbaum,Nuhn&Ambrosius,
1979).
0 Theintrinsicaffinityoffishimmunoglobulinisrelativelylowanddoesnot
increaseduringtheimmuneresponse.Thefunctional affinityhoweverishigh.
Immunoglobulin
levels
DatashowninTABLE6demonstratethatimmunoglobulinlevelsinserumoffish
arecomparabletoimmunoglobulin levelsinman.CalculationsbasedupondataobtainedfrompassivelytransferredIginsharksindicatethatthesyntheticratein
thesespeciesiscomparabletothatinman(100-150and80mgIg/kgbodyweight/day
respectively) (Small,Klapper&Clem, 1970).
At thispointthestatementofClem (1970)thattheonly"primitive"aspect
offishimmunoglobulinsisthelimitednumberofimmunoglobulin classesseemstobe
correct.
TABLE6
Serumimmunoglobulin levels infish
absoluteamount
(mg/ml)
relativeamount
(Ig/totalprotein)
reference
9
1
lemonshark
10-12
2
nurseshark
5-6
smoothdogfish
stingray
50%
3
35%
4
17
1.7
40%
5
carp
6%
6
browntrout
6-7
10%
7
0.5-1
0.5 - 1%
paddlefish
man(IgM)
man (total)
10-20
9 -20%
8
8
1)Marchalonis &Edelman (1968b);2)Clem&Small (1967);3)Fidler,Clem&Small
(1969);4)Marchalonis&Schonfeld (1970);5)Legier,Weinheimer,Acton,Dupree&
Russell (1971);6)Richter,Frenzel,Hädge,Kopperschläger&Ambrosius (1973);
7)Ingram&Alexander (1979);8)Marchalonis (1977).
Location of immunoglobulin
Inadditiontoserum,antibodieshavebeendetectedinintestinalandsurfacemucus
ofplaice (Fletcher&Grant,1969).Mucusantibodieswere similartoserum antibodies
inelectrophoreticmobilityandcarbohydrateandaminoacidcomposition.Natural
hemagglutinins foundinthewater solublefractionofPacifichagfishmucuswere
immunologically relatedtoserumhemagglutininsandshowedasimilarheat-sensitivity
(Spitzer,Downing,Koch&Kaplan, 1976). Bradshaw,Richard&Sigel (1971)reported
thepresenceofantibodytoavarietyofantigensinsurfacemucusofgar,snapper
36
andbowfin.Antibodyactivityinserumandmucuswastotallyremovedbytreatment
with 2-mercaptoethanolorrabbitanti-gar IgMserum. Inrainbowtrout
anti-Vibrio
anguillarum agglutininscouldbedetectedinsurfacemucus 3-6weeksaftermaximum
serumtiterswereattained.Serumandmucusagglutininswere identical inOuchterlony
doublediffusionandimmunoelectrophoresis.Agglutinatingantibodyto Salmonella
paratyphi was foundinskinmucusofchannelcatfishafterintraperitoneal injection
ofbacteria.Byimmunodiffusionskinmucusgaveaprecipitationlineidenticalwith
serumimmunoglobulin (Ourth,1980).
Itisnotknownbywhichmechanismantibodiesgainaccesstomucus.Itis
possiblethatantibodiesproduced inlymphoidorgansaresecretedinmucusorthat
theyareproduced locallybymucosalplasmacells.Whenplaicewerefedheat-killed
Vibrio anguillarum, highantibody titreswere foundinintestinalmucus inalmost
completeabsenceofcirculatingantibody (Fletcher&White,1973a). This observation
favorstheideathat localantibodysynthesis existsinfish.
%Immunoglobulin ispresentinserumandmucusofskinandintestine.
37
CHAPTER4
HUMORAL IMMUNITY
Following antigeninjectionittakessometimebeforethefirstantibodies
appearinserum.Antibodygradually increasesandreachesaplateaufollowedbya
decline.Afterasecondinjectionwiththesameantigenthelatentperiodisshorter,
theresponseisacceleratedandhigherantibodylevelsarereached.Asecondaryresponseisonlyobservedwhentheanimalhasformed immunologicalmemoryafterthefirst
injection.MostantibodiesbelongtotheIgMclassduringthefirstdaysofaprimary
response.IgGisthepredominant antibodyclassduringthelaterphaseoftheprimary
responseandespeciallyduringthesecondaryresponseinmammals.
Thefirststageofthehumoral immuneresponse involvestheeliminationofantigenfromthecirculation.Inmammalsandfishthiseliminationprocesisaccomplished
bymononuclearphagocytes.PhagocytosisisdiscussedinChapter2(non-lymphoid
defence).However,itisobviousthatadistinctionbetweenspecificandnon-specific
defenceisonlyarbitraryinthisrespect.Antigentrappingandpresentationare
essential stepsintheinductionofanimmuneresponse.
Antibody response
TheAtlantichagfishfailedtoproduceantibodiestothesolubleantigensBSA
andKLHandtothecorpuscularantigen Brucella abortus (Finstad&Good, 1966).
AttemptstoinduceantibodyformationinPacifichagfishwereunsuccessfulwhenusing•
awidevarietyofantigens: Brucella abortus, typhoid,paratyphoidAandBvaccine,
BSA,KLH,bacteriophageT~andactinophageMSP-8 (Papermaster,Condie&Good, 1962;
Papermaster,Condie,Finstad&Good, 1964).MorerecentlyitwasshownthatPacific
hagfishwereabletoproduce specificantibodytoKLHandSRBC,butrepeatedimmunizationwasneededtoobtainmoderateantibodytitres (Thoenes&Hildemann,1969;
Linthicium&Hildemann, 1970).AhighantibodyactivitytogroupAstreptococcalcarbohydratewasdetectedinhagfishimmunized intravenouslywithstreptococci (Raison,
Hull&Hildemann, 1978a).Hagfishsynthesizebactericidal antibodiesafter injection
withthegramnegativebacterium Salmonella typhosa (Acton,Weinheimer,Hildemann&
Evans, 1969).Thesebactericidinsshowedalesserdegreeofspecificity (sincethey
cross-reactedwithheterologousgramnegativebacteria)andashorter inductionperiod
thanotherhagfishantibodies (Acton,Weinheimer,Hildemann&Evans,1971).
Inthesealampreynoantibodiesweredetectedafterimmunizationwithdiphteria
toxoid,BSA,BGG,SRBC,rabbitRBC,KLH,typhoidHandtyphoid0antigens.Aweak
primarybutaclear-cutsecondaryresponsewasobtainedwhenusing Brucella abortus and
bacteriophage T?asimmunizingagent (Papermasteretal., 1964;Finstad&Good, 1966).
LaterstudiesofMarchalonis&Edelman (1968)andLitmanetal. (1970),whichwere
designedtocharacterize immunoglobulins,showed that lampreydidproduce antibodies
tobacteriophage V?>Brucella andhumanRBC.Inbrook lampreyprimaryandsecondary
responsestoSRBCweredemonstratedinwhichbothhaemagglutinatingandhaemolysing
39-
aîitibodieswereinvolved (Fujii,Nakagawa&Murakawa, 1979a).
# I t isconcluded thatinspiteofinitialexperimentalfailures,antibodyresponsesareinducibleinthemostprimitivevertebrates;theAgnatha.However,the
humoralimmunesystemofhagfishispoorlydeveloped.Raison,Hull&Hildemann
(1978b)proposedthathagfishlackahumoralimmunesystemequivalenttothe
usualBlymphocyte-plasmacellsystem. Immunoglobulinswouldexistprimarilyin
amembraneboundstate,functioningaslymphocytesurfacereceptors.Thepresence
ofserumimmunoglobulincouldbetheresultofsheddingofreceptorsratherthan
activesecretionofimmunoglobulin.
Chondrichthyeshavebeenshowntoproducespecificantibodyafter immunization
withavarietyofantigens.Amongthemwereviralantigenssuchasinfluenzavirus
PR8 (Clem&Sigel,1963;Sigel&Clem,1965;Clem,DeBoutand&Sigel, 1965),bacteriophageT 2 (Papermasteretal., 1964;Suran,Tarail&Papermaster,1967)andpoliovirus (Clem&Sigel,1963),furthermorebacterialantigenssuchas Salmonella
paratyphi (Clem&Sigel,1963)and Brucella abortus (Finstad&Good,1966;Frommel,Litman,
Finstad&Good, 1971).AlsoxenogeneicerythrocyteslikeSRBC (Finstad&Good,1966)
andchickenRBC (Sigel&Clem, 1966),proteinslikeBSA,BGGandKLH (Clem&Sigel,
1963; Papermasteretal., 1964;Finstad&Good,1966;Sigel &Clem,1966;Suranetal.,
1967),carbohydrateslikestreptococcalgroupAcarbohydrate (Clem&Leslie,1971;
Clem,McLean&Shankey,1975;Sledge,Clem&Hood,1974)and Pneumococcus
typeIIIandVIIIpolysaccharides (Clemetal., 1975)andhaptens(DNP)conjugatedto
proteincarriers (Leslie&Clem,1969;Voss,Russell&Sigel,1969;Voss,Rudikoff&
Sigel,1971;Sigel,Voss&Rudikoff,1972)wereused.
Inlemonsharkandnursesharkkeptat27-30C,maximumantibodytitreswere
obtained30-40daysafterimmunizationwithPR8orSendaivirus.After restimulation
withPR8virus,renewedantibodysynthesiscouldbedetected,butnotintheorderof
arealsecondaryresponse (Sigel&Clem, 1965).OtherstudiesofSigel &Clem (1966)
withnursesharkindicatedthatalthoughtheanimalsshowedaprimaryresponseto
BSAandchickenRBC,clear-cutsecondaryresponseswerelacking.Lemonshark,kept
at26-28CproduceantibodiestoBSAaftera latentperiodof 10-12days.Maximum
titreswereobtainedafter 25-40dayswithantibodylevelsremainingataplateauup
to90daysafterinjection.Noanamnesticresponsewasobservedafterasecondinjection (Clem&Small,1967).Onlyafteraweakprimaryresponseaheightenedsecondary
responsecouldbeobtained.AccordingtoSigel,Lee,McKinney&Lopez (1978)thehumoralimmuneresponseofsharks resemblesamammalianresponsetothymus-independent
antigens. In mammals thymus-independent antigens do not evoke true secondary responses
(Mer, Stastok, «augh, Prescott $toll,ffl),Sijel et al, (19Î8) m %\
CHAPTER4
HUMORAL IMMUNITY
Following antigen injectionittakessometimebeforethefirstantibodies
appearinserum.Antibodygraduallyincreasesandreachesaplateaufollowedbya
decline.Afterasecondinjectionwiththesameantigenthelatentperiodisshorter,
theresponseisacceleratedandhigherantibody levelsarereached.Asecondary responseisonlyobservedwhentheanimalhasformed immunologicalmemoryafterthefirst
injection.MostantibodiesbelongtotheIgMclassduringthefirstdaysofaprimary
response.IgGisthepredominant antibodyclassduringthelaterphaseoftheprimary
responseandespeciallyduringthesecondaryresponseinmammals.
Thefirst stageofthehumoral immuneresponse involvestheeliminationofantigenfromthecirculation.Inmammalsandfishthiseliminationprocesisaccomplished
bymononuclearphagocytes.PhagocytosisisdiscussedinChapter2(non-lymphoid
defence).However,itisobviousthatadistinctionbetweenspecificandnon-specific
defenceisonlyarbitraryinthisrespect.Antigentrappingandpresentationare
essentialstepsintheinductionofanimmuneresponse.
Antibody response
TheAtlantichagfishfailedtoproduceantibodiestothesolubleantigensBSA
andKLHandtothecorpuscularantigen Brucella abortus (Finstad&Good, 1966).
AttemptstoinduceantibodyformationinPacifichagfishwereunsuccessfulwhenusing
awidevarietyofantigens: Brucella abortus, typhoid,paratyphoidAandBvaccine,
BSA,KLH,bacteriophageT ?andactinophageMSP-8 (Papermaster,Condie&Good, 1962;
Papermaster,Condie,Finstad&Good, 1964).MorerecentlyitwasshownthatPacific
hagfishwereabletoproducespecificantibodytoKLHandSRBC,butrepeatedimmunizationwasneededtoobtainmoderateantibodytitres (Thoenes&Hildemann,1969;
Linthicium&Hildemann, 1970).AhighantibodyactivitytogroupAstreptococcalcarbohydratewasdetectedinhagfishimmunized intravenouslywithstreptococci (Raison,
Hull&Hildemann, 1978a).Hagfishsynthesizebactericidalantibodies after injection
withthegramnegativebacterium Salmonella typhosa (Acton,Weinheimer,Hildemann&
Evans, 1969).Thesebactericidins showedalesserdegreeofspecificity (sincethey
cross-reactedwithheterologousgramnegativebacteria)andashorter inductionperiod
thanotherhagfishantibodies (Acton,Weinheimer,Hildemann&Evans,1971).
Inthesealampreynoantibodiesweredetectedafterimmunizationwithdiphteria
toxoid,BSA,BGG,SRBC,rabbitRBC,KLH,typhoidHandtyphoid0antigens.Aweak
primarybutaclear-cutsecondaryresponsewasobtainedwhenusing Brucella abortus and
bacteriophage 1? asimmunizing agent (Papermasteretal., 1964;Finstad&Good, 1966).
LaterstudiesofMarchalonis&Edelman (1968)andLitmanetal. (1970),whichwere
designedtocharacterize immunoglobulins,showedthatlampreydidproduce antibodies
tobacteriophageF 2 , Brucella andhumanRBC.Inbrook lampreyprimaryandsecondary
responsestoSRBCweredemonstratedinwhichbothhaemagglutinatingandhaemolysing
39
antibodieswere involved (Fujii,Nakagawa&Murakawa, 1979a).
# I t isconcluded thatinspiteofinitialexperimental failures,antibodyresponsesareinducible inthemostprimitivevertebrates;theAgnatha.However,the
humoral immunesystemofhagfish ispoorlydeveloped.Raison,Hull&Hildemann
(1978b)proposed thathagfishlackahumoral immunesystemequivalenttothe
usualBlymphocyte-plasma cell system. Immunoglobulinswouldexistprimarily in
amembranebound state,functioningas lymphocyte surface receptors.Thepresence
ofserumimmunoglobulincouldbetheresultofsheddingofreceptorsratherthan
active secretionof immunoglobulin.
Chondrichthyes havebeenshowntoproducespecific antibodyafter immunization
withavarietyofantigens.Among themwereviral antigenssuchasinfluenzavirus
PR8 (Clem&Sigel,1963;Sigel&Clem,1965;Clem,DeBoutand &Sigel, 1965),bacteriophageT 2 (Papermasteretal., 1964;Suran,Tarail &Papermaster, 1967)andpoliovirus (Clem&Sigel, 1963), furthermorebacterial antigens suchas Salmonella
paratyphi (Clem&Sigel,1963)and Brucella abortus (Finstad&Good, 1966;Frommel,Litman,
Finstad &Good, 1971).Alsoxenogeneic erythrocytes likeSRBC (Finstad&Good, 1966)
andchickenRBC (Sigel&Clem, 1966),proteins likeBSA,BGGandKLH (Clem&Sigel,
1963; Papermasteretal., 1964;Finstad &Good, 1966;Sigel &Clem, 1966;Suranet al.,
1967),carbohydrates likestreptococcal groupAcarbohydrate (Clem&Leslie,1971;
Clem,McLean&Shankey,1975;Sledge,Clem&Hood,1974)and Pneumoaooous
typeIIIandVIIIpolysaccharides (Clemetal.,1975)andhaptens(DNP)conjugatedto
proteincarriers (Leslie&Clem, 1969;Voss,Russell &Sigel,1969;Voss,Rudikoff&
Sigel,1971;Sigel,Voss &Rudikoff,1972)wereused.
Inlemonsharkandnursesharkkeptat 27-30C,maximum antibody titreswere
obtained30-40daysafterimmunizationwithPR8orSendaivirus.After restimulation
withPR8virus,renewedantibody synthesis couldbedetected,butnot intheorderof
arealsecondaryresponse (Sigel&Clem, 1965). OtherstudiesofSigel &Clem (1966)
withnursesharkindicated thatalthoughtheanimalsshowedaprimaryresponse to
BSAandchickenRBC,clear-cut secondaryresponseswere lacking.Lemon shark,kept
at26-28CproduceantibodiestoBSAafteralatentperiodof 10-12days.Maximum
titreswereobtainedafter 25-40dayswithantibodylevels remainingataplateauup
to90daysafterinjection.Noanamnestic responsewasobservedafterasecondinjection (Clem&Small, 1967).Onlyafteraweakprimary response aheightened secondary
response couldbeobtained.According toSigel,Lee,McKinney&Lopez (1978)thehumoralimmune response ofsharks resembles amammalian response tothymus-independent
antigens.Inmammals thymus-independentantigensdonotevoke truesecondary responses
(Baker,Stashak,Amsbaugh,Prescott &Barth, 1970).Sigeletal. (1978)suggest that
sharks lackT-helpercellsand that shark "B"cellsareabletorespond to "mammalian
thymus-dependent"antigens inaT-independent way. InChapter6thequestionof
lymphocyte heterogeneity infishwillbediscussed inmoredetail.
40
Thechondrosteanpaddlefishproducedantibodiesafterimmunizationwith
abortus or Salmonella
paratyphi
Brucella
(Fish,Pollara&Good, 1966).Antibodiesremainedde-
tectableforlongperiodsaftersecondarystimulation (Pollara,Finstad&Good, 1966).
Definiteprimaryandsecondaryantibodyresponseswereobtainedwithproteinantigens
likeBSA,BGGandKLH (Finstad&Good,1966).
Initialstudiesonthehumoral immuneresponseoftheholosteangarshowedthat
theanimalsdidproduceantibodiestoBSAbutthatasecondaryresponsewasabsent
(Sigel&Clem,1965;Clem&Sigel,1965,1966). Inalaterstudy,Bradshaw,Clem&
Sigel (1969)demonstrated thatthegarproducedantibodytodiphteriatoxoidandBSA
afteralatentperiodof11-30days.Withbothantigensaclearsecondaryresponse
wasobtained (80-foldenhancedantibodytitresfordiphteriatoxoid).Antibodiesproducedduringasecondaryresponsepersisted forover275days.Thebowfinshoweda
barelydetectableprimaryantibodyresponse tobacteriophageT-,andKLH.Thesecondary
responsewasvigorousforT 2butnotimpressive forKLH (Papermaster,Condie,Finstad,
Good&Gabrielsen,1963;Papermasteretal., 1964).
Thecapabilityofteleost fishtoresponduponantigenicstimulationwithspecificantibodyproductionhasbeendemonstratedsincethebeginningofthiscentury.
Mostoftheearlystudieswereperformed inordertoobtainprotective immunity
againstepizooticdiseasesinculturedfish.Thisaspect,incombinationwithvaccin
development,willbediscussed inaseparateparagraph inthischapter.
Primaryandsecondaryantibodyresponseshavebeenobservedinteleostfish
usingprotein,cellularandcarbohydrate antigens (seereviewofCushing,1970;
Carton,1973;Corbel,1975).Threetypicalexampleswillbegiven.
1)Goldfishproduceantibodyafteralatentperiodof7-10dayswhenimmunized
withBSAat25C.Peaktitreswerereachedbyday20.Inasecondaryresponse antiBSAantibodiesweredetected 3daysafterimmunization.Peakantibody titres,reached
atday 15,exceededthoseofaprimary response (Trump&Hildemann, 1970).
2)InMozambiquemouthbrooderkeptat30C,maximumantibodytitreswereobtainedonday11afterimmunizationwithSRBC,thelatentperiodwasonly2days.
FollowingasecondinjectionwithSRBC,antibodiesweredetectedwithin2days,the
8-foldenhancedpeaktitrewasreachedonday8 (Sailendri&Muthukkaruppan, 1975).
3)Browntrout,keptat20Cwereimmunizedwith Salmonella
typhimuvium lipopoly-
saccharide (acarbohydrate antigen).Inaprimaryresponseantibodiesweredetected
after14days,maximumtitresbetweenday56and63.Inasecondaryresponsethe
latentperiodwas 14days,theenhancedpeakwasreached34-40daysafterinjection
(Ingram&Alexander, 1980).
Thelengthofthelatentperiodandthekineticsoftheantibodyresponseare
influencedbythefollowingfactors:
a)theambienttemperature.Theeffectoftemperatureontheimmuneresponseis
discussedinChapter7.Evenwhenkeeping2different speciesatthesametemperature
41
similarkineticprofilesarenottobeexpectedbecause thesespeciesmayhavedifferentpreferentialtemperatures.Forexamplepeak antibodytitresinaprimaryresponse
at15Cafter immunizationwithMS~bacteriophagearereachedinbrowntroutonday
35andincarponday42(O'Neill,1980).
b)thetypeofantigen.Inbrowntrout,keptat20°C,maximumtitresto Salmonella
typhi werereachedafter49days,toKLHafter43days (Ingram&Alexander,1976a).
c)thenatureoftheantigen.Busch (1978)injectedrainbowtroutwithpreparations
ofentericredmouthdisease (ERM)bacterium.Thelatentperiodwas shorterinanimals
injectedwithwatersolublepreparations thaninthosereceivingorganicsolvent
solublepreparations (13and28daysrespectively).Moreover,ashorter latentperiod
andhigherantibody titreswereobtainedafter injecting formalinkilledbacteriathan
afterthesameprocedurewithphenolkilledbacteria.
d)therouteofadministration.Indace,keptat18Cantibodywasdetected 10days
afteranintraperitonealinjectionofhorseserumwhereasthelatentperiodtook12
daysafter intramuscular injection.Moreoverahigherpercentageofanimals responded
to intramuscularthantointraperitoneal injectedantigen (Harris,1973a).
e)theantigendose.Alongerlagperiodwasobservedwhenimmunizing carpwithlow
dosespigserum.However,thepeakdayoftheresponsewasnotdependentofantigen
dose (Ambrosius&Schäker, 1964).
# F r o mthedatamentioned aboveitmaybeclearthatitisalmost impossibleto
drawdefiniteconclusionswhenconcerningthekineticsoftheantibodyresponse.
Onlyageneralconclusioncanbedrawn:thecapacitytomountahumoralantibodyresponseandtheformationofimmunologicalmemoryiswelldevelopedin
teleostfish.ThecapacityofChondrichthyestodevelopimmunologicalmemoryis
questionable.
Cellular aspects
Afterantigeninjectionittakesacertaintimebeforethefirstcirculating
antibodiesappear (latentorlagperiod).During thatperiodantigeniseliminated
fromthecirculation (clearance)duetotrappingbymacrophagesanddendritic cells
inlymphoidorgans (Ellisetal., 1976;Secombes&Manning, 1980). Subsequently
B-likelymphocytesstarttoproliferateanddifferentiate intoplasmacells.Plasma
cellssynthesizeandsecretespecificantibody.Infish,twotechniqueshavebeen
usedtostudythesecellularaspectsofthehumoral immuneresponse:therosette
assayandtheplaqueassay.Withtherosetteassay,originallydevelopedbyZaalberg
(1964)andBiozzi,Stiffel,Mouton,Liacopoulos-Briot,Decreusfond&Bouthillier
(1966)thenumberofantigenbindingcells (ABC)canbedetermined.Inthisassay
cellscarryingsurface immunoglobulinorsecretingantibodyspecificfortheantigenusedreact in vitro withthisantigen.Whenxenogeneicerythrocytesareusedas
antigen,cellssurroundedbyerythrocytes (rosettes)areformed.Cellswithasingle
layeroferythrocytesareABCbutcellswithamultiple layeroferythrocytesrepre-
42
sentantibody secreting cells.Intheplaque assay,originallydevelopedbyJerne&
Nordin (1963)onlyantibody formingcellsarevisualized.
Inbrook lamprey,immunizedwithahighSRBCdoseABCcouldbedetected after4
daysinspleenandblood.Maximumnumberswereobtainedonday12.Afteralow
dose immunizationmaximumABCnumberswere foundonday8.Thisobservationisin
contrasttodatamentioned abovewhereitwasshownthatlowantigendosesextendthe
latentperiod.MultilayeredABCwereobservedinspleenandblood.Thus,although
lamprey lackplasmacellsonmorphological grounds (Finstead&Good,1966)cells
whichactively secrete antibodyarepresent (Fujii,Nakagawa&Murakawa, 1979b).In
rainbouw troutABCwereobservedinspleenandpronephros afterimmunizationwith
SRBC.Cellswiththeultrastructuralmorphologyofplasmacellsweresurroundedby
multiple layersofSRBC.Unfortunatelytheimmunizationscheduleusedinthisstudy
doesnotallowustodrawconclusions aboutthekineticsoftheABCresponse (Chiller,
Hodgins,Chambers&Weiser, 1969).ABCwerefoundingoldfish thymus,spleenand
pronephros fromday2onwards,maximumnumbersbeingreachedatday8.Multilayered
ABCwerepresentinspleenandpronephros (Warr,Deluca,Decker,Marchalonis&Ruben,
1977).Alsoinstudiesonthecarrier-hapteneffecttherosetteassayhasbeenused
tomonitortheresponse.RelevantexperimentsarediscussedinChapter6.
