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Cell adherence to larvae of Dictyocaulus viviparus in vitro
References
'
CHRISTISON, G.1. & CURTIN, T.M. (1%9) Laboratory An'-'Care 19.259-262 DAVIES. A.J., FLEET. I.R.. HARRISON. F WALKER. F.M. (1979) Journal of Physiology 3 DOUGLAS. R.H. & GINTHER, O.J. (1976) Prostaglandrns 25 1-260 GLEESON. A.R.. T H ORBURN, G.D. & C(>X, R.I. (I 974) Prosta~la, ndins 5,521- 529 GURPIDE, E. (1975) Tr acer Method s in Hormonle Research. New . , York. S~nneer-vcrrag . DD 74-75 . RINO, S.P % H A N I N G ; R V . , ~ 1 LI'SZEK. F 7 SPEROFIF. L. (1977) I3rostanlandiri >gicalReview HORTON. E.W. &POYSER. N.L. (I
..
'
..
.. -"
595-651 KINDAHL, H.L.. EDQVIST, L.E., BANE, A. & GRANSTROM. ' (1976a) Acta Endocrinolonica 82, 134- ' '^ I A H L , H.L., EDQVIST, L.E., GRAl (1976b) Prostaglandins 111.871-878 CHELL, M.D., FLINT,, 4.D.F. & TLIRNBULL, I ostaglandins 11,319-329 PET1ERSON, A.J.. TERVIT, H.R., FAIRCLOUGH, R.1I., HAVIK, P..1. & SMITH, J.F. (1976) Prostaglandins 12, 551-551S SHE:MESH, M. & HANSEL, W. (1975) Proceedings of the Society Biology and Medicine 148. 123-126 ~ O Experintental I S H l l.LE. V.M.. KARLBOM, I., EINARSSON, S., LARaau1., -., NDAHL, H . & EDQV in. A 26, 169. terinarn~edizr
Research in Veterinary Science 1981,31,389-391
ktyocaulus viviparus in vitro
herence to larv: N. H. H. KNAPP-,
G . A. OAKLEY
Imperial Chemical Ind~triesLimited, Phurrrruccur Division, Alderley House. Alderley rarK, Mamesj~eru, Cheshire
Bovine eoslnopnlis survivea lor up l o 48 ho urs in vitro in a medium of undiluted bovine serum and bee9m e adherer~ t t o 1he surface of I>ict~~ocaulus viviparus larv ae earlier 1than was other cells found in peripheral blood. Cell adherence .. . associated with a hea I labile fact o r in normr11 bovine serum and a hea I stable fac lor in hypetrimmune slerum. A f a ctor associated with Ieuco,cytes in vitl.o appeared lo cause la immobilit:y. Cells fi.om calves treated wvith levamisole , .. .. to those from orner .. - sources. I : behaved iaen~ically suggested that eosino phils are an important element in defence aglainst I> viviiparus infec lion.
.
~
betvYeen protect~on ana clrculatrng eoslnopnlls ana, or, corn plement fixing antibodies has been noted (Michel and Cor nwell 1959, Oakley 1980). Possibly these factors repr esent parts of a complex system which can only P.._. lunc:tion when all components are present and working in Coolperation. It was therefc)re decided t o investiga .te the in vit ro activity d or serumI containinr eosinoph!ils against o f \t hole- - bl001 . . nnrtlr lor"
-
erials and n 1 were withdrawn in heparinised lood sampl nges trom tour I W ~g calves, two of which had been ntained wor.m free and two infected twice at a four week .us per kg rval with 210 third stat;e larvae o ueight.
1 1 has tLL,, JUBBcb,cU [.,(111
n
.
FIG 1: Larva after three hours incubation
-
The coffin bottles and contents were incubated at 37°C' for 48 hours. The base of each coffin bottle was the]n examined through an inverted microscope, but it was on1Y possible t o view larvae under low power because of th ' ickness of the plastic and poor transmission of liehI 1 rough the medium. d The fluid was discarcled and tht: coffin bo th methyl alcohol to fix larvae or cells whicl1 aanerea [I Q rhn I.-..,, , n o r h m " out of th-* ,I1 e base of the bottle. '.,,L bcjttle and us;:d as a micr oscope slid(e. Larvae a]nd cells wer e Stiained with (jiemsa and examined u~ndera micl.oscope.