Studiesofthehumoral immuneresponseusingtheplaqueassayhavebeenperformedonlyinTeleosts.Thespeciesstudiedarebluegill (Smith,Potter&Merchant,
1967),rainbow trout (Chiller,Hodgins&Weiser, 1969),goldfish (Neale&Chavin,
1971),perch (Pontius&Ambrosius,1972)andMozambiquemouthbrooder (Sailendri&
Muthukkaruppan, 1975).
# T h e cellular aspectsofthehumoral immuneresponsehavebeenstudiedina
limitednumberofteleostsspecies.
Firstplaqueforming cells (PFC)were observed2daysafter immunization.Peak
ofthePFCresponsepreceedsinallcasesthepeakofserum antibodyandis
reachedatthesamedayindifferent lymphoidorgans.Anacceleratedaswellas
enhancedPFCresponsewasdemonstrated aftersecondary immunization.
Vaccination and protective
immunity
Inearlystudiesithasbeenshownthatteleost fisharecapableofproducing
antibodies againstbacterial antigens (reviewedinRidgway,Hodgins&Klontz,1966
andAvtalion,Wojdani,Malik,Shahrabani&Duczyminer,1973).Thesestudieswere
aimedtodevelopvaccination scheduleswhichprovideprotective immunity against
epizooticdiseasesincultured fish.Most attentionhasbeendevotedtothemain
bacterialdiseases:vibriosis (causedby Vibrio anguillarwri), entericredmouthdisease (Yersinia ruckeri), furunculosis (Aeromonas salmonicida),
columnarisdisease
[Flexibacter columnaris) andbacterialkidneydisease [Corynebacterium spp.).
Injectionsof Vibrio anguillarum bacterinevokedantibody titresinchinook
salmonandcohosalmon.Immunizedanimalswereprotected againstanatural challenge
43
upto6monthspost-injection (Antipa,1976).Severalreportsdescribeantibody responsesagainst Aevomonas salmonioida
afterintramuscularorintraperitonealinjec-
tions (Krantz,Reddecliff&Heist,1963,1964;Post,1966;Maisse&Dorson,1976).No
mortalityoccurredinimmunizedanimalsuponchallengewithlivebacteria.High
agglutinationtitresagainst Flexibaoter
oolumnaris wereobtained inrainbowtrout
afterparenteralvaccination.Susceptibilityofvaccinatedanimalstochallenge
withvirulentcolumnarisbacteriawasgreatlyreduced (Becker&Fujihara, 1978).It
canbeconcludedthatvaccinationbyinjectingkilledbacteriaevokesahumoral
antibodyresponseandprovidestheanimalswithprotective immunityuponchallenge
withlivebacteria.
Inlargescalefishcultureimmunizationofindividualanimalsishardlyapplicableforeconomicalreasons.Thereforemassvaccinationmethodshavebeendeveloped:
a)Oralimmunization.Thismethodhasbeenusedforabout40yearsandconsistsof
mixingvaccinwithfood.Ahumoralantibodyresponsehasbeenobservedbothinblood
andmucusofintestineandskinafterfeedingplaicewith Vibrio
anguillarvm
antigens
(Fletcher&White,1973a). Oralimmunizationhasyieldedpositive (e.g.Braaten&
Hodgins,1976;Fryer,Rohovec &Garrison,1978)butalsonegativeresults (Schachte,
1678; Udey &Fryer,1978).Efficacyoforal immunizationisreviewed inSnieszko
(1970),Anderson (1974)andEvelyn (1977). Inmostcasesoralvaccinationhasnotbeen
satisfactorybecauseofthevariableresultsandthediscrepancybetweenlaboratory
andfieldstudies.
b)Sprayvaccination.Inthismethodtheanimalsareremovedfromwaterandunderhigh
pressurevaccinissprayedonfish (Evelyn,1977;Gould,O'Leary,Garrison,Rohovec&
Fryer, 1978).Resultsobtainedthusfarsuggestthatthistechniqueismoreeffective
thanoralimmunization.
c)Bathimmunization.Inafirstversionofthistechniquethefishwereplaced ina
vacuumchamber inahyperosmoticvaccinsolution.Duringashortperiodtheatmosphericpressurewasreduced (Amend, 1976). Inasecondversionantigenuptakewas
accomplishedbymerelyplacing theanimalsinahyperosmoticvaccinsolution
(Amend&Fender,1976).Recentlyithasbeenshownthatdirectimmersionisaseffectiveashyperosmotic infiltration (Egidius&Anderson,1979;Antipa,Gould&
Amend, 1980). Incohosalmon,highagglutinationtitreswereobservedagainst V.
anguillarum
and A. salmonioida
afterhyperosmotic infiltrationwithabivalentbacterii
(Antipa&Amend, 1977).
#Basedupontheresultsobtainedthusfar,bathimmunizationseemstobesuperior
tooralorsprayvaccination.Itisapromisingmethodinthepreventionofinfectiousdiseases inculturedfish.
Relative littleattentionhasbeenpaidtothecellularaspectsoftheantibodyresponseindevelopingvaccinationschedules.Anderson (1978)adaptedtheplaque
assaytodetectantibodyformingcells inrainbowtroutinjectedwiththe 0-antigen
of Yersinia
44
ruckevi.
Inanimalskeptat17C firstPFCweredetectedafter 7days,the
peak occurredonday11(Anderson,Roberson&Dixon, 1979b).PFCwerealso detected
after immersionoftheanimalsintheantigensolution (Andersonetal.,1979a).The
minimalantigenconcentrationrequiredtoevokearesponsewas5yg/ml (Andersonet
al., 1979c).After flushexposureofrainbowtroutat11°CfirstPFCwereobservedon
day9whilehighestPFCnumberswere foundonday14-16 (Andersonetal.,1979d).
Anothermethodtostudytheimmune statusofrainbow troutafterbath immunization
hasbeendevelopedbyChilmonczyk (1977, 1978b).Lymphocytesoftroutafter survival
ofanaturalVHSinfectionwere inducedtoblast transformation in vitro
withVHSvirus,whereas controlanimalsdidnot.Specific lymphocyte stimulationwas
alsoobtainedinvaccinatedandsubsequently challenged animals.
Anaphylaxis
Inmammalsanaphylactic reactionsarecausedbyinteractionofantigenwitha
special antibodyclass (IgE)boundtomast cells.This interaction leadstodegranulationofmastcells (releaseofhistamine).
Anaphylactic reactionsweredemonstratedinperch,sunfish,rockbass,and
goldfish (Dreyer&King, 1948). Sensitizationafter intraperitoneal injectionsof
eitherhorse serumorovalbuminelicited signsofanaphylaxis afterre-exposureto
homologous antigen followingalatentperiodofatleast10days.Theanaphylactic
reactionwas specificandcharacterizedbyfoldingandcurlingoffins,equilibrium
disturbance,sinkingtothebottomanddiminishedmovement.These signspersistedfor
6 hours.Unsensitized fishdemonstratednoanaphylatic symptoms.
Later reports failedtorepeat theseobservations.ClemandLeslie (1969) attemptedtodemonstrate anaphylaxisinthemargatebytwodifferent approaches.Inthe
firstapproach animalswere intravenously injectedwithmargate antiseratoBSA.Animalswere injected intravenouslywithBSA4to24hours laterbutnoreaction followed.
Inthecutaneous approach,animalswere injected intravenouslywithBSAandEvans
blue4to24hoursafter subcutaneous injectionofmargate antiserumtoBSA.No
obviousblueingwasnotedatthesiteofinjection.Harris (1973b)wasunableto
demonstrateananaphylactic reactionindaceandchubusinghorse serumoreggalbumin.
However,inplaiceandflounder intradermal injectionswithfungal extracts cause
immediate erythema (Fletcher&Baldo, 1974). Immediatehypersensitivity reactions
occurredinchannel catfishandgoldfish following immunizationandchallengewith
different antigens (solubilizedprotozoa,BSA,versatol).Thereactions (disorientation,vertical swimming,increased opercularmovementandgasping)were specific
forthesensitizing antigenandcouldbepassively transferredtononsensitized
recipientswith serumfrom sensitized animals (Goven,Dawe&Gratzek, 1980).
Mastcellshavebeendescribedinfish (Ellis,1977a)andithasbeenshownthat
thesecells containhistamine (Roberts,Young &Milne, 1972).AnIgE-likemolecule
hasnotbeen foundinfish.IfIgMcanmimicthehomocytotropicIgEfunctionorthat
mast cellsoffishareactivatedbyanothermechanismisnotknown.
#Anaphylaticreactionshavebeendemonstratedinanumberofteleost species.If
45
immunoglobulinisinvolved inthesereactionsisnotknown.
46
CHAPTER5
CELLULAR IMMUNITY
Cellularimmunity involvesallspecific immuneresponsesmediatedbyacombinationofliving lymphoidandmononuclearphagocytesandtheirnon-antibody effector
molecules.Cellmediatedimmunity includesthemixedleukocytereaction,migration
inhibition,delayedtypehypersensitivity, transplantation immunityandtumor
immunity.Arelatedbutnonantigenspecificreactionusedfordemonstration
ofTcellactivityisthein vitro responsetocertainmitogens.Thecapacityof
fishlymphocytestorespondto"Tcellmitogens"isdiscussedinchapter6.
Mixed leukocyte réaction (MLR)
McKinneyetal. (1976,1980)failedtodemonstratepositiveMLRusingcells
fromsharks,snappersandgars.Inxenogeneiccombinations,catfish leukocytesas
targetcellsevokedpositiveMLRbyhumanperipheralbloodcellsbutthereciprocal
combinationgavenegative results.Inrainbowtroutitwasobserved thathomologous
mixturesofperipheralblood leukocytesorpronephros cellsobtained from2individualfishrevealedmarkedproliferative responses.Maximumresponseswereobserved
onday7withpronephros cellsandonday9withperipheralbloodlymphocytes (Etlinger,
Hodgins&Chiller,1976b; 1977).Inbluegillapositivemixed lymphocyte reaction
wasobtainedwithpronephros cellsat32°Cbutnotat22°C.PositiveMLRhasbeen
observedin12outof15randomtwo-waycultures.Negativereactionswerenot
correlatedwithalowresponsivenesstophytohaemagglutinin (PHA)byeithercell
preparation (Cuchens&Clem, 1977).
# I nanumberofTeleostspositiveMLRhasbeenobservedwhileinotheresnegative
resultswere obtained.
Migration inhibition (MI)
Jayaraman,Mohan&Muthukkaruppan (1979)assessedthecellmediated immune
responseofMozambiquemouthbroodertoSRBCusingthemigrationinhibition (MI)
technique.FormalinizedSRBC,whichhavebeenshowntoactivateT-helpercells
specificallywithoutthegenerationofplaque formingcellsinmice (Dennert&
Tucker, 1972)evokedanenhancedMIresponsewithoutadetectablePFCresponse.
OptimalMIresponseswere recordedwhenusingrelativelylowSRBCdoses.Inthe
garMIreactionswere inducedinkidneyandperipheralbloodcellsafterasecondaryallograftrejection (McKinney,McLeod&Sigel, 1980).ThelectinsPHA,concanavalinA (conA)andpokeweedmitogen (PWM)alsoevokedMIreactions (McKinney,
Ortiz,Lee,Sigel,Lopez,Epstein&McLeod, 1976).Themigratingcellswereidentifiedasmacrophagesbutitwaspresumed that lymphocyteswere involvedinproducing
inhibitorysubstances.Intheelasmobranch dogfishanMIresponsetoanantigenextractofthenematode Proleptus
obtusus couldbedemonstrated (Morrow&Harris, 1978).
# M I reactionshavebeenobtainedinallfishspecies testedthusfar.
47
Delayed type hypersensitivity
(DTH)
Adultsealampreyswere injectedintramuscularwithcompleteFreund'sadjuvant
followed21dayslaterbyachallengewitholdtuberculin.Within48hoursall
sensitizedanimalsshowedtypicaldelayedtypeskinreactions (Finstad&Good, 1966;
Good,Finstad&Litman, 1972). Inhagfish,noevidenceforDTHtoBGGortuberculin
was found (Papermaster,Condie&Good, 1962). InbowfinaDTHreactionwas observed
3daysafterchallengewith Ascaris antigen.Thechallengewasgiven30daysafter
initialsensitization.Thereactionconsistedofawelldefinedinduratedareain
theaxialmusculature (Good&Papermaster,1964). Inthehomed shark,guitarfish
andpaddle fish,moderatetosevereinflammatory reactionsoccurredafterrepeated
injectionwithBGGincompleteFreund'sadjuvant.AccordingtoFinstad&Good (1966)
thereactionswereduetoDTH.Inrainbowtroutapositivedelayed cornealreaction
topurifiedproteinderivateoftuberculin (PPD)hasbeendemonstrated1monthafter
immunizationwithcompleteFreund'sadjuvant.Thereactionreachedapeakin3-5
daysandlasted 12days.Unimmunizedcontrols injectedintracorneallywithPPDonly
showedatransientandfaintcloudingreaction (Ridgway,Hodgins&Klontz, 1966).
# Delayedtypehypersensitivityreactionshavebeendemonstratedinallclasses
offish,excepthagfish.
Transplantation
immunity
Pacifichagfish-oneofthemostprimitivevertebrates-wassupposedtobeincapableofrejectingallografts (Good&Papermaster,1961;Papermaster,Condie,
Finstad&Good, 1964).However,laterstudiesrevealedthatthecapacitytorecognizeandrejectskinallograftswaswelldevelopedinthisspecies (Hildemann&
Thoenes, 1969).Allgraftdestruction involvedinflammatoryreactions,lymphocyte
infiltration,capillaryhemorrhageandpigmentcelldestruction.Inanimalskept
at 18-19Cfirst-setallograftsshowedachronictypeofrejectionillustratedbya
mediansurvivaltime (MST)ofthegraftsof72days. Second-setgraftsplacedone
monthlaterwererejectedwithin28days.Whensecond-setgraftswereplacedimmediatelyafterthefirstrejectionprocesswascompleted,anacute rejectionoccurred
within14days.Rejectionoffirst-setskinallograftsinsealamprey,keptat 1821Cstartsduringthe3thweekaftertransplantation.Between42and47allografts
arecompletelyrejected (Finstad&Good, 1966;Perey,Finstad,Pollara&Good, 1968).
Therejectionofsecond-setgrafts,placed39days afterfirst-setgraftingstarts
inthesecondweek.By18daysmostgraftswerecompletely destroyed (Pereyetal.,
1968).
Stingrays,belongingtotheclassofChondrichthyes,keptat18-28Cshowedby
day21theonsetoffirst-setgraftrejection.Survivalendpointsranged from30-53
days.Second-setgraftsevokedamoresevere inflammatoryreaction,mostgrafts
showedsurvivaltimesoflessthan12days (Pereyetal., 1968). Inthehomed shark,
keptat22°CfoursuccessivesetsofskinallograftsyieldedMST'sof41,17,9and
48
7 daysrespectively (Borysenko&Hildemann, 1970). Lymphocytes andmononuclear cells
werepresent inthe inflammatory exudatebutnoplasmacells (Finstad &Good, 1966).
Intheholostean gar,first-set allograftswere rejected inanacutemanner
(McKinney,McLeod&Sigel, 1980). Duringthe first8days aftertransplantation both
autologousandallogeneic graftswereinvadedbyinflammatory cells. Subsequently,
allograftsproceeded to focal andextensive necrosis ofthe connective
tissuebyday 10-18whilechromatophores cleared. Cells infiltrating the allografts
were identified as lymphocytes,monocytes andgranulocytes.Second-set allografts
wereallrejectedby day 12.Anotherinteresting fishisarrowanabelonging tothe
superorder Osteoglossomorpha
whichincludes fishnearorjustabovetheholostean
leveloforganization. Inarrowanasmaintained at 25Cfirst-set skin allografts
wererejected inasub-acutemanner (MST 17.9days)whereas second-set grafts showed
anacute rejectioncharacterized byanMST ofS.1 days (Borysenko &Hildemann, 1969).
Histopathologicmanifestations ofallograft rejection includehyperplasia, lymphocyte
infiltration,hemostasis,pigment cellgranulationanddonorcellreplacementbyhost
tissue (Hildemann, 1970).
IntheChondostreanpaddlefish,keptat 18-25°C,first-set skinallograftswere
rejected betweenday 42-68. Second-set graftswererejectedwithin 12days.The
second-set rejectionwas characterized bypronouncedhemorrhage,followedby necrosis
(Pereyet al., 1968).
Inteleost fish,kept atoptimal temperatures,allografts are always rejected
inanacutemanner,i.e. rejection times shorterthan 14days.Transplantation immunity hasbeenextensively studied ingoldfishvisingthescale grafttechnique.
Following transplantation both autografts andallografts become revascularized,the
time required forrestoration ofcirculation varies from 1dayat 32Cto 12-15days
at 10C (Hildemann, 1957). Allograftsbecomeovergrownwithhyperplastic host tissue
andelicitcapillary leakage andvasodilation inthe contact zonewith recipient
tissue.Theendpointofdonortissuebreakdownhasbeenestimatedbyabiological
test,i.e.regraftingscalesonthedonor. Itwas foundthatallografts survivedup
tothetimethatclearing ofthedensehyperplastic tissue grownoverthegraft
started.TheMSToffirst-set allografts ingoldfishkept at 25Cwas 7.2 days.
Second-set grafts,placed 25days after first-set grafting,induce earlyand severe
inflammation andwere rejected rapidly (MST4.7 days) (Hildemann &Haas, 1960). With
anincreasingnumberofallografts,ranging from 1-9 graftsper animal,nosignificantantigendosageeffect ontheMSToffirst-orsecond-set allograftswas observed
(Hildemann, 1957). A definite secondary immune response totissue allograftswas
obtained already at6-8 days following first grafting.
The followingexperiments havebeenperformed todemonstrate that allograft
rejection isacell-mediated immune reaction.Lyophilized graftsorgraftsheatedat
48C for20minutes didnotprovoketransplantation immunity asmanifestedby the
findingthat second-set graftswerenot rejected faster.Probablynoisohaemagglutinating antibodies areinvolved inthenormalprocess ofgraftrejection sinceno
49
alterationinserumtitreofrecipient animals towardsredbloodcellsofdonor
animalswasobserved afterscale rejection.Inanothersetofexperiments animals
were immunizedwithwholeblood fromdonor animals.Fourintraperitoneal injections
yieldedahaemagglutinationtitreof1:526 whereas2intramuscular injections gave
risetoaserumtitreof1:42.Whentheseanimals receivedscaleallograftsfrom
theirrespectiveblooddonorsnosecondary responsewas evidentinanimals injected
bytheintraperitoneal route (MST7.2days)butastrong immunitywasexpressedin
animals injectedbytheintramuscular route (MST5.6days) (Hildemann, 1958).
Sailendri (1973)studiedallograft rejectioninMozambiquemouthbrooder.In
animalskeptat25C,allografts showedswelling,hyperplasiaandsevere inflammation
ontheseconddayaftertransplantation. Laterexpansionofmelanophoresoccurred.
Inflammation disappearedatthe6thdayanddésintégrationofmelanophoreswascompleted2days latermarkingthesurvivalend-pointofthegrafts.Thegraftswere
invadedbyleukocytes fromday3onwards,withamaximumonday6.Therejection
processofsecond-set allograftswasaccompaniedbyamore severe inflammation.
Second-set allografts,transplantedaslongas4month after first-set,were rejected
inanacceleratedway,indicatingalonglivedimmunologicalmemory. Xenografts
evokedamoresevere inflammationandfaster rejection thanallografts.Antigen
dosage (3-18allograftsperanimal)hadnoeffectontheMSTofthegrafts.Secondsetallografts transplanted fromathird-party donorwere rejectedinaprimary
fashion.
# I n Agnathaandcartilaginous fish allograftsarerejectedinachronicway
(MST>30days). Sub-acute rejection (MSTapproximately20days)occursin
Chondostreans andHolosteans,whileTeleosts showanacute rejectionofallografts (MST<14days). Second-set graftsarealways rejected fasterthanfirstset. Ithasbeendemonstrated thatallograft rejectionisamanifestationof
cellular immunity.
Ontogeny of transplantation
immunity
Adult rainbowtrout,keptat16-18C,reject skingraftsin19-21 days.Firstsetgraftsareinvadedbylargenumbersoflymphocytesonday5postgrafting followedbymaximal infiltrationonday9.In26dayoldtrout lymphocytesarenotpresent
inthegraftbeforeday7reachingamaximumatday12.However,thetotalrangeof
rejectiontimes fallswithin thatofadults (Botham,Grace&Manning, 1980).
Inadultcarp,keptat22-23C,lymphocyte infiltrationinfirst-set skingrafts
starts2daysaftergrafting,reachingamaximumbyday4-6.Allograft breakdown
commencesbyday8while totalrejectionoccursbyday14postgrafting.In2-4weeks
oldanimals invading lymphocytesareseen firstbetweenday4-8aftergraftingand
graftbreakdown startsatday16.In2monthsoldcarp lymphocyte infiltration also
startsbyday4-8,butgraftbreakdown commencesbyday11.Itisconcluded thatcarp
asyoungas16daysposthatchingarecapableofmountinganallograft responsebut
completematurationofthecellular immune systemtakes over2months (Bothametal.,
50
1980).
TheMSToffirst-set allografts in4.5montholdMozambiquemouthbrooderis
thesameasinadults (5.4and5;2daysrespectively).Inyoungeranimals (1.5
months)scaleallografts survived for10.9days (Sailendri,1973).
# I n oviparous Teleosts thecapacityoflarvae torejectallografts starts
16-45days after fertilization.
51
CHAPTER6
LYMPHOCYTE HETEROGENEITY
The lymphocytesofhighervertebratescanbedividedin2major subsets accordingtotheirorigin,functionandcharacteristics.Cellsderived fromthethymus
(Tcells)areinvolvedincellular immune reactions suchas:allograft rejection,
graftversushost reaction,mixed lymphocyte reaction,delayed typehypersensitivityandhelper functioninantibody formation.B lymphocytes,whicharederived
fromthebursaofFabriciusinbirdsandfromabursa-equivalentinmammals,mediatehumoral immunitybyproducing antibodies.Tcells functionpredominantlyby
direct contactwith antigenwhileplasmacells (Bcellderived)produce antibodies
whicheitherdirectlyorindirectlyactuponantigens.InTABLE7somecharacteristicsofmammalianTandBcellsarelisted.
TABLE7
Characteristics ofTandB lymphocytes inadultmammals
Feature
T lymphocyte
B lymphocyte
bonemarrowand/orbursa-equivalent
origin
thymus
Thy-1 antigen
present
absent
membraneboundIg
absent
present
Fereceptor
absent
present
C,receptor
absent
presentonsomeBcells
mitogenresponse
PHA,ConA
LPS
"carrier-reactive"
+
-
"hapten-reactive"
-
+
function
helper cell,
suppressorce 11,
killercell
plasmacellprecursor
InthisChapter datawillbepresentedwhicharerequiredtoanswerthequestion
whether fish lymphocytescanbeclassifiedinTandBsubsetsornot.
Thy-1 antigen
Thy-1wasshownbyReif&Allen (1963)tobeasurface antigenpresentonmouse
thymocytesandnervous tissue.Laterstudies revealed thatThy-1 antigencould
beusedasamarkerformouseTcells (Raff,1971). Most anti-Thy1seraareisoantiserabutTcell specific antiseracanalsobeobtainedbyimmunizing rabbits
withmousebraincellsandabsorbingtheantiserumwithmouseerythrocytesandliver
cells (Golub, 1971;Thiele,Stark&Keeser, 1972). Strictly speaking these antisera
cannotbedenominated anti-Thy-1 serabuttheyareusefulasaTcellmarker.
Cuchens&Clem (1977)preparedarabbit antiserum againstbluegill braintissue.
Incombinationwithcomplement thisantiserumkilled about 70%ofpronephros lymphocytes. Cellssurvivingtheantiserum treatment responsedtoLPS(amammalianBcell
53
mitogen,seebelow)butnottoPHA(amammalianTcellmitogen)whereasuntreated
pronephroscellscanbestimulatedbybothLPSandPHA.Theseresultssuggestthat
bluegill lymphocytesresponsivetoPHAcarrysurfaceantigensrelatedtomurine
Thy-1antigen.
Surface immunoglobulin
(slg)
InmammalsTcells lackslginthesense thattheydonotreactwith antiseradirectedagainstserumIg(Raff,1970).Ithasbeenestablishedthatthevast
majorityoffishlymphocytes,includingthymocytesbearsurface immunoglobulin
(TABLE 8). Thisconclusionisbasedupontheobservationthatarabbitantiserum
raisedagainst fishserumimmunoglobulinreactswithsurfacematerialoflymphocytes.
Thepositivereactionoftheanti-immunoglobulinserawiththymocytesisprobably
notduetocross-reactivitywithcellsurfacecarbohydratesbecause (1)components
withanelectrophoreticmobilityofimmunoglobulinpolypeptidechainscanbeextractedfromthymocytes (seebelow)and(2)apositivereactioncanbeobtainedusing
antiseradirectedagainst immunoglobulinlightchainswhich lackacarbohydrate
moiety (Fiebig&Ambrosius, 1976;Clem,McLean,Shankey&Cuchens,1977). Interestingly,quantitativeandqualitativedifferencesinslgonlymphocytesfromdifferent
lymphoidorgansexistinfish.Carpperipheralbloodandpronephros lymphocytescarry
moreslgmoleculespercellthanthymocytes (Fiebig,Scherbaum&Ambrosius,1977).
LymphocytesofgoldfishandCrusiancarpareheterogeneousinthequantitative distributionofslg:fluorescenceintensityofspleen>>pronephros>thymus (Warr,
DeLuca,Decker,Marchalonis&Ruben,1977;DeLuca,Warr&Marchalonis,1978).Alower
quantityofslgonskatethymocytescomparedwithsplenocyteswassuggestedbyEllis
&Parkhause (1975).Inrainbowtroutthymocytespossessedatleast 10-foldlower
amountsofslgcomparedwithsplenocytes (Yamaga,Etlinger&Kubo,1977).Inblue
gill,however,quantificationofslgrevealedthattherewaslittledifferencebetweenlymphocytesfromperipheralblood,spleen,pronephrosandthymusinthisrespect (Clem,McLean,Shankey&Cuchens,1977).
Inordertofurthercharacterizeslgoncarplymphocytes,Fiebig
&Ambrosius (1976)radioiodinatedperipheralblood,pronephrosandthymuslymphocytesandextractedsurfaceproteinswithnon-ionicdetergents.Proteinwasanalysed
onsodium-dodecylsulfate-polyacrylamide-electrophoresis.SurfaceIgwasneverpresent
inatetramericform,asinserumIgMbutasmonomers (H 2 L 2 )• ThismonomericIgon
pronephros lymphocyteshadaM.W.of220,000whileslgofthymocyteshadaM.W.of
260,000.Peripheralblood lymphocytescarriedbothforms.Furthermoreslgwaspresent
onalllymphocytesasHLsubunits (M.W. 110,000).Thymocytes,apartfromHandL
chains,showed2additionalcomponentsuponelectrophoresis,withM.W.'sof100,000
and35-40,000.Thelastcomponentwasnotobservedonpronephros lymphocytes.In
goldfishslgofsplenocytesandthymocytesdifferinsolubilizationcharacteristics
withnon-ionicdetergentsandinHchainelectrophoreticmobility.TheHchainsof
splenocyteswerecomparabletomammalianychainswhereasthymocyteHchainsexpress54
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edafastermobility (Warr,DeLuca&Marchalonis,1976). Furthermore,slgofsplenocytesdiffersfromserumIginadeficiencyof10,000dintheHchainofsplenocyte
slg (Warr&Marchalonis,1977).TheHchainpeakofpronephrosslgwasverybroad
comparedwiththesharpHchainpeakobtained fromthymocyteslg (Ruben,Warr,Decker
&Marchalonis, 1977),Characterizationofslgofrainbowtroutsplenocytesshowed
thepresenceof3majorcomponentswithM.W.'sofapproximately 100,000,65,000and
25,000.IncontrastthymocytescontainedseveralhighM.W.components (70-150,000)
butlackedpeaksintheLchainregion (Yamaga,Etlinger&Kubo, 1977). Bluegillslg
isquitesimilartoserum Ig,andM.W.'sofHchains isolated fromthymocytesis
similartothatisolatedfromotherlymphoid tissue (Clem,McLean,Shankey&Cuchens,
1977).
# I t canbeconcludedthatinfishnearlyall lymphocytescarryslgwhichispresentinamonomericformorasHLsubunits.However,thereissomeheterogeneity
(bothquantitativeandqualitative)inslgamonglymphocytes fromdifferent
lymphoidorgans.
Hapten-cavviev
effect
Cellularco-operationduringanimmuneresponsebetweendifferent lymphocyte
populationscanbestudiedusingthehaptencarriereffect.Ahaptenisasmall,
initselfnon-antigenicmolecule (suchasDNP)whichiscovalentlycoupledtoa
large"carrier"molecule,generallyaprotein.Thehapten-carriereffectbasically
involvespreimmunizationwithacarrier,followedbyaboosterwiththehaptencarriercomplex.Thisscheduleresultsinanenhancedprimaryresponsetothehapten.
Inmammals,TcellsreactwiththecarriermoietywhileBcellsrespondtothehapten.
Hapten-carrierimmunizationwasusedinbrooklampreytoexplorelymphocyte
heterogeneityatthelowestphylogenetic levelofthevertebrates.Carrierpriming
didnotenhancethefollowinganti-haptenresponse (Fujii,Nakagawa&Murakawa, 1979b).
Foranumberofteleoststhehapten-carriereffect nowhasbeendemonstrated
usingtwobasicallydifferenttechniques.Afirsttechniqueinwhichthenumberof
haptenbindingcellsisdeterminedhasbeenusedingoldfish.Twooreightdays
aftercarrierpriming (HRBC)animalswereimmunizedwiththehaptentri-nitrophenol
(TNP)conjugatedtoHRBC.TheTNP-bindingpronephros lymphocytes
wereshown
tobehaptenspecificsincepreincubationofsensitizedcellswithTNP-glycinecompletelyeliminatestheresponse.Ingoldfishkeptat24C,maximumTNPbindingactivityappearsatday8afterchallenge.Highdoseprimingisineffectiveingenerating
helperactivity.Helpermemoryevokedbylowdosecarrierprimingisshortlived:
TNP-HRBCchallenge8daysafterHRBCprimingdoesnotresultinananti-TNPresponse
(Ruben,Warr,Decker&Marchalonis, 1977).
Theotherapproachtodemonstratethehapten-carriereffect (titrationofhapten
specificserumantibodies)wasfollowedinwinterflounder.Carrierpreimmunization
withchickenglobulin (CG)orKLHwas followed3weeks laterbyimmunizationwiththe
56
haptensNIP(3-iodo-4-hydroxy-5-nitrophenyl aceticacid)andNNP(3,S-dinitro-4hydroxy-phenylaceticacid)covalently coupledtoCGorK1H.Antibody titreswere
determinedbytheabilityofserumtoinactivate haptenated bacteriophages. Preimmunizationwiththecarrier enhancedtheantibodyresponsetothehaptenina
specificway:aresponsetoNIP-CGcouldonlybedetected afterpreimmunizationwith
CG,notafterpreimmunizationwithKLH(Stolen&Mäkelä, 197S,1976).Weiss&Avtalion (1977)preimmunizedcarpwitheithernativeBSA,methylatedBSAoracetylated
BSAascarriers.Threeweeks lateranimalswere immunizedwiththehaptenpenicillin
conjugatedtothecarrierinvarious epitope densities (Pen,-BSAandPen,--BSA).It
was shownthat2factorswere importantinobtainingamaximal enhancementofthe
primary anti-hapten response:a)preimmunizationwithamodified carriermolecule
ratherthanitsnative formandb)alowhaptendensityonthehapten-carrier conjugate.
Rubenetal. (1977)andWarretal.(1977)have attemptedtoseparate "carrierreactive"and"hapten-reactive" lymphocytepopulations usingthenylonwool column
adherencemethod (Julius,Simpson&Herzenberg, 1973). Pronephrosandthymus cells
ofgoldfish,whichwereprimedwithHRBCandchallengedwithTNP-HRBC,wereused.
Hapten-reactive cellswere only foundinpronephros,notinthymus.Thesehaptenreactive cellswereretainedinthenylon-wool column.Thus,hapten-reactiveand
carrier-reactivecells couldbephysically separatedbytheiradherencetonylonwool columns.
• inconclusionitisevident thatcell-cell cooperationinhumoral immuneresponsesisdemonstratedforanumberofteleost fish indicating thatthereare
at least2different lymphoidcellpopulations.Ontheotherhand thisphenomenonprobably doesnotyetoccuratthelevelofAgnatha.
Cell surface
receptors
TheonlyreportonC,receptorsoncellsurfacesinfishdealswithafibroblastcell lineofgoldfish.Itappears thatthegoldfish fibroblasts haveareceptor
formouse (AKR)C,butthefunctionofthis receptorisunknown (Ueki,Fukushima,
Hyodoh&Kimoto, 1978).
# A s farasweknownothingispublishedsofaraboutC,andFc receptors
on fishlymphocytes.
Mitogen response
Inhighervertebrates,TandBlymphocytes canbedistinguishedbytheir in
vitro
responsetodifferentmitogens.Incontrasttoaparticular antigen,which stimulates
onlyasmall fractionoflymphocytes,mitogens stimulate substantial partsoflymphocytepopulations non-specifically.After stimulation large lymphoblastsareformed,
whichwillundergomitosisandfinally develop intoeffector cells.Inmammalsthe
mitogensphytohaemagglutinin (PHA)andconcanavalianA (ConA)stimulatepredominantlyTcells;lipopolysaccharide (LPS)andpolyvinylpyrrolidone (PVP)onlyBcells.
57
BloodcellsofPacifichagfish
,synthesizeENAandproliferateinresponsetoPHA.Relative longincubationperiodsandhighPHAconcentrationarerequiredcomparedwithmammalian lymphocytecultures (Tam,Reddy,Karp&Hildemann,
1977).
Peripheralbloodlymphocytes fromnursesharkrespondtoConA,butnottoPHA
(Lopez,Sigel&Lee, 1974). SeparationonFicoll-Isopaquegradientsyielded2populationsoflymphocytesofwhichonerespondedtoPHAandConAandtheotherpopulationonlytoConA.Itwasconcludedthatsharkspossess lymphocytescapableof
respondingtomitogenswhicharestimulatorsformammalianTcells.
SinceitwaspossibletoinhibitPHAresponsive cellsbytheadditionofConAresponsivecellstheauthorssuggestthatnursesharkpossessedsuppressorcells
(Sigel,Lee,McKinney&Lopez, 1978).
Withintheteleost family,mostworkonmitogeninduced lymphocytetransformationhasbeencarriedoutwithrainbowtrout.Bogner&Ellis (1977)demonstrated
that in vivo peripheralblood lymphocytesrespondtoPHA,ConAandLPSwiththe
formationoflymphoblasts. In vitro, peripheralbloodlymphocytes couldbestimulated
byPHA,ConA,LPSandPPD(Etlinger,Hodgins&Chiller,1975,1976a;Chilmonczyk,
1978a). SimultaneousexposuretobothLPSandPHAledtosignificanthigherstimulationsthanbyeachmitogenalone,suggesting thatthereare2differentsubpopulationsofperipheralblood lymphocytes (Chilmonczyk, 1978a). Rainbowtroutthymocytes
respondtotheTcellmitogenConAbutnottotheBcellmitogenLPS.Leukocytes
frompronephrosrespondtoLPSbutnottoConAandPPD,whilesplenocytescouldbe
stimulatedbyLPS,PPDandConA (Etlingeretal., 1976a).
Peripheralblood lymphocytesofsnappercouldbestimulatedbyPHA(McKinneyetal.,
1976),Incontrasttotheclear-cutorgandistributionofmitogen responsiveness
inrainbowtrout,bluegill lymphocytes frompronephros,thymusandspleencanbe
stimulatedbyPHA,ConAandLPS(Cuchens,McLean&Clem, 1976).
Morphological consequencesoflymphocyte transformationhavebeenstudied
inrainbowtroutsplenocytes (Etlinger,Hodgins&Chiller, 1978).ConAinduced
blastcellsarelargerandpossessalighter (May-Grünwald/Giemsa)stainingnucleus
thanlymphocytes,haveahighercytoplasm-nucleus ratioandanincreasedamountof
mitochondriaandroughendoplasmicreticulum (RER).Plasmacellsoccur infrequently
inConAstimulated cellcultures.Blast cellmorphologyofLPSandPPDculturesis
similartothosewithConA.LPSandPPDinducemitogenesisrevealedbythepresence
ofabout 7% plasmacellsafter7daysofculture (Etlinger,Hodgins&Chiller, 1976b).
©Lymphocytesoffishrespond in vitro tomitogensbyblasttransformation.A
differentorgandistributionforcellsrespondingtoT-andB-cellmitogens
is foundinsomespecies.
Do fish possess T and B lymphocytes?
Intheliterature3different answershavebeengiventothequestionabove
mentioneda)yes,theydo,b)no, onlyBcells,andc)no,onlyTcells.
58
a) Taking theabovementioneddataintoaccount,Marchalonisconcludedthatfish
lymphocytescanbedividedinto2subsets,onewithT-likeproperties andtheother
withB-likeproperties (Marchalonis,Warr &Ruben,1978;Warr &Marchalonis,1978)
Themajorargument againstthisconclusion isthatinfish (andinlowervertebrates
ingeneral)both"T"and"B"cellscarryslgwhereas inmammalsTcellsdonot.This
argument iscounteredby statingthatmammalianTcellsdocarryslg. Infish,thymocyteslgdiffers fromserumIgandpronephros slg.Thymocyteslgcross-reactwith
rabbitanti-Igserabecauseofthephylogenetic distancebetweenmammals andfish.
Indeed,whenusingchickenantiserumagainst Fabfragmentsofserum Ig,thymocytes
ofmanandmousecarrycross-reactingslg (Jones,Graves &Orlans,1976;Marchalonis,
Warr,Bucana,Hoyer,Szenberg &Warner,1977;Szenberg,Marchalonis &Warner, 1977).
Thereverseexperiment,goldfishanti-carp serum Igincubatedwithcarpthymocytes,
shouldconsequentlyyieldnegativeresults.
b) OntheotherhandMcKinneyetal. (1976)suggest thatnoheterogeneity exists
infishlymphocytes.Infacttheystatethat fishdonotpossess Tlymphocytes.Their
argumentsarethefollowing.
1)Organculturesofthymussynthesizeantibody. 2)Duringahumoral immuneresponse
infishnoshiftfromIgMtoIgGoccurs.ThisshiftisstimulatedbyTcells inmammals
(Katz&Benacerraf, 1972). 3)Althoughthehapten-carriereffecthasbeendemonstratedinfish,itremainstobeproventhatcarrierreactivecellsareTcellsor
lymphocytesatall.4)Theassumptionthatevery fishlymphocytestimulatedbya
mammalianTcellmitogen (PHA)isarealTcellispremature.Forinstance,blood
cellsfromthetunicate, Ciona intestinalis,
whichlacksathymusrespondtoPHA
(Tarnetal., 1977). Furthermore,undercertaincircumstances,PHAcanstimulate
mammalianBcells (Greaves,Owen&Raff, 1974).
ThereforeMcKinneyet al. (1976)provisionally concludedthat fishlymphocytes
actprincipallyasBcellsandthattheobservedTcellfunctions reflectmultipotentiality.
c) Reciprocal transplantationofthymicprimordiabetweendiploid andtriploid
amphibianembryos {Rana pipiens) suggestedthata)thymuslymphocytes arisewithin
thethymus;b)virtuallyall lymphocytes arethymusderived (Turpen,Volpe&Cohen,
1973; 1975). Ifthisistrue forlowervertebrates ingeneral,fish lymphocytes
shouldbeclassifiedasTcells.
ThedifficultyinassessingTandBequivalents inlowervertebratescanbe
illustratedbysummarizing relevantdataconcerningthemostprimitiveclassoffish:
theAgnatha.Onmorphological groundtheseanimals lackadefinitethymus (Good,
etal., 1966;Riviere,Cooper,Reddy&Hildemann, 1975);
Allograftsarerejected (Perey,Finstad,Pollara&Good,1968;Hildemann&Thoenes,
1969);Peripheralblood lymphocytes respondtoPHA (Cooper,1971;Tarnet al., 1977);
Hapten-carriereffectisabsent (Fujiietal.,1979b). Theanimalsrespondto (a
limitedvarietyof)antigensbytheproductionofspecificantibody (Linthicium&
Hildemann,1970;Fujiietal., 1979a); nomammaliantypeplasmacellswereobserved
59
(Goodet al., 1966).
# A t thismoment itisimpossible toclassifyfishlymphocytes indefinite T
andBcellsubsets,althoughdataformoreadvanced speciesaremoreconsistent.
Moredataarerequiredonontogenyoflymphocytes,effectsofthymectomyand
behaviourofseparated "T"and"B"cellsinreconstitutionexperiments.Until
thenitispreferred tospeakintermsof functionalT-andB-likefish
lymphocytes
60
CHAPTER7
FACTORSAFFECTINGTHEIMMUNERESPONSE
Temperature
Since fisharePoikilothermievertebrates,theambient temperaturewillinfluenceallmetabolicprocesses includingtheimmune response.Fromatheoretical
viewpoint thisphenomenonoffers interestingpossibilities,e.g. dissociationof
the immune response intodiscrete stepsbyamere variationinwater temperature.
Thetemperature dependenceoftheimmune systemalsohassomepractical consequences.
Non lymphoid defence
Ithasbeenshownincarpthatphagocytosis occurredatarelative slow rate
attemperatureswhichprevented antibody synthesis (seebelow) (Avtalionetal.,
1973; Wojdani,Katz,Shahrabani &Avtalion, 1979).TherateofMS2 bacteriophage
clearance fromthebloodofcarpandrainbowtrout decreaseswith lowering temperatures (O'Neill, 1980).Thesameeffectwasobservedinnurse sharkandlane snapper
(Russell,Taylor&Sigel, 1976).Thecomplement systeminfish functions verywellat
lowtemperatures (Chapter 2).Naturalagglutinins toSRBCand Salmonella
typhosa
inthegardisplayahigheractivityat4°Cthanat22°C.Inpaddlefish also "cold"
agglutininswere found (Legier,Weinheimer,Acton,Dupree&Russell, 1971). Itis
possible that"cold"agglutinins representacompensatory mechanismfortheimmunosuppressive effectoflowtemperatures infish (Bradshaw&Sigel,1969 ) .
• Nonlymphoid defence systemsarerelative temperature independent.
Cellular immunity
InChapter5datahavebeenpresentedwhich indicate that allograft rejection
isamanifestationofcellular immunity.Inanumberofpublications ithasbeen
shownthat allograft rejectionisdelayedatlowtemperatures (Goss,1961;Stutzman, 1967;Botham,Grace&Manning, 1980). Hildemann (1957)studiedtheeffectof
water temperatureonthemedian survival timeofallograftsingoldfish.Atall
temperatures studiedaclear-cut secondary responsewere observed (Table 9 ) .Both
firstsetandsecondsetallograft rejectionwas strongly temperature dependent.
InMozambiquemouthbrooderthetemperature effectoncellular immunitywasmuch less
pronounced (Table9)(Sailendri, 1973). Itwasfoundingoldfish thattherelationshipbetweenMSTandtemperaturewasnotlinearbutthatabreak occurred between
20and25C (Hildemann&Cooper, 1963). Similarobservationshavebeenmadeinamphibians (Cohen,1966)andreptiles (Borysenko, 1970). Grafted frogs (Macela&Romanovsky, 1969)andturtles (Borysenko, 1970),maintainedatlowtemperatures,reject
allograftsinanaccelerated fashionwhentransferredtohighertemperatures during
allograft rejection.Therefore antigenprocessingandrecognitionmusttakeplaceat
lowtemperatures.Borysenko (1979)postulated thatatlowtemperaturesanearlybut
post recognitionevent (cellproliferation?)isprimarily affectedwhileathigher
61
temperatureseffectorfunctionsarethelimiting factor.Ifthisholdstruefor
fishremainstobeinvestigated.
TABLE9
Influenceoftemperatureonallograftrejectioninteleostfish
Median Survival Time (days)
Temperature
(
(°C)
Mozambique mouthbrooder
Goldf ish '
First-set
Secondset
10
40.5
19.5
16
17
20.5
13.9
19
12.6
8.0
21
8.3
5.4
25
7.2
4.7
27
28
6.3
4.4
4.3
3.2
First-set
30
32
Second-set
9.4
N.D.
7.8
5.8
7.0
5.0
5.2
4.2
5.1
N.D.
Data takenfromHildemann, 1957 (1)andSailendri,1973(2).
N.D. =notdone.
Humoral immunity
Earlystudiesontheeffectoftemperatureonthehumoral immune responsesuggestedthatfishwerenotabletoproducecirculatingantibodybelow10C (reviewed
inAvtalionetal., 1973).More recentreportsdemonstrated alsothatcertain species
lackahumoralresponsebelow9-12C:IntheJapaneseeel anti-Vibrio
anguillarum
titreswereobtainedmorerapidlyathighertemperatureswithinthe15-23Crange.
Noantibodyproductionwasdetectedat11°C(Muroga&Egusa, 1969). Carpkeptat
12Cdidnotproduce antibodies againstBSA(Avtalion, 1969a).
Veryinterestingstudieshavebeenperformedontheimmuneresponsivenessof
cold-water fish.Sablefish,ahabituantoftheNorthPacific,produced agglutinating
antibodiesattemperaturesof5-8Cwithin1month afterinjectionofforeignerythrocytes (Ridgway,Hodgins&Klontz, 1966).IntheAntarctic teleost,theicefish,
keptat2°Cneutralizing antibody couldbedetected42-56daysafter immunization
withMS2bacteriophage (O'Neill,1979,1980).Itthereforeismoreappropriateto
statethatthetemperature range overwhich antibodyproductioncantakeplaceis
relatedtothenormalenvironmental temperature rangeofthespecies considered.
Themostextensive studiesontheeffectoftemperatureonhumoral immunity
havebeenperformedbyAvtalionandco-workers.Carpkeptat25°C (high temperature)
produceagglutinatingantibodies againstBSAafteralagperiodof10days.Whenkept
at12-14C (lowtemperature)noantibody couldbedetectedupto77daysafterimmunization.Whencarp,immunizedandsubsequently keptat14°Cfor35days,were
62
transferredto25°C,antibodiesweredetected 7days later.Animals immunizedand
kept at25°Cfor8daysbeforetransferto 14Cproduceantibodies atthis lowtemperature (Avtalion, 1969a). Similarresultshavebeenobtainedwhenusing Aeromonas
punctata asimmunizingagent (Avtalion,Wojdani,Malik &Shahrabani, 1973). Later
studies showedthatatemperature sensitiveeventduringtheresponse,was situated
betweenthe 3rdand4thdayafterprimary stimulation.Thiswas illustratedbythe
factthatonlycarpkeptathightemperatures formorethan3days after immunization
produce antibodies inthecold (Avtalion,Weiss &Moalem, 1976). Carpwereableto
mountasecondary responseagainstBSAatlowtemperaturesprovidedthatpriming
tookplaceat2S°C (Avtalion,1969b,1969c;Avatalion,Malik,Lefler &Katz, 1970).
Animalswereprimedwiththe0-antigenof Salmonella abortus (OSA)athightemperaturesandtransferredto 12°Cbefore theappearanceofcirculating antibody.After
a simultaneous immunizationwithOSAandBSA,onlyantibodies againstthebacterial
antigenwereproduced.Thus,memory formationat lowtemperatureswas immunologically
specific (Avtalionetal., 1973).
Threedifferentapproacheshavebeenusedtodemonstrate thatthe functionof
helpercellswasprimarily affectedby lowtemperatures.
a)Carp,whichwereinjectedwithrabbitgammaglobulinasacarrier,before
theirtransfer tolowtemperatures,were abletoproduce anti-hapten (penicillin)antibodies following immunizationwith thehapten-carrier conjugate inthe
cold (Avtalionet al., 1976).
b)AcetylatedBSA (AcBSA)doesnotevokeantibodyproductionincarp.AninjectionwithnativeBSA,given40daysafterAcBSApriming,givesrisetoa
secondarytypeanti-BSAresponse.AcBSAhas lost itsabilitytostimulateantibodysynthesisbutretained itspotentialmemory formation (Weiss &Avtalion,
1977).Theanti-BSAresponse in Tilapia (hybridof T. aureus x T.
nilotioa)
keptatlowtemperatureswasofasecondary typewhenprimingwithAcBSA
occurredathightemperatures (Avtalion,Wishkovsky &Katz, 1980).
c)Penicillinconjugated toBSAat lowepitopedensity (PenrBSA)stimulatecarp
forbothanti-haptenandanti-carrierantibodies (Avtalion &Milgrom, 1976).
PreimmunizationwithBSAenhancedtheprimaryanti-haptenresponse atlowtemperatures (Weiss&Avtalion, 1977).
Theoptimaltemperature forLPSstimulationofbluegillpronephros lymphocytes
was 22C,while responses toPHAandConAwereoptimalat32°C (Cuchens &Clem,
1977).TheseresultsareconsistentwithAvtalion'shypothesis thatT-likecells
requireahighertemperature thanB-likecells (Avtalion,Weiss,Moalem&Milgrom,
1975).However,studies inrainbowtroutrevealed thattheoptimal temperature for
stimulationby PHA,ConA andLPSwasalways 28C (Chilmonczyk, 1978a).
Incarpkeptat25°Cboth lowdose (0.01-0.1 mg/kg)andhighdose (10-50mg/kg)
tolerancetoBSAcouldbe induced (Serero&Avtalion, 1978).At lowtemperatures,
onlyhighdosetolerance couldbe induced (Avtalionetal., 1980).
63
OnbaseofthedatadiscussedaboveAvatalionet al. (1973)proposedamodel
fortemperaturesensitivestagesduringthehumoral immuneresponse.Following
primaryantigenic stimulation,theinitial stepsoftheresponsesuchasphagocytosis, primingandtolerance inductionarerelativetemperature insensitive.Thefirst
temperaturesensitiveeventmightbecellularinteractionand/ordifferentiation.
Cellularmultiplicationofmemorycells andantibodyformingcellsaretemperature
insensitive inthismodel.However,theeffectorphaseofthehumoralresponse (antibodysynthesisandrelease)isagainarelativetemperaturesensitiveevent.
• Bothcellularandhumoral immuneresponses offisharetemperaturedependent.
Immuneresponsescantakeplacewithinthenormal temperature rangeofthe
species.Therearestepsintheimmuneresponsedifferingintemperaturesensitivity.
Fromthepracticalpointofviewthetemperaturedependence oftheimmuneresponseisimportantbecause ithasbeenshownthatseasonalvariations intheincidenceofinfectiousdiseases infisharecorrelatedwithchangesinwatertemperature (Besse,Levaditi,Guillon&deKinkelin,1965;Schaperclaus,1965;Roberts,
1975). Theoptimaltemperatures forgrowthandreplicationofinfectiousmicroorganismsvaries (Reichenbach-Klinke, 1976). Forinstancethegrowthrateof Aeromonas salmoniaida and A . hydrophila (causative agentoffurunculosis)was optimal
atwatertemperaturesofabout 20°C (Groberg,McCoy,Pilcher &Fryer, 1978).Maximum
mortalityinsalmonidsinfectedwiththecausativeagentofbacterialdisease,
Corynebaeterium spp.occursintherangeof7-12°C (Sanders,Pilcher&Fryer,1978).
Theoptimal temperature forthe -in vitro replicationofpathogenic rhabdoviruses
specific forsalmonids is 14°C,forrelatedvirusesspecific forwarmwaterfishes
itis20-22°C (Ahne, 1978).
9 Fromtheimmunologicalpointofview,animalsinlargescale fishcultureshould
bekeptattheiroptimal temperature.Loweringthetemperaturewilldiminish
orinhibittheimmune capacityoffishwhile thecircumstancesmightbecome
favourable forcoldadaptedpathogenicmicro-organisms atthesametime.
Stress
The importanceofsocialfactorsinfishpopulationshasbeenunderestimated
inthe firstreportsonfishimmunology.ThenegativeresultsofPapermasteret al.
(1962)andGood&Papermaster (1964)inobtainingimmune responsesinAgnatha
wereprobablycausedbypooranimalhusbandrysince laterinvestigators (Thoenes&
Hildemann, 1969;Linthicium&Hildemann,1970)succeededindemonstrating antibody
production.
Fishundercrowdedconditionsproducepheromone-likecrowdingfactors (Pfeiffer,
1974).Thesecrowdingfactors inhibitgrowthandreproductionanddepresstheheart
contractionrate (Pfuderer,Williams &Francis, 1974).Moreover,theimmuneresponse
ofcrowdedblue gouramitoinfectiouspancreaticnecrosisviruswasdepressed.It
64
wasdemonstratedthatpheromone-likefactors,releasedincrowded fishpopulations,
were reponsiblefortheobservedimmunosuppression (Perlmutter,Sarot,Yu,Filazzola
& Seeley, 1973). Carpkeptundercrowdedconditionsshowedarelativelyhighsusceptibilitytoexperimentalinduced Aeromonas infections (Avtalionetal., 1973). Miller
& Tripp (1979)studiedtheeffectofcaptivityontheimmune responseofthekillifish.Fishmaintainedatthelaboratoryfor1-2monthsshowedaloweranti-SRBC
responsethanfreshlycapturedspecimens,whilenoeffectonallograftrejection
wasobserved.Thesuppressive factor-specificforhumoral immuneresponses-was
presentinserumsinceitcouldbetransferredfromlaboratory animalstofreshly
captured fish.Atpresentitisclearthatphysiologicalandmorphologicaleffects
ofstressinfisharetoalargeextenthomologouswith thoseinmammals (Mazeaud,
Mazeaud&Donaldson,1977;Peters, 1979).
#Stresscancause immunosuppression.Thisshouldbekeptinmind,notonlywhen
studyingimmuneresponsesunderlaboratoryconditions,butalsowhendevising
rearingsystemsforlargescale fishculture.
Antibiotics
and
antimetabolites
Antibioticsarefrequentlyusedinanimalhusbandryforthepreventionand
controlofinfectiousdiseases.Dataontheimmunosuppressiveeffectofantibiotics
inmammalsaregiveninappendicesVandVI.Reportsontheeffectsofantibioticsin
fishdealwithcellular immunity.Inkillifishtheantibioticschloramphenicol, tetracycline,puromycinandcycloheximideprolongedtheMSTofallografts (Goss, 1961;
Cooper, 1964). Furthermore,corticosteroids,nucleicacidandaminoacidanalogues
andfolicacidanaloguesalsoprolongedallograftsurvival (Goss,1961;Hildemann&
Cooper, 1963). Similarobservationshavebeenmadeingoldfish (Levy,1963;Stutzman,
1967).
• Thelimitedexperimental dataavailable todaysuggestthatantibioticscan
exertimmunosuppressiveeffectsinfish.
Environmental pollution
and
irradiation
Althoughimmunosuppressive effectsofenvironmentalpollutantsinmammalsand
birdsarewelldocumented (Vos,1977;Vos,Faith&Luster,1980)littleisknown
abouteffectsofthesecompoundsinfish.IthasbeenobservedthatDDTcansuppress
bothhumoralandcellularimmune responsesingoldfish (Zeeman&Brindley,197S, 1976,
1979)whenusedindoseswhichareimmunosuppressiveinmammalsandbirds (Vos, 1977).
Amarkedreductioninthenumberoflymphocytesinthespleenofguppyandbrown
troutwasobservedafterexposuretoDDT(Walsh&Ribelin,1975). Zincappearedto
suppresstheimmuneresponseofzebrafishagainst Proteus
vulgaris
butnotagainst
infectiouspancreaticnecrosisvirus (Sarot&Perlmutter, 1976).
Fewreportsexistdealingwitheffectsofradiationontheimmunesystemof
fish.Severeatrophyinkidney,spleenandthymuswasfoundinchannelcatfish
65
A careful use of t h i s drug i s recommended since oxyTC can exert immunosuppressive
effects during a primary response.
78
SAMENVATTING
Dit proefschrift behandelt enkele aspecten van het c e l l u l a i r e en humoral immuunsysteem van karperachtige vissen.
In appendix I wordt de ontwikkeling van het c e l l u l a i r e en humorale immuunsysteem
b i j de prachtbarbeel (Barbus aonohonius) beschreven. In 3-4 maanden oude dieren
kon een humorale anti-schape rode bloedcellen (SRBC) reactie worden aangetoond, maar
de hoogte van de r e a c t i e was lager dan in 9 maanden oude volwassen dieren (90 en
700 plaque vormende cellen(PVC)/10 w i t t e cellen, r e s p e c t i e v e l i j k ) .
Cellulaire immuniteit werd onderzocht met de schubtransplantatie techniek. Tussen
6 en 9 maanden oude dieren bestond geen signifikant verschil in de gemiddelde afs t o t i n g s t i j d van allogeen getransplanteerde schubben (respectievelijk 8 en 8.3
dagen).
Uit deze experimenten kan de conclusie worden getrokken dat b i j 3 t o t 4 maanden
oude dieren het vermogen om op SRBC te reageren aanwezig i s , maar dat het humorale
immuunsysteem pas 5-6 maanden l a t e r volgroeid i s . Cellulaire immuniteit b e r e i k t het
volwassen niveau na 6 maanden of eerder.
In het vervolg van het onderzoek werd om een aantal redenen de karper (Cyprinus
aarpio') gekozen als proefdier. Allereerst moest de plaque t e s t van Jerne, waarmee
het aantal antilichaam-producerende cellen kan worden bepaald, worden aangepast
voor het werk met de karper (appendix I I ) . Het bleek dat serum van de brasem
(Abramis brama) een betrouwbaarder complement bron vormde dan allogeen karper serum.
Gebruik makend van de plaque t e s t werd de kinetiek van de anti-SRBC respons onderzocht (appendix I I I ) . Het bleek dat b i j de karper de n i e r (pro- en mesonephros) een
belangrijk antilichaam-producerend orgaan i s . De milt droeg slechts voor 5% aan het
t o t a l e aantal PVC b i j . Wanneer karpers b i j hoge temperaturen gehouden werden (2024 C) vertoonden zij een k a r a k t e r i s t i e k e primaire en secundaire respons. Bij lagere
temperaturen (10-18 C) wordt de piek van de primaire PVC respons l a t e r b e r e i k t ,
maar de hoogte van de respons b l i j f t g e l i j k . De r e l a t i e tussen de temperatuur en
het t i j d s t i p waarop de piek-respons bereikt wordt, suggereert dat e r b i j de karper
tenminste 2 stappen in de immuunrespons zijn met een verschillende temperatuurgevoeligheid. De k a r a k t e r i s t i e k e eigenschappen van een secundaire respons verdwijnen
geleidelijk b i j lagere temperaturen. De vorming van immunologisch geheugen zou
daarom ook temperatuur afhankelijk kunnen zijn. De invloed van antigeen dosering en
de route van antigeen toediening op het ontstaan van immunologisch geheugen werd
onderzocht in appendix IV. Immunisatie met een lage antigeen dosering toegediend
langs de intramusculaire weg was optimaal voor de vorming van immunologisch geheugen.
Dit vermogen om een secundaire respons op te wekken was specifiek voor het antigeen
dat gebruikt werd b i j de primaire immunisatie, en bleef gedurende ten minste 10
maanden op een hoog niveau.
In appendix V en VI werd het effect van het antibioticum Oxytetracycline (oxyTC)
79
op het immuunsysteem van de karper onderzocht. Het voeren van oxyTC had geen
effect op de a f s t o t i n g van allogeen getransplanteerde schubben,, maar in dieren die
met oxyTC werden ingespoten was de a f s t o t i n g s t i j d signifikant verlengd van 8.5 naar
11-20 dagen. Het remmend effect van oxyTC op de humorale immuun respons kon worden
aangetoond op het niveau van antigeen bindende én antilichaamproducerende cellen.
Gedurende een primaire respons werden de PVC aantallen teruggebracht t o t 5% van
normaal. Secundaire responsen werden n i e t signifikant geremd door oxyTC. Een zeer
s e l e c t i e f gebruik van dit antibioticum wordt aanbevolen omdat het onder bepaalde
omstandigheden immunosuppressief kan werken.
80
CURRICULUM VITAE
Ger Rijkers werd op 13 oktober 1952 als Gerrit Tjalling Rijkers te Zeelst (N.Br.)
geboren. Het voorbereidend wetenschappelijk onderwijs werd gevolgd aan het Peelland
College (voorheen Pius XII College) te Deurne alwaar in 19 71 het diploma Atheneum B
werd behaald. In dat zelfde j a a r werd de studie biologie aangevangen, de p l a a t s i n g s commissie bepaalde dat d i t aan de Landbouwhogeschool te Wageningen zou z i j n . Het
ingenieursexamen in de (cel)biologie werd in a p r i l 1977 afgelegd, met moleculaire
biologie en celbiologie als hoofdvakken en plantkunde a l s bijvak.
Op 11 j u l i 1977 werd een begin gemaakt met een promotieonderzoek aan de afdeling
Experimentele Diermorfologie & Celbiologie onder (bege)leiding van Prof. Dr. J . F .
Jongkind en Dr. W.B. van Muiswinkel. Dit onderzoek werd mogelijk gemaakt door een
3-jarig promotie-assistentschap van de Landbouwhogeschool. Het betreffende project
(000.772) had als t i t e l : "De bouw en functie van het immuunsysteem b i j de karper
(Cyprinus ecœpio)". In de week van 11 j u l i 1980 werd het manuscript van d i t proefs c h r i f t b i j de drukker ingeleverd.
Vanaf half augustus 1980 i s h i j werkzaam in de klinische immunologie aan het
Wilhelmina Kinderziekenhuis b i j Dr. B.J.M. Zegers te Utrecht.
81
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101
APPENDIX I
103
Developmental Irwninobioloyy,
© 197? Klseviei'/'.lorth-üGllarid
J.H. Solor.on andJ.D. Horton eds,
Fiomediaal Preoa, Amsterdam.
THE IMMUNESYSTEMOFCYPRINID FISH
THEDEVELOPMENTOFCELLULARANDHUMORAL RESPONSIVENESS
INTHEROSYBARB (BARBUS CONCHONIUS)
G.T.RijkersandW.B.vanMuiswinkel
DepartmentofExperimental AnimalMorphology andCell Biology,
Agricultural University,Wageningen,TheNetherlands
INTRODUCTION
Thebasicpropertiesofmammalian lymphocytes-therecognitionofnonselfand
thesubsequentdifferentiationandproliferation-appeartobecommontolymphocytes fromallvertebrates.Althoughanumberofinvestigations concerningphylo1 2
genetic aspectsoftheimmuneresponseareknown ' comparatively little informationisavailable aboutstructureoflymphoid organsandfunctionoflymphocyte
populations inPoikilothermievertebrates.Mostobservations inthisrespecthave
34
beendoneonAmphibia ' .Somedataaboutthekineticsoftheimmuneresponsein
fishhavebeenpublished ' .Thereareindications thatco-operationbetween2
populationsoflymphoid cellsisneededduringthehumoral immuneresponsein
7 8
9
fish ' .Rejectionofallograftsisacommon featureinectothermicvertebrates.
Inteleost fishanacuterejectionisobservedwithin2weeks after grafting.The
immunological natureoftherejectionisdemonstratedbyasecond-set rejection
timeof4-6days
Information aboutthedevelopmentoftheimmune systeminfishislackingor
11 12
very scarce ' .Thepresentstudywasperformedtoaddsome informationabout
thefunctional developmentofthecellularandhumoral immunesysteminCyprinid
fish.
MATERIALSANDMETHODS
Animals:Rosybarbs (Barbuscohchonius)ofbothsexeswereusedatanagebetween3and30months (table I).Theanimalswerebredatthelaboratoryandkept
at 24°Cinfullglassaquaria.Daily feedingwasperformedwithTrouvitpellets
(Trouw&Co,TheNetherlands)ortubifexworms.Theywere acclimatized totheexperimental aquariaforatleast1weekbefore injectionorgrafting.
Antigen:Sheepredblood cells (SRBC)wereobtained fromtheDepartmentofCell
BiologyandGenetics,Erasmus University,Rotterdam.Forimmunizationtheywere
washed3timeswithphosphatebuffered saline (PBS)andadjustedtoaconcentrationof2x10/ml.
Q
Immunization:Allanimalswere immunizedwith 10 SRBCin5ulPBSusinga50ul
Hamiltonsyringe.Inpreliminary experiments themosteffective immunizationturned
outtobedorsal,intramuscular injection.
105
TABLEI
SomecharacteristicsofBarbus conchonius
Age inmonths Numberof Standard length* Weight
animals
(cm+s.e.)
(g +_ s.e.)
Number ofwhite spleen
cells (x 10 +_s.e.)
3
14
1 .45 + 0.06
0 . 0 8 5 + 0.01
4
28
3.27 + 0 . 0 5
1.13
+ 0.07
1.4+0.1
2.41
+0.14
2.8 + 0.3
+0.3
1 1 . 2 + 0.2
9
24
4.45 + 0.08
30
3
6.46 + 0 . 0 3
11.1
1.0 + 0 . 2 * *
* Standard length is total lengthwithout tail.
**Determined in3specimen.
Cellsuspensionsandbleeding:AnaesthesiawasperformedbymeansofMS-222
(tricainemethanesulfonate,Sandoz Ine,150mg/1)inaquariumwater.Fishwerebled
by cuttingthetail.Theamountofblood obtained fromoneanimal ranged from1pi
(4months old)to20ul (9months old).Subsequently thespleenwas removedand
brought intocoldHank'sbalanced saltsolution (HBSS,Difco,Detroit) supplementedwith 5%newborncalfserum.Thespleenwasmincedwith scissorsandsqueezed
throughanylon-gauze filtertogiveasinglecellsuspension.Serawere storedat
-90°C.
Complement:Apreliminary experimentwas carriedouttoselectasuitable complementdonor.Weprepared fresh complement fromseveralBarbus species (B.conchonius,B.lateristriga,B.filamentosusandB.nigrofasciatus),grass carpand
rainbow trout.Theisologous complementofB.conchonius turnedouttocontaina
high levelofhaemolyticfactorstolyseSRBCinthepresenceofB.conchoniusantibodies.Thecomplementwasabsorbedfor15minutesat0°Cwithpacked SRBC.Aftercentrifugationthesupernatantwasabsorbedforanother10minutesat20°C.Aftercentrifugationthesupernatantwascollectedandstoredat-90°C.
Serology:Haemolysing(HL)andhaemagglutinating(HA)antibodiesweredetectedin
seraofindividual animalsusing standard techniques .Theserawere dilutedwith
PBStoyieldastartingvolumeof30ul.Microtitreplates were incubatedfor2-3
hoursat24°C.
Plaque forming cell assay:Todetectthepresenceofantibody formingcellsthe
1—
—
e
14
plaque—techniqueofJerneinanagar-freemediumwasused .Themonolayer plaque
assayslideswereproduced according tothetechniqueofMajooret al. . A mixQ
tureof0,1mlcellsuspension,0,1mlSRBC (5x10/ml)and10ulcomplementwas
preparedandincubatedasamonolayerbetweentwoglass slides.After anincubation
of2hoursat24°Ctheslideswerescoredforthenumberofhaemolyticplaques
106
usingadissectionmicroscope.Partofthespleencellsuspensionwasusedfor
white cellcounting.Tolysetheerythrocytes thesuspensionwas incubated for
2 x 1 5 minutes in0,7S?„NH4C1 in0,17M tris-HClpH 7,2 16 .
Scaletransplantation:Forscale transplantationamodificationoftheHilde17
wasused.Repeated anaesthesiawithMS-222 (150mg/1)causeda
manntechnique
considerable lossofanimals.Forgraftingandsubsequentdaily inspectionwe
placed thefishinapetridish (Fig.1 ) .Thebottomplateofthedishisfilled
with cottonwool,drained inaquariumwater.The fish isplaced onthecottonwool
and thelidisclosed immediately. Inadditiontooneautograft,asacontrol,
threetosixallograftsperrecipientaremade.Grafting isdoneintherowof
scalesjustabovethelateral line.Daily inspectionofthescaleswascarriedout
using alow-powermicroscope.
Fig. 1.A simpledevice for scale transplantation infish.
A)Hole tosupply aquariumwater.
B)Ovalhole enabling scale transplantation.
RESULTS
PFC-response tosheeperythrocytes:A groupof64male,9monthsoldanimals
were immunizedwithSRBC/Onday 2,4,6, 7,8,9, 11,14and 17afterimmunization
4 animalswerekilled fordetectionofPFC inthespleen.Aplaquewith anantibodyproducing cell inthecenter isshowninfig.2.Thefirstplaques appeared on
day4afterimmunization (Fig.3 ) .Thereafter thenumberofplaques increasedand
reachedamaximumonday 7 (700PFC/10 whitespleencells (WSCJ). Inthesubsequentperiod thenumberofplaquesdecreased toless than10PFC/10 WSC.Twenty
eightdays aftertheprimary injection 18animals received asecondary injection
withSRBC.Groupsof3animalswerekilledonday3,5,8and 16after theantigen
injectiontodetermine thenumberofPFC's inthespleen.Thespleens of6animals
werepooled andtestedonday6.Thefirstplaquesappeared after3days,thepeak
responseoccuredonthefifthday (4200PFC/10 WSC).Qnday 16,thelastday tested,therewere 11PFC/10 WSC.A groupof 28,4monthsoldanimalswere immunized
withSRBC.Onday 2,4,6, 8,9, 10and 13after theinjectionoftheantigen4
107
MB°¥^0uà* * * $ & .
h
n
St P
UqUe
Pr dUCed
a l a u ef
Ip?;en'cen
?PFCr
Iro,
H ^-0miCr0
?r!Ph° f*Pcell
^ sheep
P 1 °™ing
spieencell (PFC)
Around
thisantibody
producing
an'area°oflysed
erythrocytes rsvisible.Fish erythrocytes (FE)andthrombocytes (T)areaîso
10240 .
d
5120 . Ö
2560.
O 9MONTHS OLDPRIMARY
RESPONSE
5
UJ
• 4 MONTHS OLD PRIMARY
RESPONSE
si
1280 _ m
640 . o
320 .
I
*
/
a.
/
160 .
80 .
40.
20 .
10.
1
™
1
108
3
•
•
5
7
1
T
1
1
1
9
11 13 15 17
DAVYS AFTER I NUECTION
Fig.3.Primary immuneresponse
ofBarbus conchonius toSRBC.
Eachpoint representsthemean
value from4animals+s.e..
TABLEII
Immuneresponse ofBarbus conchonius toSRBC
Age inmonths
PFC/10 WSC
HA-titre
3
30+ 15
4
91 + 43
80+ 53
9
699 +434
987 +683
-
Plaque forming cells (PFC)weredetected,7-8 days after injectionof theantigen,
thehaemagglutination (HA)titrewas determined onday 13-14.Thedatarepresent
themeanvaluefrom 3-4 animals +standard error.
animalswere testedforthepresenceofPFCinthespleen.Thefirstplaquesappearedonday8(91PFC/106WSC,seefig.3 ) .Day8alsoturnedouttobethepeak
dayofthePFC-response.Duringthesubsequentperiodthenumberofplaquesdecreasedanddroppedunder10PFC/10 WSConday13.Additionallyagroupof3animals (3months old)were testedforthepresenceofPFCinthespleen7daysafter
immunizationwithSRBC.Atthat time30PFC/106WSCwere detected (tableII). The
animalswerehomogenized inHBSSandcentrifuged afterremovalofthespleen.The
HA-titreinthesupernatantofthehomogenate turnedouttobe1:58+21.Comparinganimalswiththeageof4months (young)and9months (old)weseethatthe
PFC-peakresponseisreached
aboutthesametimeafter immunization (fig. 3 ) .
However,thenumberofPFC/10 WSCorPFC/spleenishigherinoldanimals.Furthermore,thetime intervalinwhichPFC'scanbedetectedisshorterforyoung animals. Itisworthwhiletomentionthatsomeyounganimalsdidnotrespondatall,
evenatthepeakday.AsvisualizedintableI,3monthsoldanimalshavearelativelyhighnumberofwhite spleencells.TheirpeakPFCresponsewaslowerthanin
4monthsoldanimals.
Antibody responsetoSRBC:Seraofimmunizedanimalswereanalysedforthepresenceofagglutinatingandlyticantibodies.HAandHL-titresin9monthsoldanimals reachedamaximum (1:987and1:40respectively)onday14oftheprimary response (fig.4b).TheHA-titresurpassedthedetectionlimit (dilution1:20)for
thefirsttimeonday11.TheHL-titrewasalways lowerthantheHA-titre.FollowingasecondaryinjectionofSRBC,28daysafterprimary immunization,HAantibodiesweredetectableforthefirsttimeonday3andreachedamaximumonday5
(1:2229).ElevendaysafterthepeaktheHA-titrestillwas1:80.HLantibodies
appearedonday5andamaximumwasreachedonday8 (1:213).HAandHLantibody
responsesin4monthsoldfishwereverylow(fig.4a).ThehighestvalueforHA
antibodieswasobservedonday13(1:80),thelasttestday.HLantibodies first
appearedonday9,amaximumwasreachedonday10 (1:35).
Scale transplantation:Seven9monthsoldanimalsandsix6monthsoldanimals
109
b)PRIMARYRESPONSE
9MONTHSOLD
a)PRIMARY RESPONSE _,
4 MONTHS OLD
2048
11 13 15 17 19 21
DAYSAFTERINJECTION
10 12 14 16
Fig.4.Primary antibody responseofBarbus conchoniustoSRBC.Eachpointrepresentsthemeanvalueof4animals +_s.e..
wereusedforscale transplantation.After transplantation thefollowing stagesof
rejectionoftheallografts were distinguished:
a)scalesbecome overgrownbyhyperplastic host-tissue.This resultsinawhite
appearance.
b) orangepigment cells turn intored.
c)blackpigment cellsarebranching.
d)clearanceofthehyperplastic host-tissue.
Thebeginningoftheclearingphenomenonisconsidered tobethesurvival endpoint
17
(S.E.P.)ofthegraft .Themediansurvival time (M.S.T.)wascalculated fromthe
S.E.P.'s.Autografts survivedwellinallcases.TheMSTofBarbus conchonius(8,0
days) fallswithintherangeofMST'sreportedforothercyprinid fish.Barbus filamentosushasanMSTof8,6days,Carrasius auratusof7,2days,bothat25°C .
TheMST'sof6and9monthsoldanimals showednosignificantdifference (table
III).
TABLE III
Allogeneic scale graft rejectioninBarbus conchonius
Age inmonths
110
Numberofgrafts
per experiment
Temperature
(°C)
MedianSurvivalTime
(days+ s.e.)
6
17
24
8,3 + 0,2
9
6
24
8,0 + 0,5
DISCUSSION
Thepresent studydescribes thefunctionalmaturationoftheimmunesystem in
Barbus conchonius.Thehumoral immunesystemwas studiedby injecting the animals
with SRBC.Thepeak responsewas determined by counting thenumber ofPFC/10 WSC
andmeasuring HAandHL-titres inserum. Itisknown thatthe immunocompetent
cells inteleost fishare localized notonly inthespleen,but also inthe thymus
18
andhead-kidney .Therefore theHAandHL titrationdataprovide abetterestimationof the immunecapacity of theanimal than thenumber ofPFC/10 WSC.
19
20
Smithetal. andChiller reported that infish isologous complement orcomplement fromcloselyrelated species isaprerequisite forhaemolysing sensitized
SRBC.Ourresultswith isologous complement ofBarbus conchonius confirm theirobservations,however,closely related species asB.filamentosus,B. nigrofasciatus
andB. lateristriga failed as asuitable complement donor. Inour experiments serumofSalmogairdneriwas asactive asB.conchonius complement.This isastriking observationbecause these 2species arenotrelated.
Theprimary response showed amaximum of 700PFC/10 WSC onday 7afterinjectionofSRBC.This figure iscomparablewith other teleost fishasTilapiamossambica (287 PFC) 6 , Salmo gairdneri (165PFC) 2 0 andPerca fluviatilis (848PFC) 5 . A
second injectionwith thesameantigen elicited ahigher PFCresponse (6-foldincrease)atday 5.This illustrates the immunenatureofthePFCresponse.Young
animals (4months old)showed a lowerPFCresponse (90PFC/10 WSC)andHAand
HL-titres. Itisconcluded that 4months oldanimalshave the competence torespond toSRBCbuttheirhumoral immunesystem isstillnot full-grown.Plaqueswere
detected inthespleenof 3months old animals 7days after injectionof theantigen. However,thenumber ofplaqueswasvery low (0-6plaques/spleen).
Forstudying cellular immunitywemodified thescale transplantation technique
17
ofHildemann .No significant differencebetween theM.S.T.'s ofadult (9months)
andyoung-adult (6months)animalswas detected (table III).Takingallograftrejectionasaprobe forcellular immunity itisconcluded thatthecellular immune
system inB.conchonius reached theadultstageat6monthsorearlier. Inviviparous Cymatogaster,newborn fish-with anage of 5months -have anMSTof 13days,
11
whereas theMST foradult animals is 7days .Thedifference inthematuration of
cellular responsiveness betweenCymatogaster andBarbusmightbeexplained by a
difference inambient temperature (17°Cand 24°Crespectively).
Studies inmice indicate thatthematurationofhumoral responsiveness takesat
least 2months (ref. 21,22andownobservations).Therefore itistempting tosay
thatthedevelopment of the immuneresponsiveness incyprinid fish iscomparable
withthesameprocess inmammals taking intoaccount thetemperature difference
betweencold andwarmblooded vertebrates.
111
ACKNOWLEDGMENTS
Weare grateful toDrs.L.P.M.Timmermans,L.H.P.M.Rademakers andE.Egberts
for reading themanuscript, toMr.W.J.A.Valenfordrawing thefigures andto
Mrs.A.E.M.Tapilaha-Wattilete for typing.
REFERENCES
1. Good,R.A.andPapermaster,B.W. (1964)Adv. Immunol.4,1-115.
2. Cooper,E.L. (1973)inContemporary Topics inImmunobiologyVol.2(Davies,
A.J.S. and Carter,R.L.,eds.)pp. 13-38,PlenumPress,NewYork.
3. Manning,M.J.andHorton,J.D. (1969)J.Embryol.Exp.Morph.22,265-277.
4. Turpen,J_B.andVolpe,E.P. (1975)Amer.Zool. 15,51-61.
5. Pontius,H. andAmbrosius,H. (1972)ActaBiol.Med.Germ. 29,319-339.
6. Sailendri,K.-andMuthukkaruppan,Vr. (1975)J. Exp.Zool. 191,371-382.
7. Siu-Stolen,J. andMäkela,0. (1975)Nature 254,718-719.
8. Yocum,D., Cuchens,M. and Clem,L.W. (1975)J. Immunol. 114,925-927.
9. Hildemann,W.H.and Cooper,E.L. (1963)Fred.Proc. 22,1145-1151.
10. Hildemann,W.H.andHaas,R. (1960)J. Cell.Comp.Phys.55,227-233
11. Triplett,E.L.and Barrymore,S. (1960)Biol.Bull. 118,463-471.
12. Kallman,K.D. (1970)Transpl.Proc.2,263-271.
13. Hudson,L.andHay,F.C. (1976)inPractical Immunology pp. 125-137,Blackwell ScientificPublications,Oxford.
14. Zaalberg,O.B.,vanderMeul,V.A.andvanTwisk,M.J. (1968)J.Immunol.
100,451-458.
15. Majoor,G.B.van 'tVeen,M.B,and Zaalberg,O.B. (1975)J. Immunol.Methods
7,301-304.
16. Boyle,W. (1968)Transplantation 6,761-764.
17. Hildemann,W.H. (1957)Ann.NYAc.Sciences 64,775-791.
18. Corbel.M.J. (1975)J.FishBiol.7,539-563.
19. Chiller,J.M., Hodgins,H.O.,Chambers,V.C.andWeiser,R.S. (1969)
J. Immunol. 102,1202-1207.
20. Smith,A.M.,Potter,M. andMerchant,E.B. (1967)J. Immunol.99,876-882.
21. Shalaby,M.R.andAuerbach,R. (1973)Differentiation 1,167-171.
22. Haaijman,J.J., Schuit,H.R.E.and Hijmans,W. (1977)Immunology 32,427-434.
112
APPENDIX II
113
Journal of Immunological Methods, 33 (1980) 79—86
© Elsevier/North-Holland Biomedical Press
THE HAEMOLYTIC PLAQUE ASSAY IN CARP (CYPRINUS
CARPIO)
G.T. RIJKERS, E.M.H. FREDERIX-WOLTERS and W.B. VAN MUISWINKEL
Department of Experimental Animal Morphology and Cell Biology, Agricultural
University, Zodiac, Marijkeweg 40, 6709 PG Wageningen, The Netherlands
(Received 31 July 1979, accepted 21 October 1979)
A haemolytic plaque assay for the enumeration of antibody forming cells in the carp
(Cyprinus carpio) is described. Serum of bream (Abramis brama) turned out to be a more
reliable complement source than allogeneic carp serum. The addition of 3—5% bream
serum to a mixture of immune lymphoid carp cells and xenogeneic erythrocytes gave
optimal results. Higher amounts of bream complement inhibited plaque formation.
Plaque formation was also suppressed when inactive or heat-inactivated bream or carp
serum was added to a mixture containing normal bream complement.
INTRODUCTION
For many years the plaque assay of Jerne and Nordin has been a useful
tool for in vitro quantitation of antibody secreting cells or plaque forming
cells (PFC). Although it was originally developed to detect cells which
secrete antibodies against cellular surface antigens of erythrocytes, it has also
been used to detect PFC against soluble antigens by coating indicator erythrocytes with proteins, polysaccharides or haptens (Jerne et al., 1974).
Recently the technique has been adapted for use with cells from vertebrate classes other than mammals such as amphibians (Horton et al., 1976),
reptiles (Rothe and Ambrosius, 1968; Kanakimba and Muthukkaruppan,
1972) and birds (Jankovic et al., 1972).
The haemolytic plaque assay has been used in the following fish species:
blue gill, Lepomis macrochirus (Smith et al., 1967), rainbow trout, Salmo
gairdneri (Chiller et al., 1969), perch, Perca fluviatilis (Pontius and Ambrosius, 1972), Mozambique mouthbrooder, Tilapia mossambica (Sailendri and
Muthukkaruppan, 1975), goldfish, Carassius auratus (Warr et al., 1977) and
the rosy barb,Barbus conchonius (Rijkers and Van Muiswinkel, 1977).
Carp, (Cyprinus carpio) together with Tilapia and a few salmonids constitute the basis for large scale fish culture (Weatherly and Cogger, 1977).
Address for correspondence: Ir. G.T. Rijkers, Department of Experimental Animal
Morphology and Cell Biology, Agricultural University, Zodiac, Marijkeweg 40, 6709 PG
Wageningen, The Netherlands.
115
For economical reasons the study of the immune system of carp under mass
culturing conditions is of great importance (Avtalion et al., 1976; Fiebig
and Ambrosius, 1977; Rijkers et al., 1980). Research has been hampered by
lack of a sensitive method for monitoring the humoral immune response
in carp. In the present study a haemolytic plaque assay in liquid medium is
described.
MATERIALS AND METHODS
Animals
Carp (Cyprinus carpio), ranging from 6 to 18 months of age and 75 to
300 g in weight, were either bred at the laboratory or obtained from the
Organisation for Improvement of Inland Fisheries (Nieuwegein, The Netherlands). They were kept in aquaria with running tap water at temperatures of
18 or 24°C and fed daily with pelleted food (Trouvit K30, Trouw and Co.,
Putten, The Netherlands).
Swiss (Cpb : SE(S)) random-bred female mice, 20 weeks old were used.
They were purchased from the Centre for Laboratory Animals (Wageningen,
The Netherlands).
Sheep red blood cells (SRBC)
Blood was collected from the jugular vein of 6 sheep into heparinised
vacutainers® (B-D). The blood samples were pooled and an equal volume of
Alsever's solution added. The SRBC were stored at 4°C for a maximum
period of 2 weeks. For the plaque assay, SRBC were washed 3 times with
phosphate buffered saline (PBS) and finally diluted in Hank's balanced salt
solution (HBSS, Difco, Detroit, U.S.A.) to a concentration of 5 X10 8
SRBC/ml. HBSS was supplemented with 5% newborn calf serum (NCS) and
antibiotics (40 I.U. mycostatine, 200 I.U. penicillin and 0.2 mg streptomycin
per 100 ml). Carp were injected intramuscularly (i.m.) in the dorsal region
with 10 9 SRBC (0.05 ml of 2 X 10 1 0 SRBC/ml PBS). Mice were injected
intraperitoneally (i.p.) with 5 X 10 8 SRBC.
Complement sources
Pooled sera of pig, goat, cow, sheep, horse and dog were obtained from
the Foundation for Blood Group Studies, Wageningen. The chicken serum
was a gift from the Department of Animal Husbandry, Agricultural University, Wageningen. The amphibian and fish sera and sera or coelomic fluid of
invertebrates (indicated in Table 1) were collected from animals kept in our
laboratory. Bream serum was collected at the Tjeukemeer Field Station of
the Limnological Institute (Oosterzee, The Netherlands). Blood was collected by caudal puncture and allowed to clot for 2 h at 0°C. Sera with a
high natural haemolytic activity against SRBC were absorbed for 20 min at
0°C with an equal volume of packed SRBC. All sera were stored at —20°C.
Inactivation of bream or carp complement was accomplished by incubating
serum for 10—30 min at 42°C.
116
TABLE 1
INFLUENCE OF COMPLEMENT SOURCE ON THE DEVELOPMENT OF PLAQUES
BY IMMUNE CARP CELLS
Complement activity was determined in the haemolytic plaque assay with a head-kidney
suspension of carp immunised with 1 0 9 SRBC.
Complement source
Plaques
Positive/total
Mammals
including rabbit, guinea pig, man,
cow, goat, sheep, horse, dog
—
pooled sera
—
pooled sera
Birds
chicken
Amphibia
axolotl
Fish
carp
bream
chub
nase
barbel
rosy barb
goldfish
grass carp
roach
bleak
tench
rainbow trout
labyrinthic
catfish
perch
Invertebrates
earthworm a
locust a
pond snail a
Ambystoma
mexicanum
Cyprinus carpio
Abramis brama
Leuciscus cephalus
Chondrostoma nasus
Barbus barbus
Barbusconchonius
Carassius auratus
Ctenopharyngodon
idella
Rutilus rutilus
Alburnus alburnus
Tinea tinea
Salmo gairdneri
Clarias lazera
—
+
+
+
+
+
—
—
—
—
—
—
—
—
Perca fluviatilis
—
Lumbricus terrestris
Locusta migratoria
Lymnaea stagnalis
—
—
—
0/3
31/56
55/58
5/8
2/2
2/4
pooled sera
0/7
0/4
0/4
0/3
0/3
pooled sera
0/5
0/3
pooled coelomic fluid
pooled coelomic fluid
pooled haemolymph
Haemolymph or coelomic fluid tested.
Plaque assay
Cell suspensions were prepared as described previously (Rijkers and
Van Muiswinkel, 1977) and suspended in HBSS supplemented with NCS
and antibiotics. The plaque technique in agar-free medium as described by
Zaalberg et al. (1968) was used. Monolayer plaque assay slides were produced according to the technique of Majoor et al. (1975). Briefly, 0.1 ml cell
suspension, 0.1 ml SRBC (5 X10 8 /ml) and complement were mixed and
incubated between two glass slides at 25°C for 2 h. The number of haemolytic plaques was scored using a low-power dissecting microscope with dark117
field illumination. Doubtful plaques were examined under a high-power
phase-contrast microscope.
RESULTS
/
Complement sources
Sera of 28 animal species were tested for their ability to lyse SRBC in
the plaque assay with immune carp cells. Only five complement sources
were capable of lysing sensitised SRBC (Table 1). These were sera from carp,
barbel (Barbus barbus), bream (Abramis brama), chub (Leuciscus cephalus)
and nase (Chondrostoma nasus), all fish belonging to the teleost family of
the Cyprinidae.
Among the sera with complement activity, bream serum was superior even
to allogeneic carp serum. Fifty-five out of 58 individually tested bream sera
were able to lyse sensitised SRBC. A plaque developed with bream serum is
shown in Fig. 1. The highest number of plaques was observed with cells from
the main lymphoid organs of carp (head-kidney, kidney, spleen) when bream
serum was used.
Complement concentration
The optimal complement concentration in the plaque assay of carp was
Fig. 1. A phase-contrast photomicrograph of a plaque forming cell (PFC) from the headkidney of carp. Several carp red blood cells (CRBC) and head-kidney leucocytes (L) are
found within the area of lysed sheep red blood cells.
118
determined by adding increasing amounts of bream serum to a mixture
of SRBC and head-kidney cells of an immunised carp (Fig. 2). Low numbers
of haemolytic plaques were developed when 1% bream serum was added.
Three to 5% serum appeared to be optimal, while higher concentrations
inhibited plaque formation.
In a comparative experiment the complement dependence of SRBC lysis
after sensitisation with mouse antibody was determined using rabbit serum
as complement source. The minimal amount of rabbit complement needed
to develop any plaques was 3% (Fig. 2). Higher plaque numbers were
observed with increasing complement concentration (optimum 10%). Inhibition with super-optimal complement levels as observed in carp was not
found in mice.
200
200
150
100
50
5
10
PERCENTAGE COMPLEMENT
0
5
10
15
PERCENTAGE INACTIVE BREAM SERUM
Fig. 2. Effect of complement concentration on number of plaques. • = Plaques developed
by a head-kidney cell suspension of carp, injected twice with 10 9 sheep red blood cells
(SRBC) i.m. Secondary response, 10 days after injection. Pooled bream serum was used as
complement source. 0 = Direct plaques developed by a spleen cell suspension of mice,
immunised with 5 X1 0 8 SRBC i.p. Primary response, 4 days after injection. Pooled rabbit
serum was used as complement source. Each point represents the arithmetic mean (±1
standard error, n = 4) of the number of plaques in 0.2 ml cell suspension.
Fig. 3 . Inhibition of plaque formation in carp by inactive bream serum. Plaques developed
by a head-kidney cell suspension of carp, injected twice with 1 0 9 sheep red blood cells
(SRBC) i.m. Secondary response, 10 days after injection. Increasing amounts pooled heatinactivated (20 min, 42°C) bream serum (•) or 2 individual inactive normal bream sera
(o, a) were added to a mixture containing lymphoid cells, SRBC and 5% bream serum.
Each point represents the arithmetic mean (±1 standard error, n = 4) of the number of
plaques in 0.2 ml cell suspension.
119
PFC inhibition
In carp, the inhibition of plaque formation was also observed when
normal inactive or heat-inactivated bream serum was added to a mixture
containing optimal bream complement concentration (Fig. 3). Inhibition was
more pronounced when natural inactive bream serum was used. PFC inhibition was also observed when carp serum was used as complement source;
20% inactive or inactivated carp serum added to a mixture containing 5%
active carp serum completely inhibits plaque formation.
In mice, the addition of inactivated rabbit complement did not alter the
number of plaques developed by an optimal complement concentration.
DISCUSSION
In all plaque assays for lower vertebrates, allogeneic complement is used,
with the exception of the clawed toad, Xenopus laevis, where guinea pig
complement is satisfactory (Horton et al., 1976). Conversely, amphibian
sera are able to serve as complement source for SRBC sensitised with rabbitanti-SRBC antibodies (Ruben et al., 1977).
Other claims for the cooperation of lower vertebrate antibodies with
mammalian complement have not been tested in the haemolytic plaque
assay (Inebi and Home, 1979). In the present study in carp, xenogeneic
serum from a related species, the bream, was used as complement source.
Ability to haemolyse sensitised SRBC in vitro was present in 95% of the
bream sera tested, while for carp the figure was only 50%.
Whereas mammalian complement requires a temperature of 56°Cfor heatinactivation, complement of lower vertebrates is more heat labile. For teleost
fishes inactivation temperatures from 42 to 53°Care reported (Feldman and
Miller, 1960; Hodgins et al., 1967; Chiller et al., 1969; Gigli and Austin,
1971). In our experiments sera of bream and carp lost the ability to develop
plaques after incubation at 42°C for 10 and 30 min respectively. Surprisingly, incubation for 60 min at 37°C had no effect upon the resulting number of plaques.
Reduced plaque numbers may be due to failure of antibody secretion by
plasma cells or inhibition of complement-mediated haemolysis. In a tube
haemolysis experiment, with SRBC sensitised with carp-anti-SRBC antibodies, 3—5% bream serum was optimal, while higher concentrations
inhibited haemolysis as measured by 541 nm absorbance. This suggests that
reduced plaque numbers may be caused by inhibition of complement
activity.
In mice, the addition of inactivated serum to a mixture of SRBC, immune
spleen cells and complement had no effect upon the number of plaques. In
frogs, serum complement levels are reduced during hibernation (Green and
Cohen, 1977). However, addition of serum from hibernating frogs had no
inhibitory effect on the high complement titres observed in sera from
non-hibernating animals. Thus, inhibition of plaque formation in carp by
120
inactive serum seems to be a unique finding. With super-optimal complement
concentrations reduced plaque numbers were also observed. Since complement cannot be the limiting factor an inhibiting or "anti-complementary"
factor must be assumed. This assumption is strengthened by the finding
that when the concentration of "anti-complementary" factor israised artificially by addition of inactive or inactivated serum to an optimal amount of
active complement, the same suppression phenomenon is observed. The
nature of this 'anti-complementary' factor is not clear. It may be that
specific inhibitors resembling the C r esterase inhibitor or C 3b -inactivator of
mammals, are present in higher concentrations.
While individual carp sera may be a reasonable complement source in the
haemolytic plaque assay of carp, 4 vol. of inactive carp serum added to
1 vol. active carp serum completely inhibit plaque formation. The development of haemolytic plaques was completely inhibited when equal volumes
of pooled bream complement and naturally inactive bream serum were
mixed. Therefore it is not advisable to pool fish sera as a complement source.
Although complement is critical, the haemolytic plaque assay in carp
presented here offers the possibility of monitoring the immune response in
this species in a sensitive and quantitative way.
ACKNOWLEDGEMENTS
We thank Professor J.F. Jongkind and Dr. J.L. Molenaar (General Hospital, Delft) for advice during the performance of this study and preparation
of the manuscript.
We thank Mr. R. van Oosterom for skilful technical assistance,
Mr. M.G.B. Nieuwland and Mr. W. Hazeleger (Dept. of Animal Husbandry,
Agricultural University) for collecting the sheep erythrocytes, Dr. W. van
Densen (Limnological Institute, Tjeukemeer) for providing bream serum,
Mr. W.J.A. Valen for drawing the figures and Mrs. G. van Malsen-Eggink for
typing the manuscript.
REFERENCES
Avtalion, R.R., E. Weiss and T. Moalem, 1976, in: Comparative Immunology, ed. J.J.
Marchalonis (Blackwell Scientific Publications, Oxford) p. 227.
Chiller, J.M., H.O. Hodgins and R.S. Weiser, 1969, J. Immunol. 102, 1202.
Feldman, H.A. and L.T. Miller, 1960, Science 1 3 1 , 3 5 .
Fiebig, H. and H. Ambrosius, 1977, Acta Biol. Med. Germ. 36, 79.
Gigli, I. and K.F. Austen, 1 9 7 1 , Ann. Rev. Microbiol. 25, 309.
Green, N. and N. Cohen, 1977, Dev. Comp. Immunol. 1, 59.
Hodgins, H.O., R.S. Weiser and G.J. Ridgway, 1967, J. Immunol. 9, 534.
Horton, J.D., J.J. Rimmer and T.L. Horton, 1976, J. Exp. Zool. 196, 2 4 3 .
Inebi, W. and M.T. Home, 1979, J. Fish Dis. 2, 159.
Jankovic, B.D., K. Isakovic and S. Petrovic, 1972, Eur. J. Immunol. 2, 18.
121
Jerne, N.K., C. Henry, A.A. Nordin, H. Fuji, A.M.C. Koros and I. Lefkovits, 1974, Transplant. Rev. 18, 130.
Kanakimba, P. and VR. Muthukkaruppan, 1972, J. Immunol. 109, 4 1 5 .
Majoor, G.B., M.B. Van 't Veer and O.B. Zaalberg, 1975, J. Immunol. Methods 7, 3 0 1 .
Pontius, H. and H. Ambrosius, 1972, Acta Biol. Med. Germ. 29, 319.
Rijkers, G.T. and W.B. Van Muiswinkel, 1977, in: Developmental Immunobiology, eds.
J.B. Solomon and J.D. Horton (Elsevier/North-Holland, Amsterdam) p. 2 3 3 .
Rijkers, G.T., A.G. Teunissen, R. Van Oosterom and W.B. Van Muiswinkel, 1980, Aquaculture, in press.
Rothe, F. and H. Ambrosius, 1968, Acta Biol. Med. Germ. 2 1 , 525.
Ruben, L.N., B.F. Edwards and J. Rising, 1977, Experientia 3 3 , 1522.
Sailendri, K. and VR. Muthukkaruppan, 1975, J. Exp. Zool. 1 9 1 , 3 7 1 .
Smith, A.M., M. Potter and E.B. Merchant, 1967, J. Immunol. 99, 876.
Warr, G.W., D. DeLuca, J.M. Decker, J.J. Marchalonis and L.N. Ruben, 1977, in: Developmental Immunobiology, eds. J.B. Solomon and J.D. Horton (Elsevier/NorthHolland, Amsterdam) p. 2 4 1 .
Weatherley, A.H. and B.M.G. Cogger, 1977, Science 197, 427.
Zaalberg, O.B., V.A. Van der Meul and M.J. Van Twisk, 1968, J. Immunol. 100, 4 5 1 .
122
APPENDIX I I I
123
The immune system of cyprinid fish. Kineticsand temperature dependence of
antibody-producing cells incarp(Cyprinus carpio)
G.T. RIJKERS, ELIZABETH M. H.FREDERIX-WOLTERS& W.B. VAN MUISWINKEL Departmentof ExperimentalAnimal MorphologyandC'ellBiologyAgriculturalUniversity.Wageningen, TheNetherlands
SUIvMARY
After immunizationofcarpwith sheepredbloodcells spleenaccountsfor
only 51ofthetotalnumberofplaque forming cells (PFC). Inaddition,
thymus,peripheral bloodandheartcontainedlownumbersofPFC(<0.5,
1and0.51respectively).Pronephrosandmesonephroswerethemajorantibodyproducingorgans (53and4050oftotalPFCrespectively).Thetemperaturedependenceoftheantibody forming cellresponseinspleen,pronephros
andmesonephroswasstudiedinanimalskeptat12-24C.Loweringtemperatures inducedadelayinthepeakoftheprimaryresponsebuthadnoeffect
onthemagnitudeoftheresponse.Thetemperature-peakdayrelationshipindicated thattherearestepsintheprimary immune responseofcarpdifferingintemperature sensitivity.Theanamnesticcharacterofthesecondary
responsewasclearly demonstratedat24and20Cbutlostat18C.
INTRODUCTION
Bothcellularandhumoral immune responsesinPoikilothermie vertebratesare
temperature dependent.Atlowtemperatures allograft rejectionproceedsatarelative slowrate (Hildemann, 1957;Cohen, 1966;Borysenko, 1970)andantibodyproductionisdiminished (Pliszka,1939;Ambrosius&Schäker, 1964;Harris,1973;Paterson&
Fryer, 1974)orcompletely absent (Nybelin, 1935;Barrow, 1955;Avtalion, 1969a,b;
Avtalion,Malik,Lefler&Katz, 1970). Upperandlowertemperature limitsofthe
immune responseareclosely relatedtotheecological temperature rangeofthespecies considered (Ridgway,Hodgins&Klontz, 1966;Tait,1969;O'Neill, 1980).The
relationship betweentemperatureandallograft survival timeingoldfish indicated
that therewereatleast2temperature sensitive stagesintheprocessofallograft
rejection (Hildemann, 1957).Accordingtostudiesindifferent animals,thefirst
phaseoftheimmune response includingantigenprocessingandrecognitionisrelative
temperature independent (Cone&Marchalonis, 1972;Avtalion,Wojdani,Malik,
Shahrabani&Duczyminer, 1973).Thefirsttemperature sensitive eventmightbethe
subsequent interactionbetweenT-andB-like cells (Avtalion,Weiss&Moalem, 1976;
Marchalonis, 1977;Cuchens&Clem, 1977).Apossibleexplanationisablockin
"T-helper"activity (Cuchens,Mclean&Clem, 1976)oran,increasein'T-suppressor"
activityatlowtemperatures (Avtalion,Wishkovsky&Kati, 1980).Thefollowing
cellularmultiplicationanddifferentiationofB-like celjlsareconsideredtobe
temperature independent (Avtalionetal., 1973).Accordingtothesameauthorsthe
second temperature sensitiveeventisthesynthesisandreleaseofantibodiesby
125
plasmacells (Avtalionetal., 1973).
Most studiesmentionedabovearecenteredaroundtheinfluenceoftemperature
ontheeffectorphaseoftheimmune response,e.g.antibody levelsinserumor
allograftsurvivaltime.Inthispaperthekineticsoftheplaque formingcell
responseatdifferent temperatureswasstudiedinordertoobtainmore insightin
thetemperaturedependenceoftheinductiveandproliferativephaseofthehumoral
immunerespuseinpoikilotherms.
MATERIALSANDMETHODS
Animals
Outbredcarp (Cyprinus oarpio) wereobtained fromtheOrganizationforImprovementofInlandFisheries (O.V.B.,Nieuwegein,TheNetherlands)orbredinourlaboratory.Animals from6and18monthsold,weighing 75-300g,wereused.Theywerekept
inaquariawithaeratedwater (pH7.4-7.8,N0 ? <0.3ppm,4DH)andfeddailyon
pelleteddryfood (K30,Trouw&Co,Putten,TheNetherlands).Animalswerekeptat
12,16,18,20or24C.Theywereacclimatizedforatleast2weeksateachtemperaturebeforeantigeninjection.
Antigen and immunization
Sheepredbloodcells (SRBC)wereobtained from6animals.Pooledbloodwas
mixedwithanequalvolumeofAlsever's solution.Thecellswerewashed3times
withphosphatebufferedsaline (PBS,pH7.2)beforeuse.Carpwere immunizedby
g
anintramuscular (i.m.)injectionof10 SRBCin0.05mlPBSinthedorsalregion.
Plaque forming cell
assay
Plaque formingcells (PFC)weredetectedusingthemethoddescribedpreviously
(Rijkers,Frederix-Wolters&vanMuiswinkel, 1980a).Bream (Abramis brama) serum
wasusedascomplement source.Plaquesweredevelopedat25Cfor2hunless stated
otherwise.Viabilityoflymphoidcellswasdeterminedwiththedyeexclusionassay
(0.21trypanblueinPBS).
RESULTS
Organ distribution
of PFC
Inordertoobtainanideaabouttherelativeimportanceofdifferent lymphoid
organsofcarp,thenumberofPFCinspleen,pronephros,mesonephros,heart,
peripheralblood,thymus,liverandintestinewasdetermined. Pronephrosandmesonephroscontributeforabout90°softhetotalPFCnumberafteri.m. injectionwith
SRBC (Table I). Spleenaccountsonlyforabout 5°swhilethymus,heartandperipheral
bloodcontainverylowpercentagesofPFC(<H ) .PFCwerenotdetectedinliver
andintestine.Similarresultswereobtainedwhencarpwere injected intravenously
126
TABLEI. Organdistributionofplaque formingcells (PFC)incarpat20C.
Organ
PFC/10 whitecells
Thymus
0. 3+0.
PFC/organ
04*
%oftotalPFC
120
< 0.5
17,100
5
Pronephros
548
+30
184,000
53
Mesonephros
87
+4
138,500
40
Heart
71
+7
1,950
0.5
Peripheralblood
15
+1
3,000
1
-
Spleen
25
+1
Liver
0
0
Intestine
0
0
9
Animalswere injected twicewith 10 SRBC (i.m.)withanintervalof 1month.
ThenumberofPFCwasdetermined 12daysafter thelastinjection (peakof
theresponse).
*Arithmeticmean+ 1S.E. (n=4).
withthesameantigen.Afteroral administrationofSRBCconsiderable numbersof
PFCweredetectedinpronephrosandmesonephrosandtoalesserdegreeinspleen
butnotinintestine.
PFC
kinetica
Thenumberofantibody formingcellsinsuspensionsofspleen,pronephrosand
mesonephroswasdeterminedatsuccesive days afteri.m.injectionwith10 SRBCin
carpwhichwerekeptat24°C (Fig. 1).ThefirstPFCcouldbedetectedatday6.
g
Peak responsewasreachedonday9(93+43PFC/10 WCinpronephros). Following
a secondary injection1monthafterthefirst,PFCwerealreadyobservedonday3.
Thepeakoftheresponseonday8wasa50-foldhigher thanintheprimary response
(4800+2360PFC/10 WCinpronephros).PFCnumbers diminishedintheperiod after
day8butupto20daysafter injectionPFCwere observed.
To assesspeakprimaryandsecondary anti-SRBC responseincarpwhichwere
keptat20C,animalswere testedonafewselected days (Fig.2 ) .Peakofboth
theprimary andsecondary responsewasreached around 12daysafter injection.
The secondary injection gaverisetoonly10timesmorePFC(95+28and1104+_
582PFC/10 WCinpronephros respectively).
Incarpkeptat18°C,thefirstPFCappearedinspleen,pronephrosandmesoq
nephros 11days afterprimary injectionwith 10 SRBCi.m.(Fig. 3).Peak response
was reachedonday17(379+185PFC/10 WCinpronephros).AfterwardsPFCnumbers
rapidly declinedbutPFCremainedpresentinpronephrosupto30days afterinjection.Whenasecondary injectionwas given45days afterprimary thisdidnot result
inasignificant higherpeak responseinmesonephrosorpronephros (304+_101PFC/
10 WC),although firstPFCappeared3-4daysearlierandremainedforalonger
127
period.Peaksecondaryresponsewasreachedinspleenandmesonephrosonday15,
inpronephrosonday 17.
At 16C,maximumnumbersofPFCcouldbedetectedinspleen, pronephros
(131 +_29PFC/10 WC)andmesonephrosataround27daysafterinjection(Fig. 4 ) .
1"
daysafterinjection
Fig. 1. Kineticsofprimary (closed symbols)andsecondary (opensymbols)
anti-SRBC response inspleen (a),pronephros (b)andmesonephros (c)ofcarp
(6monthsold)keptat24C.Animalswere injected twicewith 10 SRBC.The
intervalbetween2i.m. injectionswas 1month.PFC/10 WC=plaqueforming
cellsper 10 viablewhitecells.Eachpointrepresents thearithmeticmean
+ 1S.E. (n=4).
Ü 10*.
daysafter injection
Fig. 2. Kineticsofprimary (closed symbols)and secondary (opensymbols)
anti-SRBC response inspleen (a),pronephros (b)andmesonephros (c)ofcarp
(6monthsold)keptat20°C.Animalswere injected twicewith 10 SRBC.The
intervalbetween2i.m. injectionswas lmonth.PFC/IO WC=plaqueforming
cellsper 10 viablewhitecells.Eachpointrepresents thearithmeticmean
+ lS.E. (n=4).
128
10'_
'O 3 -,
a.
10'.
10'.
-H
10
25
30
daysafter injection
Fig.3. Kineticsofprimary (closed symbols)andsecondary (opensymbols)
anti-SRBC response inspleen (a),pronephros (b)andmesonephros Lc) ofcarp
(18monthsold)keptat 18°C.Animalswere injected twicewith 10 SRBC.The
intervalbetween2i.m. injectionswas45days.PFC/10 WC=plaqueforming
cellsper 10 viablewhitecells.Eachpointrepresents thearithmeticmean
+ 1S.E. (n-4).
129
10 2 _
ü
ä
I ' '' ' I'
25
36
25
"I
30
25
daysafterinjection
Fig.4. Kineticsofprimaryanti-SRBC response inspleen (a),pronephros (b)
andmesonephros (c)ofcarp (6monthsold)keptat 16C,Animalswere i.m. injected
with 10 SRBC.PFC/10 WC=plaque formingcellsper 10 viablewhitecells.Each
pointrepresents thearithmeticmean+ 1S.E. (n=4).
n -1
55
60
50
50after injection
days
Fig.5. Kineticsofprimaryanti-SRBC response inspleen (a),pronephros (b)
andmesonephros (c)ofcarp (6monthsold)keptat 12C,Animalswere i.m. injected
with 10 SRBC.PFC/10 WC=plaque formingcellsper 10 viablewhitecells.Each
pointrepresents thearithmeticmean+ 1S.E. (n=4).
Peakprimaryresponsewasreachedaroundday49incarpkeptat12C (91+
35PFC/106 WCinpronephros;Fig. 5 ) .
ToascertaintheobservationthatcarpareabletodevelopPFCatlowtemperatures,PFCslideswereincubatedat16or12°C-ambienttemperatureofthe
animalsconsidered-insteadoftheususal25°Cforplaqueincubation.Itappeared
130
55
thatbothat16and12°Cplaqueswere formed.However,thespeedofdevelopment
waslowandattainedlevelsdidnotmeetvaluesobtainedatthe25Cincubation
(Table II).Itisconcludedthatantibodyproductionandsecretioncontinuesat
lowtemperatures,butatareducedrate.
TABLEII.Effectof incubationtemperatureonplaquedevelopment.
Ambient temperature
( C)
PFC incubation
PFC incubation
temperature (C) time (hours)
PFC/10 whitecells
i n pronephros
16
25
2
61 + 20*
16
16
5
26 + 20
12
25
2
19 +
3
12
12
5
4 +
1
22
_6 +
1
12
12
Animalskeptat 16Cwere tested forthepresenceofplaqueformingcells (PFC)
inpronephros 25daysafterani.m.injectionof 10 SRBC.Animalskeptat 12C
were tested 44daysafterinjection.
*Arithmeticmean +_ 1S.E. (n=4).
Insummarizingtheresultsincanbeseenthatwithloweredtemperaturesthe
peakoftheprimary responseindelayed (Fig.6)butthemagnitudeoftheresponse
isunaltered.Theanamnesticcharacterofthesecondaryresponsehowever,is
graduallylostwhenloweringwatertemperatures.
DISCUSSION
Theroleofthepronephrosinantibodyproductioninteleostfishhasbeen
demonstratedinanumberofstudies (Smith,Potter&Merchant,1967;Chiller,
Hodgins,Chambers&Weiser,1969;Chiller,Hodgins&Weiser,1969;Pontius&
Ambrosius,1972;Sailendri&Muthukkaruppan, 1975).Inthetoad Bufo mavinus ,plasma
cellsweremostabundantinintertubular lymphoidtissueofthekidneyafterinjectionwithbovineserumalbumin (BSA) (Cowden,Dyer,Gebhardt.&Volpe, 1968).
Itwassuggestedthatalsoinotherlowervertebratesthekidneymightbethe
majorsiteforantibodyproduction.However,inthetortoise Agrionemys
hovsfieldii,
thekidneyplayednoroleinantibodyproductionafterimmunizationwithSRBC
(Rothe&Ambrosius, 1968).Inrainbowtrout [Salmo Gairdneri) PFCwerefoundin
mesonephrosafterimmunizationwith0-antigenofthebacterium Yersinia
ruakeri,
butnumberswerenotashighasinspleen (Anderson, 1978). Inperch (Perca
fluviatilis)
mesonephroscontained lowerPFCnumbersthanspleenandpronephros after
injectionwithSRBC (Pontius&Ambrosius, 1972). Ingoldfish [Carassius
auratus)
131
50-,
40.
I 30.
E
a 20
10-
10
Fig. 6.
15
20
watertemperature(°C)
25
Temperature dependenceofhumoral anti-SRBC response incarp.
pronephrosandmesonephrosaremajorsitesofantibodyproductionagainst
Salmonella.
typhi (Neale&Chavin, 1971). Inourexperimentswithcarp,highPFCnumberswere
foundinmesonephros.Thiso.-gantogetherwithpronephros contributed for90°;of
thetotalPFCillustratingtheimportanceofthekidneyasawholeforantibody
formation.
InmosthigherandlowervertebratesthespleenisanimportantPFCcontaining
organ.IncarponlylowPFCnumberswerefoundinspleen.However,itcannotbe
excludedthatthisorganplaysanimportantroleinthedifferentiationofB-like
cellsintoplasmacells.SincePFCwereobservedinperipheralblood itispossible
thatproliferationanddifferentiationofB-likecellstakesplaceinspleenand
thatmaturatingplasmacellsmigratethroughthebloodstreamandsubsequentlyhome
inotherorgans (e.g.kidney). Toverifythisassumptionsplenectomyshouldbe
performed.Resultsofsplenectomyexperiments inotherteleostfishareconflicting.
Splenectomy ingraysnapper (Lutjanus
gviseue)
didnotaffecttheantibodyresponse
againstBSA (Ferren,1967).StudiesofYu,Sarot,Filazzola&Perlmutter (1970)on
theeffectofsplenectomy inbluegourami (Tvichogastev
triohopterus)
suggestthat
thespleenisamajorlymphoidorganinthisspecies.Unfortunatelysplenectomyin
carpisnotpossiblebecausethespleenisinterspersedwithliverandpancreasand
inmostcasesnotadistinctorganbutfragmentedalongtheintestine.
Harris (1973)showedthatdace [Leuoisaus
132
leuciscus)
arecapableofproducing
antibodiesovertheircompleteenvironmentaltemperaturerange (2-18C ) .Theinfluenceoftemperatureuponantibodyproductioncouldbeageneralphysiological
effectasdescribed forothermetabolicprocesses (Huisman,1974;Brett,1979).
A logarithmicrelationbetweentemperatureandoxygenconsumptionorstandard
metabolismwasobservedbyBrett (1976). Inourexperimentsitturnedoutthat
a linearrelationshipexistsbetweenpeakdayandlowtemperatures (12-18C)but
theslopeofthegraphchangedat20Candhigher (Fig.6 ) .Thepossibilityofa
generalphysiological effectoftemperatureontheimmuneresponseasawholeis
thereforeunlikely.Inthisrespectourresultsareinagreementwiththeideas
ofAvtalionetal. (1976)andMarchalonis (1977)indicatingthatthereareless
andmoretemperature sensitivestepsintheimmuneresponse.
Inallinstancesplaqueassayslideswereincubatedat25C.Forcarpkept
atlowtemperatures (12-16C)thisiswellbeyondtheirambienttemperature.To
assurethatcarpwerecapableofperforminganimmuneresponseat 12and 16C
PFCslideswerealsoincubatedatthesetemperatures.About 30%oftheplaques
werevisualizedatlowtemperaturescomparedwiththesituationat25C.Itis
admittedthatalongerincubationperiod forplaquedevelopmentwasneeded.It
ispossiblethatat 12ChigherPFCnumbers canbereached in vivo becauseour
in vitro systemwasnotdevisedforlongincubationperiods.
IncontrasttoAvtalionetal. (1970)whodidnotshowantibodyformation
incarpat 12Cusingtitrationtechniques,wewereabletodemonstrateaclear
PFCresponseatthistemperature (maximumday49).Sincenoexponentialrelationshipbetweentemperatureandpeakdaywasobservedandthemagnitudeoftheresponseremainedconstantoverthewholetemperaturerangewithcarpofthesame
age,weexpectthatcarpareabletomountanimmuneresponseattemperatures
below 12C.However,inthosecaseslagperiodswillbecomeverylongandthe
protectiveeffectofsucharesponseisthereforequestionable.
Criteriatoassess immunologicalmemory invertebratesareanincreasedrate
ofantibodyproductionandhigherlevelsofantibody (Marchalonis,1977).At24C
a truesecondaryresponsewasfound,illustratedbyanearlyappearanceofthe
firstPFCanda50xhigherpeakthanintheprimaryresponse.At20C,a10x
highersecondaryresponse isreachedonthesamedayasduringtheprimaryresponse.
Duringthesecondaryresponseat 18CthefirstPFCappearearlierbutthepeak
coincideswiththeprimaryresponse.Itmustbementionedthattheanimalswere
g
primedwithhighantigendosis (10 SRBC/animal)andthattheintervalbetween
primaryandsecondaryinjectionwasabout 1month.Lowerprimingdoseandprolonged
interval betweenprimaryandsecondaryinjectionleadtoahighersecondaryresponse (Rijkers,Frederix-Wolters&vanMuiswinkel, 1980b).Whereasthemagnitude
oftheprimaryresponseisnotaffectedbylowertemperatures,theanamnestic
characterofthesecondaryresponseisgraduallylost.Thismight indicatethatthe
formationofmemorycellsisalsoarelativetemperaturesensitiveprocess.
133
Therelationshipbetweentemperatureandpeakdayofthehumoralimmuneresponseincarp (Fig.6)closelymatchedtherelationshipbetweentemperatureand
cellularimmuneresponse(allograftsurvivaltime)ingoldfish(Hildemann,1957).
Itisconcludedthatinbonyfishhumoralandcellularimmunityareaffectedby
temperatureinasimilarway.
ACKNOWLEDGEMENTS
ThetechnicalassistanceofMr.R.vanOosteromandMr.J.Wijnenandthe
biotechnicalsupportofMr.S.H.Leenstraisgratefullyacknowledged.Wethank
Mr.W.J.A.ValenfordrawingtheillustrationsandProf.J.F.Jongkindforhis
interestandvaluablesuggestionsduringperformanceoftheexperimentsandpreparationofthemanuscript.
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136
APPENDIX I V
137
© 1980 Elsevier/North-HoHand BiomedicalPress
Phytogeny of Immunological Memory
M.J.Manning,editor
THE IMMUNE SYSTEM OF CYPRINID FISH.
THE EFFECT OF ANTIGEN DOSE AND ROUTE OF ADMINISTRATION ON THE
DEVELOPMENT OF IMMUNOLOGICAL MEMORY IN CARP ( C y p r i n u s c a r p i o )
GERT. RIJKERS, LILY M.H. FREDERIX-WOLTERS and WILLEM B. van MUISWINKEL
Department of Experimental Animal Morphology and Cell Biology,
Agricultural University, Zodiac, Marijkeweg 40, Wageningen (The Netherlands)
INTRODUCTION
One of the most characteristic features of the immune system is the phenomenon of immunological memory. Memory means that a second contact with the same
antigen evokes an enhanced response. Immunological memory in mammals applies to
both cellular and humoral immune responses. Memory in cell-mediated reactions is
manifested by the phenomenon that second-set grafts are rejected faster than
f i r s t - s e t grafts. Criteria to assess immunological memory in humoral immune responses are an earlier rise in antibody formation, attainment of higher antibody
levels and predominance of IgG . Moreover, memory is specific for a given a n t i gen and not a general enhancement of immune competence.
Tne capacity to discriminate between "self" and "non-self" is present
2
throughout a l l vertebrates and invertebrates . A cell-mediated immune system is
found in annelids, e.g. earthworms which are capable to reject transplanted
3
xeno- and allografts . Upon repeated grafting transplants are rejected faster
but this primitive form of memory is not donor specific and only short-lived .
I t is not clear whether invertebrates possess a specific humoral immune system
5
since immunoglobulins have not been demonstrated . This does not imply that i n vertebrates are devoid of humoral defence mechanisms since in snails serum factors have been demonstrated which promote recognition and subsequent phagocytosis of foreign material , while in s i l k moths substances with a complement-like
a c t i v i t y were found in hemolymph .
In the f i r s t vertebrates (jawless and cartilaginous fish) allografts are reQ
jected in a chronic way . However, the most advanced bony fish (teleosts) show
an acute rejection of f i r s t - s e t allografts and accelerated rejection of secondset grafts '
. When cyprinid fish are kept at 25°C - a normal ambient tempera-
ture for the species - cellular immune reactions proceed even faster than in
11 12
mammals ' . At the level of cellular immunity, secondary responses seem to be
comparable in teleosts and mammals. As far as humoral immunity is concerned
there have been d i f f i c u l t i e s in demonstrating clear-cut secondary responses in
139
in lower vertebrates
13-15
. In goldfish the magnitude of the secondary response
was not enhanced but differences in the kinetics of antibody production were
taken as evidence for immunological memory . In other studies secondary responses were obtained which met the usual c r i t e r i a for immunological memory
19
. Since teleost fish possess only one IgM l i k e class of immunoglobulins
20-21
,
the criterium of predominance of IgG antibodies during the secondary response
is not valid for this group.
In mice the apparent paradox exists that priming with a low antigen dose
which evokes no or only a low primary response, is optimal for memory development. Priming with a high antigen dose, which results in a clear primary re22 23
sponse, gives a poor secondary response ' . I t has been shown that antibody
produced during the primary immunization, acts through a feedback mechanism that
limits the magnitude of the secondary response to subsequent antigenic challenge,
24 25
both in vivo and in vitro
'
In this paper duration and specificity of immunological memory in carp, a teleost f i s h , is compared with the same process in mammals. The effect of d i f f e r ent routes of administration and antigen dosages on the development of immunological memory is studied.
MATERIALS AND METHODS
Animals. Common carp (Cyprinus aarpio) were obtained from the Organization
for Improvement of Inland Fisheries (Nieuwegein) or bred in our laboratory. They
were kept in aquaria with running tap water at 18°C and fed daily with dry food
(K 30, Trouw & Co, Putten) with a "Scharflinger" automatic feeder. Animals,
weighing 200-600 g, were 1year old at s t a r t of the experiments.
Antigens. Sheep red blood cells (SRBC) were obtained from the Department of
Animal Husbandry, Agricultural University (Wageningen). Horse red blood cells
(HRBC) were purchased from the National Institute of Public Health (Bilthoven).
Cells were washed 3 times with phosphate buffered saline (PBS) pH 7.2 before use.
Immunization. Animals were injected with SRBC either intramuscular (i.m.) or
•"~""^
5
7
9
intravenous ( i . V . ) . A priming dose of 10 , 10 or 10 SRBC was i.m. injected in
the dorsal region (0.05 ml) or i . v . in the caudal vein (0.5 ml). Control animals
were i.m. injected with 0.05 ml PBS. At 1, 3, 6 or 10 months after primary i n Q
j e c t i o n , 5 animals out of each group were i.m. injected with 10 SRBC. Fourteen
days after the challenge the animals were k i l l e d and the number of antibody producing cells in different lymphoid organs was determined.
Plaque forming cell assay. The number of antibody producing cells in spleen,
pronephros and mesonephros was determined using the haemolytic plaque assay
2fi
adapted for carp . Bream (Abramis brama) serum was used as complement source.
140
After incubation for 2 hours at 25°C the plaque forming cells (PFC) were scored
under a low-power dissection microscope. The number of viable cells in cell suspensions was counted in a haemocytometer using a dye exclusion assay (0.2% Trypan Blue in PBS). Results are expressed as number of plaque forming cells per
106 viable white cells (PFC/106 WC).
RESULTS
Primary response against SRBC
The results with carp injected with different dosages of SRBC are shown in
Fig. 1. The number of PFC in spleen, pronephros and mesonephros was determined
17 days after injection (peak of the response). Similar experiments with nonimmune carp showed no background PFC. Animals injected i . v . or i.m. with 10
5
SRBC poorly responded (0.2-2 PFC/106 WC). With increasing SRBC dose a marked i n 7
9
crease in PFC response was observed. Animals i.v. injected with 10 and 10 SRBC
gave rise to higher PFC levels compared with i.m. injected animals, although d i f ferences were only significant for spleen at the highest antigen dose. Thus, after a primary injection the magnitude of the peak response at day 17 is correlated with the antigen dose.
o
9
When animals kept at 18 Care i.m. injected with 10 SRBC with an interval of
1 month between 2 injections, the same peak value is reached on the same day (day
17, Table 1.). However, in the secondary response the f i r s t PFC appear 3 days
e a r l i e r than in the primary response resulting in a ten-fold difference on day
14. In order to allow an assessment of immunological memory a l l PFC assays in
U103-
%:
S
10t
8
7
8
Log,0SRBCdOM
ig.
1. Dose-dependency oftheprimary anti-SRBCresponseincarp.Animals,
*(,« *» t»v/ö*. u t . | / i . i i u b i i \ . j i w i L u c j'l i.tuaLy a i m . UIUJV i c s | / u u o c J.II t a L J I . t\ll
tedwith
;eptat 18°C,were intramuscular (0,A,0)orintravenous (O^,!)inject
rosandme0 5 , 10'or I09 SRBC.Onday 17thenumberofPFC inspleen,pronephro
:onephroswasdetermined.Eachpointrepresents thearitmeticmean+ 1S.E.(n«4)
141
TABLE1
IMMUNE RESPONSEOFCARP AGAINST SRBC
PFC/10 white pronephros cells
day 14
day17
day20
Typeofresponse
primary
secondary
22+ 12
379+185
230+75
304+101
24+13
128+16
Animals,keptat 18°Cwere injected 2times#with 10 SRBC (i.m.).Thesecondinjectionwasgiven 1monthafterthefirst. Arithmeticmean+ 1S.E. (n=4).
ted withtheantigen dose.
g
When animals keptat18°Cwerei.m.injected with 10 SRBC withanintervalof
1month,thesame peak valueisreachedonday17(Table 1).However,inthesecondary responsethefirstPFCappear3days earlier thanintheprimary response
resultinginaten-fold differenceonday14.In ordertoallowanassessmentof
immunological memoryallPFCassaysinthefollowing experiments were performed
14days afterthelast injection.
101
intramuscular
intravenous
K^
ioi
10-
10
interval between first andsecond injection (months)
Fig.2 . Developmentof immunological memory incarp spleen.Animalswere
with1 0 5 (0,#), 107 (A,A)or 109SRBC (DM) either intramuscularorintra'
At 1,3,6or 10monthsafterpriming 5animalsoutofeachgroupwerechallengedwith 109SRBC (i.m.).Thenumber ofPFC inspleenwasdetermined 14daysafter
challenge.Eachpoint represents thearithmeticmean+ 1S.E..
142
primed
intravenous.
intramuscutw
intravsnous
O 101
10?.
I
10
1
intervalbetweenfirst andsecondinjection(months)
Fig.3. Development of immunological memory incarppronephros.For furtherexplanationseeFig.2.
Developmentofimmunological memory
Inordertostudytheeffectofdifferent dosagesandroutesof antigen administrationontheformationofimmunological memory, animals were divided into7
groups.Three experimental groupswere injected i.v.and3other groupsi.m..
5 7
9
Different dosages SRBC (10,10 and10)were injected along each route.Agroup
ofPBSinjected animals servedascontrol.
g
Five animalsoutofeach group receivedani.m.injection with10 SRBC1
month after primary injectionandwere testedforPFC14days later.Controlanimals developed only3+1,24+15and6 + 3PFC/10 WCinspleen, pronephros
and mesonephros respectively.Theresponseof10 SRBCi.m.primed animalsdid
g
not exceed these values significantly (Figs.2-4).The10 SRBCi.m.group gave
a clear secondary response (e.g.254+75PFC/10 WCinpronephros)butthe
highestPFClevelswere obtained with animals primed with only10 SRBCi.m.
(664+350PFC/10 WCinpronephros; Figs.2-4).Inalli.v.primed groupsasecondary responsewasobserved. After priming along this routePFClevelswere
higherwith increasing priming dosages.
Challengeat3months after priming producedthefollowing picture.Control
animals developed 17+12,49+12and29+17PFC/10 WCinspleen, pronephros
—
—
—
5
and mesonephros respectively. Interestingly,theresponseof10 SRBCi.m.primed
animals showedforthefirst timeaclear secondary characterandwascomparable
withtheresponseofthe10 SRBCi.m.primed group (227+99and171+80
143
lOi,
g
10»
10-
10
intervalbetweenfirstandsecondinjection(months)
Fig.4. Development of immunological memory incarpmesonephros.For further
explanationseeFig.2.
PFC/10 WCinpronephros respectively).Theresponseofthe10 SRBCi.m.group
remainedataquite high level (899+106PFC/10 WCinpronephros).Inthei.v.
primed animalsPFClevels slightly declined compared withthesituation2months
earlier.Theresponseofthe10 SRBC i.v. groupwas comparable with control
values.
Another3months laterthewhole procedurewas repeated. Incontrol animals
4 + 2 , 17+4and8+2PFC/10 WCwere observed in spleen,pronephrosandmesonephros respectively. Amazingly theresponseofthe10 SRBCi.m.groupwasstill
increasingandbecameequaltothehighPFClevelof10 SRBC i.m. primed animals (784+218and829+139PFC/106WCinpronephros).Inthe10 9SRBCi.m.
andalli.v.groupsPFClevels remained nearly constant.
Inaddition,theanimalswhich were primed i.m. receivedabooster 10months
after priming.PFClevelswere similartothesituationat6months after priming, indicatingalong-lived memory. Highest secondary responseswere achieved
5
with 10 and
10 7SRBC i.m. primed animals (1094and828+158PFC/10 6WCin proq
—
nephros).Animals primedwith 10 SRBCi.m.remainedatalevelofabout100
PFC/10 WCinpronephros.
Ingeneral thedevelopmentofimmunological memoryinspleen (Fig.2) and
mesonephros (Fig.4)runsparallel withthepicture described forpronephros
(Fig.3).
144
Specificityofthe humoral immune response
Inordertotest the specificityofthe immune response and memory formation
SRBC primed and control animalswere injected with HRBC.Itappeared that only
PFC directed against the booster antigen were present (Table 2.). The immune response against SRBC and HRBC turned outtobehighly specific andnocrossreactivity between SRBC and HRBCwas observed inthis system. The magnitudeof
the anti-HRBC responsein10 SRBC i.v. primed animals was not enhanced compared
with the responseofcontrol animals which were injected with PBS.Itis concluded that the developed memory cellswere specific for the priming antigen.
No indications forageneral enhancementofimmune competence were found.
TABLE2
SPECIFICITYOFTHE HUMORAL IMMUNE RESPONSE INCARP
First
injection
Second
injection
Test
antigen spleen
PBSi.m. 10 9HRBC i.m. HRBC
6.4 + 2.4* 16.0+8.1
SRBC
10 7 SRBC i.v. 10 9SRBC i.m.
HRBC
HRBC
0.2+0.2
0.1+0.1
SRBC
10 9 SRBC i.v. 109 HRBC i.m.
PFC/106WC
pronephros
7.3+2.8
SRBC
0.3+0.1
0
32.0+10.5
13.7+7.6
0.4+0.3
4.5+0.9
1.1+0.7
0.9+0.3
9.8+5.2
mesonephros
15.0+5.1
5.0+1.5
0.8+0.6
0.1+0.1
Carp,whichwerekeptat 18°C,were intravenous (i.v.)injectedwith 10 or10
SRBCorintramuscular (i.m.)with0.05mlPBS.Tenmonths laterallanimalsreceived ani.m. injectionwith 109 SRBCorHRBC.Thenumber ofPFC inspleen,
pronephrosandmesonephroswasdetermined 14daysafter thelast injection.
"Arithmeticmean+ 1S.E. (n=3).
9
7
DISCUSSION
Our resultswith carp show that the priming routeisimportant for the magnitudeofthe secondary response. Moreover, differencesinantigen priming dosesecondary response relationship were observed. When carpwere primed i.v. the
magnitudeofthe primary and secondary response increased with increasing antigen
g
doses.After i.m. priming withahigh antigen dose (10 SRBC)ahigh primary but
a poor secondary response was observed. Primingwith10 SRBCwas optimalfor
5
memory formation.Alow priming dose (10 SRBC)resultedinalow primary butin
145
a high secondary response when the booster injection was given at least 6 months
later.
Itappeared that the priming dose-secondary response relationship in carp
27 28
after i.m. priming is similar tomammals after i.v. priming ' .It is tempting
to speculate that antibody feedback inhibition is responsible for this phenomenon. Ithas been shown in rodents that IgM ismore susceptible to suppression
29
than IgG .Yet,antibody feedback cannot fully explain the observed differences
inmemory development after priming with different antigen doses. E.g. the secondary response of 10 SRBC i.m. primed animals should increase when the interval
between primary and secondary injection is prolonged. A second phenomenon which
might play a role is antigen persistence. Since the primary response after low
dose priming is very meager, antigen is not eliminated by antibody induced lysis
or phagocytosis.Antigen(fragments)may persist and stimulate the formation of
5
memory cells.This might be the reason why it took so long for the 10 SRBC i.m.
primed animals to develop a high secondary response.
In trout, the primary immune response against MS2bacteriophage increased
with higherantigen dose.The same dose dependency was found after a secondary
19
stimulation .Incarp, immunized with bovine serum albumin (BSA)ranging from
0.1-5 mg/kg,no significant differences in titres during the primary response
30
were obtained .High BSA doses were found to be tolerogenic in primary and secondary response.At low temperatures (14°C)low BSA doses (0.04-0.2 mg/kg)
30
turned out to be tolerogenic too .In the teleost Tilapia mossambioa the primary PFC response to SRBC was found to be proportional to the amount of antigen
31
19 30
injected .Inall studies the antigen was administered intraperitoneal ' or
31
i.v. .This might explain the observed antigen dose-secondary response relationship,especially since i.v. priming in our experiments resulted in the same relationship.
The controversial data on secondary responses in lower vertebrates is centered around at least 2factors: the antigen used and the period between first and
second contactwith the antigen. In this respect it is stated that characteristic secondary responses can only beobtained with antigens which induce a true
32
primary response .The second factor is the period between first and second
antigen contact. This is visualized by our finding that no anamnestic response
5
was observed in animals primed with 10 SRBC i.m. when a booster injection was
given 1month later. However, asecond injection 6 months after the low dose
priming resulted ina clear secondary response. It is evident that temperature
is important too for primary and secondary responses inectothermic vertebrates
30
. Results of studies in our laboratory concerning the effect of temperature
on the immune response in carp at 12-24°C will be presented elsewhere.
146
The presented results might have practical consequences in the field of prophylaxis of diseases, especially vaccination schedules. However, it must be kept
in mind thatwe used SRBC which is a corpuscular, non-pathogenic antigen. The
immune system will probably respond in another way to proliferating pathogens.
g
The most striking observation was that a high priming dose (10 SRBC)was not
optimal for memory formation; in other words: high dose vaccination does not
always result in optimal protection. Priming with a 100 x lower antigen dose
resulted ina 10 xhigher secondary response; the developed memory remained at
5
this high level for at least 10months.A low priming dose (10 SRBC)can lead
to a high secondary response when the second injection is given at least 6
months after priming. When dealing with expensive vaccins it isworthwhile to
consider that low priming doses can be used if not an immediate protection is
required.
If similar results can be achieved when a vaccin is administered by routes
applicable in mass fish culture (e.g. oral immunization or hyperosmotic infiltration) is still an open question.
ACKNOWLEDGEMENTS
We are grateful to Mr. R. van Oosterom and Mr.J. Wijnen for technical assistance, toMr. S.H. Leenstra for biotechnical support and to Mr. W.J.A.Valen for
drawing the figures.Furthermore we acknowledge Prof.J.F. Jongkind for his
stimulating interest and valuable suggestions during the performance of the
experiments and the preparation of the manuscript.
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Grantham, W.G. and F i t c h , F.W. (1975) J . Immunol., 114, 394-398.
25.
Möller, G. and Wigzell, H. (1965) J . exp. Med., 121, 969-989.
26.
R i j k e r s , G . T . , Frederix-Wolters, E.M.H, and Muiswinkel, W.B. van (1980) J .
Immunol. Meth. ( i n press).
27.
Hanna, M.G. and Peters, L.C. (1971) Immunology, 20, 707-718.
28.
Sercarz, E.E. and Byers, V.S. (1967) J . Immunol., 98, 836-843.
29.
Uhr, J.W. and Möller, G. (1968) Adv. Immunol., 8 , 81-127.
30. Avtalion, R.R., Wojdani,A., Malik, Z., Shahrabani, R. and Oucziminer,M.
(1973)Curr. Topicsin Microbiol, and Immunol., 61, 1-35.
31. Jayaraman,S.,Mohan, R. and Muthukkaruppan, VR. (1979) Dev. Comp. Immunol.,
3,67-75.
32. Ambrosius, H. (1976)in:Comparative Immunology (J.J. Marchalonis, Ed.)
Blackwell Scientific Publ.,Oxford, 298-334.
148
APPENDIX V
149
Aquaculture, 19 (1980) 1 7 7 - 1 8 9
© Elsevier Scientific Publishing Company, Amsterdam — Printed in The Netherlands
THE IMMUNE SYSTEM OF CYPRINID FISH. THE IMMUNOSUPPRESSIVE
EFFECT OF THE ANTIBIOTIC OXYTETRACYCLINE IN CARP
(CYPRINUS CARPIO L.)
G.T. RIJKERS, A.G. TEUNISSEN, R. VAN OOSTEROM and W.B. VAN MUISWINKEL
Department of Experimental Animal Morphology and Cell Biology, Agricultural
Zodiac, Marijkeweg 40, 6709 PG Wageningen (The Netherlands)
University,
(Accepted 28 August 1979)
ABSTRACT
Rijkers, G.T., Teunissen, A.G., Van Oosterom, R. and Van Muiswinkel, W.B., 1980. The
immune system of cyprinid fish. The immunosuppressive effect of the antibiotic
Oxytetracycline in carp (Cyprinus carpio L.). Aquaculture, 19: 177—189.
The effect of Oxytetracycline (oxyTC) upon the immune system of carp was
investigated. OxyTC was administered by feeding with oxyTC-containing pellets or by
intraperitoneal injection. In order to study cellular immunity, allogeneic scale transplantation was carried out. Oral administration of oxyTC had no influence upon the
median survival time (MST) of the scales. However, injections with oxyTC significantly
prolonged the MST from 8.5 to 11—20 days. Thus, cellular immunity was not affected by
oral administration of oxyTC, but injections did have a dramatic immunosuppressive effect.
To investigate the effect of oxyTC upon humoral immunity, animals were injected with
rabbit red blood cells (RaRBC). During the primary and secondary response the number
of rosette forming cells (RFC) in the spleen was determined. Control animals (not treated
with oxyTC) developed an anti-RaRBC response up to 2 5 0 0 0 RFC/10 6 white spleen cells
but oxyTC-treated animals always showed reduced RFC numbers. In some cases the
RFC number in oxyTC-treated animals was comparable with background levels in nonimmunized control animals (4000 RFC/10 6 white spleen cells). Thus, irrespective of the
route of administration, the humoral immune response is depressed by oxyTC. It is concluded that both humoral and cellular immune responses of carp are suppressed during
treatment with oxyTC.
Preliminary observations showed an increased number of granulocytes in the spleen of
oxyTC-treated animals. It is tempting to speculate that in those cases where specific
lymphoid defence mechanisms are blocked, the phagocytic defence system becomes more
active.
INTRODUCTION
There are several ways for a diseased animal to eliminate pathogenic microorganisms. Under normal circumstances the pathogen is attacked either by
phagocytic cells (granulocytes, mononuclear phagocytes) or in a more specific
way by lymphoid cells (humoral and cell-mediated immunity).
151
In addition, antibiotics and chemotherapeutics can be used for the prevent]
and control of diseases (Braude et al., 1953;Guest, 1976). At present,
penicillins, tetracyclines, macrolides, aminoglycosides, chloramphenicol,
methylenblue, nitrofurans and antifungals are employed (Sanford, 1976).
It is known that antibiotics and chemotherapeutics can evoke acute toxic
effects in animals (Martin, 1973) and increase the risk of raising resistant
pathogens (Swann, 1969; Finland, 1975). Moreover, some antibiotics can
interfere with certain steps in eukaryotic protein synthesis (Watson, 1975).
Most probably the mitochondrial protein synthesis is inhibited (De Vries and
Kroon, 1970).
Numerous studies in mammals and birds have been performed on the interaction between antibiotics and the immune system, with contradictory results
A stimulatory effect of antibiotics upon the immune response is reported by
Popovic et al. (1973). Their results show that pigs treated with Oxytetracycline (oxyTC) produce significantly higher agglutinin titers after injection
with a brucella vaccine than non-treated controls. According to other reports
antibiotics have no effect upon the immune response. In birds, feeding with
oxyTC did not interfere with antibody production after bovine serum
albumin (BSA) injection (Glick, 1968). Several studies deal with immune
suppression after treatment with antibiotics. In pigs, oxyTC depressed the
formation of immunity against experimental Erysipelothrix infection
(Fortushnyi et al., 1973). In mice injections with oxyTC depressed the antisheep erythrocyte response (Nikolaev and Nazarmukhamedova, 1974).
Feeding of oxyTC to rats and mice interfered with the antibody production
against Salmonella enteritidis (Slanetz, 1953).
Antibiotics are in common use for the prevention (Schaperclaus, 1967;
Bauer et al., 1969) or control of diseases in cultured fish (Schaperclaus, 1969;
Ghittino, 1972; Van Duijn, 1973). In order to prevent the development of
drug resistant bacteria the amounts of antibiotics used are generally high
(Van Duijn, 1973;Roberts and Shepherd, 1974).
In contrast to the data mentioned above, virtually nothing is known about
the effects upon fish. Kreutzmann (1977) demonstrated that erythropoiesis
in the eel was disturbed after treatment with chloramphenicol or oxyTC.
Reports dealing with the immune system describe effects on cellular immunit;
Antibiotics and antimetabolites can delay allograft rejection in fish (Hildemann and Cooper, 1963; Levy, 1963; Cooper, 1976). At present no data
concerning the effects of antibiotics upon humoral immunity in fish are available.
This study was performed in order to obtain information about the possible
side effects of antibiotics upon the immune system of fish. We have chosen
oxyTC because of its frequent use in fish culture (Herman, 1969).
MATERIALS AND METHODS
Animals
Carp (Cyprinus carpio, Linnaeus 1758) were obtained from the Organization
for Improvement of Inland Fisheries (OVB) at Nieuwegein, The Netherlands.
They were kept in aquaria with running tap water at a temperature of 20 ±
1°C. Animals were fed with pelleted dry food (Trouvit K30, Trouw &Co,
Putten, The Netherlands). The daily amount of food (2.5%of their body
weight) was supplied by means of a "Scharflinger" automatic feeder. At the
start of the experiment two size classes were selected from a group of 800
sarps, aged 6 months: (1) small animals with a total length between 11 and
13 cm ( 1 5 - 4 9 g); (2) large animals between 15 and 17 cm (62—105 g).
Antibiotic
Oxytetracycline (oxyTC) was administered either by intraperitoneal (i.p.)
injection of a solution of 50 mg/ml Engemycine® (Mycofarm, De Bilt, The
Netherlands) or orally by feeding fish with Trouvit-pellets supplemented
with 2000 ppm oxyTC.
Scale transplantation
It is known that repeated MS-222 anaesthesia causes a considerable loss
Dfanimals in this species (Rijkers and Van Muiswinkel, 1977). Therefore
arps were not anaesthetized during scale transplantation and subsequent
iaily inspection. Five scales in the row above the lateral line were removed
with a pair of fine forceps and kept for 5 min at maximum in sterile phosahate buffered saline (PBS). Each fish received four allografts, taken from
wo donors, and one autograft as a control. After transplantation allogeneic
jcales became overgrown by hyperplastic host tissue, which resulted in a
white appearance. At the same time some scales exhibited vasodilatation and
jranching melanophores. The beginning of the clearance of the hyperplastic
lost tissue is considered to be the survival end point (SEP) of the graft
Hildemann, 1957). The median survival time (MST) was calculated from
he SEP's.
r
mmunization
Rabbit red blood cells (RaRBC) were obtained from the Foundation of
îlood Group Studies, Wageningen, The Netherlands. The cells were washed
hree times with PBS before use. Each animal received 10 9 RaRBC, injected
ntramuscularly (i.m.) in the dorsal region. In some cases a second injection
was given 24 days later.
153
Rosette test
Cell suspensions were prepared as described earlier (Rijkers and Van Muiswinkel, 1977). The number of rosette forming cells (RFC) in the spleen
was determined according to a slight modification of the method of Zaalberg
(1964) and Biozzi et al. (1966). Briefly, 4 X 10 6 white spleen cells and
24 X 10 6 RaRBC were mixed and adjusted to a volume of 1 ml. The medium
used was Hank's Balanced Salt Solution (HBSS, Difco, Detroit, U.S.A.)
supplemented with 0.4% New Born Calf Serum and 1%antibiotics (20 I.U.
mycostatine, 100 I.U. penicillin and 0.1 mg streptomycin per ml). The cell
suspension was incubated overnight at 4°C and subsequently rotated for
10 min (10 rpm) at room temperature (Multi Purpose Rotator, Wilten, EttenLeur, The Netherlands). Rosettes were counted under a phase-contrast
microscope (objective 20 X ). Two types of rosettes were distinguished:
(1) rosettes with a single layer of RaRBC, probably cells with receptors for
RaRBC on their surface; (2) rosettes with a multiple layer of RaRBC representing cells which secrete antibodies against RaRBC.
Blood sampling and testing
Blood samples were collected by caudal puncture (Steucke and Schoettger,
1967). The hematocrit value was determined using a standard technique for
fish (Amlacher, 1976). Sera were stored at —20°C. Serum levels of oxyTC
were determined by the agar well diffusion method using Bacillus cereus
(ATCC 11778) as assay organism (Bennett et al., 1966). A calibration curve
was prepared using oxyTC dissolved in normal carp serum. The bacteria were
a gift from the National Institute of Public Health (Bilthoven, The Netherlands
Experimental
set-up
Animals were divided into five groups, which were kept in equal sized
aquaria. Control animals received no treatment (group I, n = 28). Others
received intraperitoneal (i.p.) injections of phosphate buffered saline (PBS)
every 3 days (group II). Group III was fed with pellets containing 2000 ppm
oxyTC. Groups IV and V received oxyTC by i.p. injection every 3 days (dosagi
in Table I). Groups II—V consisted of 50 animals each.
In scale transplantation experiments each group consisted of six fish (three
large and three small). In rosette tests, six (primary response) or four (secondary response) animals were used for each determination.
Student's f-test was used to test the significance of differences between
groups.
154
RESULTS
Growth effects
No difference in growth rate was observed between non-treated and PBSinjected animals (Table I, groups I and II). Feeding with oxyTC-containing
pellets had a significant growth promoting effect (P < 0.005; group III). However, injections with oxyTC diminished growth ( P < 0.005; groups IV and V).
The growth inhibition was more pronounced with higher oxyTC doses.
Differences between the two weight-classes became smaller during the experiment, because small animals grew faster than large ones in all experimental
groups, except group I.
TABLE I
Oxytetracycline (oxyTC) treatment and growth in carp
Experimental group
Size
class
Weight at
start of the
experiment
(g)
Weight
after
treatment
for one
month (g)
Percentile
increase
in weight
None
None
Small
Large
39 ± 2*
75 ± 2
50 ± 4
101 ± 4
28
35
II: PBS-injection
0.15 ml PBS/animal
0.40 ml PBS/animal
Small
Large
30 ± 2
77 ± 1
41 ± 2
96 ± 4
37
25
III: OxyTC-feeding
Pellets supplemented
with 2000 ppm oxyTC
Small
Large
35± 1
77 ± 2
54 ± 2
112 ± 3
55
45
IV: OxyTC-injection 60 mg oxyTC/kg fish
low dose
60 mg oxyTC/kg fish
Small
Large
31 ± 1
77 ± 2
39 ± 2
89 ± 3
25
15
V: OxyTC-injection 180 mg oxyTC/kg fish
high dose
180 mg oxyTC/kg fish
Small
Large
35± 2
84 ± 2
42 ± 2
94 ± 3
20
12
I:
Control
Treatment
All experimental groups (28—50 animals/group) were fed daily with pellets weighing 2.5%
of their initial body weight. Control animals received no treatment (group I). Group II
received an intraperitoneal (i.p.) injection of phosphate buttered saline (PBS) every 3 days.
The other groups received the antibiotic daily by feeding with oxyTC-supplemented
pellets (group III) or by i.p. injection every 3 days (Groups IV and V). Weighing of the
animals was performed at the start of the experiment and 1 month later.
•Arithmetic mean ( ± 1 standard error).
After 48 days of feeding with antibiotics, animals in group III showed for
the first time demonstrable levels of oxyTC in their serum; the maximum concentration observed was 1.25 ng oxyTC/ml. Injections with oxyTC gave rise
to an earlier appearance of higher antibiotic levels in serum. After 16 days of
treatment animals of group IV had 5ng oxyTC/ml and group V 10—15
Hg oxyTC/ml serum. In general oxyTC levels were influenced by the time
between the last oxyTC injection and blood sampling.
155
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Differential blood cell counts in Oxytetracycline (oxyTC)-treated carp
Experimental group
I:
Control
V: OxyTC-injection**
high dose
Number of cells (X 10 7 ) per ml*
Erythrocytes
Lymphocytes
+ monocytes
135 ± 2
110 ± 1
Granulocytes
Thrombocytes
4.6 ± 0.3
0.58 ± 0.20
0.32 ± 0.02
3.0 ± 0.3
0.66 ± 0.06
0.30 ± 0.03
* Arithmetic mean (± 1 standard error) of five animals.
** Cell counts were performed 37 days after start of oxyTC treatment.
TABLE IV
Granulocytes in the spleen of carps treated with Oxytetracycline (oxyTC)
Experimental group
Percentage granulocytes of splenic white cells
9 Days after
7 Days after
primary injection secondary injection
13 Days after
secondary injection
I:
Control
n.d.*
7.0 ± 0.6
4.2 ± 0.6
II:
PBS-injection
n.d.
4.5 ± 0.3
6.9 ± 2.5
III: OxyTC-feeding
n.d.
5.9+
1.0
5.0 ± 0.7
IV: OxyTC-injection
low dose
13.4 ± 1.0
19.0 ± 2.7
15.5 ± 1.7
V:
21.7 ± 2.1
32.6 ±'12.2
n.d.
OxyTC-injection
high dose
Animals were injected with 1 0 ' rabbit red blood cells (RaRBC) intramuscularly (i.m.) at
35 days after start of PBS injection or oxyTC treatment. A secondary injection ( 1 0 '
RaRBC, i.m.) was given 24 days later. Cells were counted in a hemocytometer under a
phase-contrast microscope (objective 20 X ). Each point represents the arithmetic mean
(±1 standard error) of six (primary response) or four (secondary response) animals.
* n.d. = not done.
splenic leucocytes were granulocytes. However, the low dose oxyTC-injection group (IV) had 10—20%and the high dose oxyTC-injection group (V)
showed even higher numbers of granulocytes (20—30%).
| DISCUSSION
|
The effect on the immune status of animals israrely taken into consideration
when chosing an antibiotic for therapeutic use. Yet, adverse sideeffects are
159
unwanted because the immune system has to co-operate with the antibiotic.
The antibiotic generally inhibits growth of the pathogen allowing the immune
system to eliminate the invaded micro-organisms.
The data about the effects of antibiotics upon the immune system are
rather confusing. Many reports are known describing positive, negative or no
effects on the immune system. However, this picture is based on effects of
different antibiotics studied in different animal species. Moreover, the parameters used (e.g. susceptibility to diseases) reveal overall effects on the wellbeing of an animal, which is not exclusively dependant on the immune status.
No studies are available concerning the effects of antibiotics upon basic
immunological processes such as antigen recognition and antibody productior
Besides being used for prevention and control of diseases, antibiotics are
used in animal husbandry because of their growth promoting effect. Growth
of freshwater fish such as Labeo and carp is promoted by supplementing fooc
with oxyTC (Mitra and Ghosh, 1967; Sukhoverkhov, 1967). In our experiments with carp the same growth promoting effect was found after feeding
with oxyTC-containing pellets. This growth promotion might be caused by
changes in the intestinal flora or gut epithelium allowing a more efficient
food uptake.
In order to study humoral immunity, animals were injected with RaRBC
instead of the usual sheep red blood cells (SRBC). SRBC and carp lymphocytes look quite alike under the phase-contrast microscope. Therefore RaRBC
which are smaller than SRBC, were used, allowing easy detection of the centr
lymphoid cell in a rosette. No data are available about the kinetics of antigen
recognizing and antibody producing cells after injection of this antigen in
cyprinid fish. The time schedule for the rosette test was based upon the work
of Warr et al. (1977), who showed that maximum numbers of splenic RFC
appeared at day 8 after injection of goldfish with horse erythrocytes.
In the scale transplantation experiments we observed no effect of orally
administered oxyTC, but injections of oxyTC delayed allograft rejection.
This observation is in agreement with that of Hildemann and Cooper (1963)
who found that in the teleost Fundulus heteroclitus, injections with tetracycline prolonged survival times of allografts.
Antibiotics have an influence not only on the peak height of the humoral
immune response but also on the kinetics of the cells involved. For instance,
the peak day of antibody producing cells after immunization of mice with
SRBC is influenced by streptomycin (Toshkov and Slavcheva, 1968); in
chickens Chlortetracycline affects the kinetics of the antibody response to
Salmonella cholerasuis (Prochâzka et al., 1968). These facts must be taken
into account when comparing our data on RFC in different experimental
groups during the primary and secondary response against RaRBC.
In control groups, injected with RaRBC, maximum percentages of RFC
scored were 1.5—3. This is in the same order of magnitude as reported by
Chiller et al. (1969) for rainbow trout and Warr et al. (1977) for goldfish.
OxyTC suppressed the number of RFC independent of the route of administration.
160
Taking oxyTC serum levels into account when regarding the effects of
oxyTC on the humoral and cellular immune response, the humoral immune
system might be more sensitive to low levels of antibiotics. This is suggested
by the observation that serum levels of oxyTC were low and humoral immunity
was suppressed while cellular immunity was not affected in animals which
were fed with oxyTC-containing pellets.
Animals injected with oxyTC showed raised levels of granulocytes in their
spleen. The function of granulocytes in fish is not yet clear. A mast cell
function is suggested by Ellis (1977) and Barber and Mills Westermann (1975,
1978). It is known that mammalian granulocytes fulfill an important role in
non-specific defence. It is tempting to speculate that in those cases where
specific lymphoid defence mechanisms are blocked, the phagocytic system
becomes more active.
The presented results have shown that oxyTC dramatically suppresses the
immune system of carp. Cellular immunity is suppressed when injecting the
antibiotic, but not by oxyTC feeding. Humoral immunity however is
suppressed in all cases. Thus, bacterial growth and the specific defence
mechanism of fish are suppressed by oxyTC at the same time. Consequently
any non-bacterial infection or an invasion by resistant bacteria causes serious
problems.
Since the concentration of oxyTC used in commercial fish farming is the
same as that used in our feeding experiments we recommend a selective and
very cautious use of this antibiotic. It may be useful to develop proper
vaccination methods for the prevention of the major diseases in large-scale fish
culture. In this respect more basic research on issues like raising long-term
immunological memory in fish is needed.
ACKNOWLEDGEMENTS
We are grateful to Professor J.F. Jongkind and Professor E.A. Huisman
(Agricultural University, Wageningen) for helpful advice during the
performance of this study and preparation of the manuscript. Furthermore,
we thank Dr. R. Bootsma (Veterinary Faculty, University of Utrecht) for
his interest and valuable suggestions.
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Hildemann, W.H. and Haas, R., 1960. Comparative studies of homotransplantation in
fishes. J. Cell. Comp. Physiol., 5 5 : 227—233.
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163
APPENDIX VI
165
or by a decomposition product (Sanford, 1976). An adverse effect of antibiotics
which is rarely taken into account is the effect on the immune system. I t has been
demonstrated that antibiotics can induce immunosuppression in man (Daikos and
Weinstein, 1951; Daniel et a l . , 1964), laboratory animals (Stevens, 1953; Weisberger
et a l . , 1964), domestic animals (Fortushnyi et a l . , 1973; Lyashenko, 1966), birds
(Lakhotia and Stephens, 1972; Panigrahy et a l . , 1979) and fish (Hildemann and Cooper,
1963; Levy, 1963).
Reports on the effects of antibiotics in fish mainly deal with cellular immunity.
I t appeared that antibiotics can delay allograft rejection (Hildemann and Cooper,
1963; Levy, 1963). In a previous paper (Rijkers et a l , 1980a) we reported the
immunosuppressive effect of Oxytetracycline (oxyTC) in carp. I t was shown that both
cellular and humoral immunity were depressed after feeding or injecting the
antibiotic. In this paper the effect of oxyTC upon the regulation of humoral
immunity in fish is studied in more d e t a i l . On base of the differential effects of
oxyTC on primary and secondary immune responses we propose a model for t h e c e l l u l a r
interaction during the humoral immune response in f i s h .
MATERIALS AND METHODS
Animals
Carp (Cyprnnusoavpio) were bred in our laboratory. They were kept in aquaria
with running tap water at 20°C. Animals were fed daily on pelleted dry food (K30,
Trouw & Co, Putten, The Netherlands), amounting 2.5% of their body weight, by means
of a "Scharflinger" automatic feeder. Eight to twelve months old animals, weighing
100-300 g were used.
Antibiotic
Oxytetracycline (oxyTC) was administered either by intraperitoneal ( i . p . )
injection of a 50 mg/ml Engemycine^solution (Mycofarm, De B i l t , The Netherlands)
or orally by feeding pellets supplemented with 2000 ppmoxyTC.
Antisera
Goat antiserum to rabbit IgG conjugated to peroxidase (GAR/IgG/P0) was obtained
from Nordic (Tilburg, The Netherlands). Rabbit antiserum to pike immunoglobulin
heavy chains (RAP/Ig/H) was prepared and described by Clerx (1978).Rabbit antiserum
to carp immunoglobulin (RAC/Ig) was prepared as described previously (Davina et a l . ,
1980).
168
Quantitation of immunoglobulin and protein levels
The enzyme-linked immunosorbent assay (ELISA) and rocket electrophoresis were
used to quantitate immunoglobulin levels using normal carp serum as a standard. In
the ELISA, essentially the procedure of Voiler et a l . (1976) was followed. Carp
serum was coated to polystyrene microhaemagglutination plates in two-fold serial
dilutions starting with 1:12,000. RAP/Ig/H (1:100) and GAR/IgG/PO (1:100) were used
as antiserum and enzyme labeled antiglobulin respectively. After incubation with
substrate (hydrogen peroxide and 5-aminosalicylic acid) 450 nmabsorbance was
measured with a Titertelc^Multiskan (Flow, McLean, USA).
In the rocket electrophoresis according to Laurell (1972) gels made up of 1%
RAC/Ig and 1% agarose (No. 4, Nordic) in high resolution buffer, pH 8.8 (Gelman, Ann
Harbor, USA) were used.Prior to the electrophoretic run (110 V, 21 h, room temperature) samples were carbamylated according to weeke (1968).
Total serum protein was determined according to Lowry et a l . (1951) using bovine
serum albumin (BSA, Organon Teknika, Oss, The Netherlands) as a standard.
Antigen and immunization
Sheep red blood cells (SRBC) were obtained from the Department of Animal Husbandry,
Agricultural University, Wageningen. Cells were washed 3 times with phosphate
buffered saline (PBS, pH 7.2) before use. 10 SRBCwere injected intramuscularly (i.m.)
in the dorsal region. I f needed a second injection was given 1 month later.
PFC assay
Antibody forming cells (PFC) were determined with the haemolytic plaque assay
adapted for carp as described previously (Rijkers et a l . , 1980b). Bream{Abramis
brama) serum was used as complement source. PFC siides-were incubated at 25°C for 2 h.
Experimental set-up
Animals were divided into 3 groups. Control animals received no oxyTC. OxyTCfeeded animals received pellets containing 2000 ppm oxyTC (50 yg oxyTC/g fish/day).
OxyTC-injected animals received an i . p . injection of Engemyciné^every 3 days
(20 yg oxyTC/g fish/day). Primary and secondary anti-SRBC responses and serum
analysis were carried out after treatment of the animals for 6 weeks. During the
anti-SRBC response oxyTC treatment was continued.
169
RESULTS
Serum analysis
Feeding oxyTC had no effect on total serum protein concentration, while
oxyTC-injection reduced protein levels only by 20%. In contrast to total protein,
serum immunoglobulin levels were markedly reduced after oxyTC feeding (decrease
40-75%) and oxyTC injection (55%) (TABLE I ) .
TABLE I
The effect of Oxytetracycline on serum protein and immunoglobulin levels in carp
Protein
Immunoglobulin (U/ml)
(mg/ml)
Rocket
ELISA
Control
4.63 + 0.42
150 + 36
116 + 11
OxyTC-feeding
4.72 + 0.30
40 + 10
102 + 19
OxyTC-injection
3.71+0.10
67 + 37
n.d.
Treatment
Protein and immunoglobulin (lg) determinations were carried out in sera of normal
carp (control) and animals treated with Oxytetracycline (oxyTC) for 6 weeks. Ig
concentration was determined with the rocket and ELISA technique and expressed as
arbitrary units (U/ml). "Arithmetic mean + 1 S.E. (n=4), **n.d.= not done.
Primary and secondary anti-SRBC response
g
Animals were injected with 10 SRBC ( i . m . ) . At day 12 after injection (peak of the
response) spleen, pronephros and mesonephros were removed for determination of the
number of antibody forming c e l l s . Control animals showed PFC levels comparable with
results obtained earlier (Rijkers et a l . , 1980c). PFC numbers in spleen, pronephros
and mesonephros were reduced by approximately 85%after feeding with oxyTC. OxyTC
injection caused an even stronger inhibition (95%, Fig. 1).
To investigate the effect of oxyTC on the formation of immunological memory and
the secondary response, animals which were treated for 2 weeks were injected with a
g
priming dose of 10 SRBC ( i . m . ) . A second injection was given 4 weeks later. Control
animals tested on day 12 (peak of the response) showed a 10-fold enhancement of
PFC numbers compared with the primary response. Surprisingly, the secondary response
in oxyTC-feeded and oxyTC-injected animals was not significantly different from the
response in control animals (Fig. 2).
In conclusion oxyTC caused a strong inhibition of the primary anti-SRBC response
170
PFC/10 6 WC
10 2
ifi
ft
10'.
p
n
1J
pronephros
spleen
1
mesonephros
Fig. 1. Primary anti-SRBC response in carp. All animals were given an intramuscular
injection with 10-? SRBC after treatment for 6 weeks. The number of plaque
forming cells (PFC) per 106 white cells (WC) in spleen, pronephros and mesonephros of
control (open bars), oxyTC-feeded (shaded bars) or oxyTC-injected animals (hatched
bars) was determined 12 days after injection. Each bar represents the arithmetic mean
+ l.S.E. (n=4).
PFC/106WC
104_
103_
A
A
i
2
10
II
A
A
A
I
10'_
1J
spleen
i
y
pronephros
A
mesonephros
Fig.2. Secondary anti-SRBC response in carp. All animals were given a primary
intramuscular (i.m.) injection with 109 SRBC after treatment for 2 weeks.
The response was measured 12 days after a second i.m. injection which was given 4
weeks after priming. See Fig. 1 for further explanation.
171
whereas the formation of immunological memory and the secondary response i t s e l f
were apparently not affected.
Regulation by specific antibody
Antigen presentation at the start of the secondary response might be different
from the same process during a primary response due to the presence of some specific
antibody at the i n i t i a t i o n of the secondary response.
In order to investigate this hypothesis carp were i.m. injected with SRBC
simultaneously with an i.v. administered anti-SRBC serum simulating a "secondary"
antigen presentation during a primary response. In preliminary experiments various
dosages of carp anti-SRBC serum were tested. Dilutions of 1:10 and 1:100 were
inhibitory but a dilution of 1:1000 caused a significant increase in the number of
PFC in pronephros and mesonephros compared with control animals which were injected
with SRBC and normal carp serum.
As expected oxyTC depressed the primary anti-SRBC response when normal carp
serum was injected in combination with the antigen. However, anti-SRBC serum (1:1000)
abolished completely the immunosuppressive effect of oxyTC on the primary response
in oxyTC-feeded animals. In oxyTC-injected animals the same phenomenon was observed
but to a lesser extent (TABLE I I ) .
TABLE I I
The effect of antibody on the primary anti-SRBC response in carp
Treatment
Plaque forming cells / 10 white cells
Spleen
NCS
Control
Pronephros
anti-SRBC
NCS
Mesonephros
anti-SRBC
NCS
anti-SRBC
29+4
37+9
59+7
179+45
29+5
76+23
OxyTC-feeding
4+2
67+28
27+11
151 +34
15+6
79+11
OxyTC-injection
2+1
24 + 5
5
94+31
2+1
34+11
o
Carps treated for 6 weeks were injected intramuscular with 10 SRBC. At the same
time the animals were injected intravenously either with 0.5 ml carp anti-SRBC serum
(agglutination-titre: 2 1 " ) , diluted 1:1000 in phosphate buffered saline (PBS) or
with 0.5 ml normal carp serum (NCS, diluted 1:1000 in PBS). The number of plaque
forming cells in spleen, pronephros and mesonephros was determined 12 days after
injection.
Arithmetic mean + 1 S.E. (n=4).
172
I t is concluded that the differential oxyTC sensitivity of primary and secondary
immune responses is probably a reflection of differences in antigen presentation in
rfhich specific antibody plays an important role. However, other factors (macrophages,
suppressor cells) might be involved too since anti-SRBC serum can not f u l l y restore
the primary anti-SRBC response in oxyTC-injected animals.
DISCUSSION
Our findings that serum immunoglobulin levels were reduced by 40-75% after
DxyTC treatment (TABLE I) whereas the antibiotic had no effect on the secondary
anti-SRBC response (Fig. 2) are apparently in contradiction with one another. One
nay assume that under natural circumstances the vast majority of serum immunoglobulin
is produced as consequence of secondary responses against so-called "environmental
antigens" entering through epithelial barriers. In oxyTC treated animals these
environmental antigens (as far as drug sensitive bacteria are concerned) are
eliminated by the a n t i b i o t i c , causing diminished antigenic load and consequently
reduced immunoglobulin levels. A comparable situation exists in germfree and
specific pathogen free mice were low immunoglobulin levels were observed compared
«ith conventional mice (Sell and Fahey, 1964; Van Snick and Masson, 1980). The
secondary anti-SRBC response was evoked by a high antigen dose while secondary
responses against environmental antigens are probably caused by low antigen doses.
The influence of antigen dose in a secondary response w i l l be discussed in more
detail when dealing with the regulation of the immune response.
A discrepancy between immunoglobulin levels was observed when comparing the
rocket and ELISA technique (TABLE I ) . This difference may be caused by the use of
2 different antisera for these techniques. Moreover, in rocket electrophoresis only
precipitated complexes are visualized while in ELISA the a f f i n i t y of the antiserum
for the antigen is important.
The immunosuppressive effect of oxyTC on a primary anti-SRBC response as measured
by PFC numbers confirmed our earlier results with the rosette assay (Rijkers et a l . ,
1980a). However, under the experimental conditions during this study, secondary
anti-SRBC responses were not inhibited by oxyTC. I t appeared that high doses of
passively transfered specific antibody i n h i b i t the primary anti-SRBC response (feed
back inhibition) whereas low antibody doses have a stimulating effect (TABLE I ) . The
same phenomenon has been encountered in mice (Möller and Wigzell, 1965) and chickens
(Morgan and Tempeli s , 1977).
Following a primary injection with SRBC cell interaction is necessary for the
development of a proper immune response. SRBC is generally accepted as a "T-dependent"
antigen in mammals (Greaves et a l . , 1974) but also in fish (Sailendri, 1973). Nonlymphoid cells such as monocytes, macrophages and dendritic cells are involved in
the regulation of the immune response and memory formation (Unanue, 1975; Van Rooijen,
1980). I t has been shown that antibiotics interfere both quantitatively and
quantitatively with monocytes (Kreutzmann, 1977) and macrophages (Rhodes and Hsu,
1974; Alexander, 1975). These are interesting observations because macrophages
play an essential role in antigen presentation (Mosier and Coppleson, 1968).
Differences in antigen presentation during primary and secondary responses are
caused by the formation of immune complexes. These complexes are an important
factor in the regulation of memory formation and the secondary response i t s e l f .
Depending on the antigen:antibody ratio and the antibody class involved immune
complexes have an immunostimulating or an immunosuppressive effect (Eardley and
Tempeli s , 1975; Gordon and Murgita, 1975; Klaus, 1978). Wehave shown that a primary
anti-SRBC response is inhibited most clearly by oxyTC injections (Fig. 1.). As a
consequence the antigen:antibody ratio in immune complexes w i l l be different for
control, oxyTC-feeded and oxyTC-injected animals which may result in differences
in memory formation. In addition, a second antigen injection in control and oxyTCtreated animals w i l l result in immune complexes with a different antigen:antibody
r a t i o . Wehave shown that a second injection with a high antigen dose evoked a
secondary response which was not affected by oxyTC (Fig. 2 . ) . However, a conclusion
that a l l secondary responses are oxyTC insensitive is premature. More refined
immunization schedules and antibiotic treatment regimens, as well as information
on antigen trapping and persistence are needed to c l a r i f y the role of oxyTC in
memory formation and secondary immune responses in' f i s h .
On base of the data discussed above we propose the following model to explain
the effect of oxyTC upon the immune response:
During a primary response oxyTC reduces antigen presentation by non-lymphoid
cells to a suboptimal level resulting in a depressed humoral response. Whena
"secondary" antigen presentation is simulated by injecting specific antibody, antigen
is presented in a supraoptimal way. The final antigen processing remains at an
optimal level even after oxyTC interference. Therefore oxyTC has no inhibiting
effect on normal or simulated secondary responses. Another possibility is an
adverse effect of oxyTC upon cell interaction between lymphoid c e l l s . During the
primary response cell interaction might be a prerequisite for differentiation of
B-like cell into plasma cells. However, during the secondary response another type
of antigen presentation permits a T-independent B cell response. I f this is true,
oxyTC has no effect upon the secondary response.
In conclusion a careful use of the drug oxyTC is recommended since oxyTC exerts
an immunosuppressive effect under certain conditions. On the other hand the data
presented here have shown that this drug can be a valuable tool for studying the
regulation of the humoral immune response in lower vertebrates.
174
ACKNOWLEDGEMENTS
The technical assistance of Mr. J.A.M. Wiegerinck and biotechnical support of
Mr. S.H. Leenstra are gratefully acknowledged. Furthermore we thank Mr. W.J.A.
Valen for drawing the figures, Mr. D. Gubbins (Cambridge, Mass., USA) for his
work on the rocket electrophoresis and Or. J.P.M. Clerx (Dept. of General Virology,
State University, Utrecht) for his generous g i f t of rabbit anti-pike immunoglobulin
heavy chain serum. We are grateful to Prof. J.F. Jongkind for helpful advice
during performance of this study and preparation of the manuscript.
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