..%.% A range of incubation times were used :one, two, wrr*l*sed. three, f'our, five, six. 12. 24 and 48 hours. UQ
: study4I
..,
,..I
udy 2
-. . ..
ntooa cells In v.> ml wnole o ~ o o awere wasnea tnree tirnes by centrifugation and resuspension in serum and made up to a final volume of 5 ml. This was transferred by Stl:rile Pasteur pipette to a 34 by 45 mm perspex box containing a 20 by 40 mm coverslip (replicates with bot glass and plastic coverslips were prepared). Othe constituents were then added so that the final culture war 0.5 ml whole blood taken, the cellular fraction washed i rum and re)suspended in a final 1rolume of 5 ml; 5.0 a rum (duplilcate samplts were pre*pared with either fet: df serum ()r hyperim mune seru m); 0 . I ml antibiotic - t ~ u u uunits per ml); 10 thir lutamine; (1.2 ml hepar~n st;age larvae of D vivipams; and one piece of !rtrand nylo th read. Control samples in which 1cells from whole bloo WIere omitted were also prepared. Cultures were incubated for thre:e hours at 37°C. Fluid .. wer as then removed by pipette and discarum. I ne.ooxes ~ o d e dwith methyi kc; )hol to fix aIny materiall adherent t e cover s lips. ~ Cover!dips were removed, !stained wit iemsa and etxamined urider the mic:roscope. ,.A,.,.
..--2..J
VL
L.
'udy3 Cultures w=,, p,Lp,.
Cult'ures were preparea as ~n stuay J except rnar one s l y worm free calf was infected with D viviparus (20 previo~ third stage larvae, per kg) and the infection treated with 24 hours later. Blood samples were taken ...- .. 14 1~vnmlsoIe .-.-.... days a fter levamisole treatment. Also replicates I~f each rl were serum unheated and heated (56°C for one hou~ includebd in the cultures prepared. To check survival of eosinophils a difrctcrtuat LCucocyte .e use and iafter 48 was made o~nblood saliples befo~ incubation. on culturt: fluid fro1n control (:ultures of D vivipa'ms. ~
'
rae incubated in the absence of blood were mobile ~cubationamd before fixing. Those incubated in the ce of blood were immobile (irrespective of the source ~ - - > .. Im usea) and on stainine found to be surrounded bv a halo ()f densely packed m lononuclear, and poly onuclear cells. F...-l..
.. .
.., .
1
L a r{ae ~ incubat ea In tne aasence or a ~ o o acells were mobile: before fix ing. Those incubated in the presence of blood cells were i mmobite and partially surrounded by an ' ' ~ncoml prere naro of cells. After fixation it was fouina tnat larvae adhered to plastic coverslips but not t() glass. Examination after staining showed that cells adhl:rent to larvae were eosinophils. No cells adhered to nylon.
.. . .
cell aanerence ro D vivil 3d of prepa ration) wou Id be accorcied toany c:ell type. was. ..found, .despite . . a. 1(oss. in. all. cc211 types, thlat many. eoslnioph~lssurvlvect In vltro In aovlne serum anaI ala not -ently require special additives t o prolong viability. Adhe rence to larvae of D viviparus was not thle special prerogative of any cell type, but eosinophils we!re found -AL...ing earlier than other cells. Adherence was mca.. a-r;1.r :d when a plastic micrc,scope slide was used, as larvae viviparus did not adher,e to glass alFter fixationI. No cell .ence to nylon was noted. >.-. - method of quantifying rne uegrer V I cru atdherence was etvolved. Estimation of percentage of parasite surtace covered (MacKenzie et al 1978) did not prove practicable as larval: were invariably coiled and the surface partially o b s c!red ~ in places. Eosinophils were always involved in .ed the p i c tIre. ~ adhe1,ence beforc:other cell t ypes obscu~ At least two f actors associated with bovine ser um were invollved, one be ing heat la1~ i l eand the other a he,at stable facto r found on1y in hyperir nmune seru~ m It. was 1:resumed -. --> >~ that t he former was complemenr anu rne latter anti1oouy. probable that eosinophils in a non imm .eems une host It s FIG 2: Larva after four hcJurs incubatic may c:ombat invading D viviparus larvae in the prc:sence of compilement and that this killer cell activity urould be ^^I.^. t Iced by circulating antibody in an immune ho!>,. Leitamisole used 24 hours after infection did ncbt appear Larvae i~ncuoatea ~n rne aosence of blood cells wc're t o exc:rt any influence on eosinophil adherence to larvae in mobile afte r 48 hours incubation. Those incubated in I.he vitro. Where eosinophils were adherent to larvae, t he larvae ,rr v l c ~ c l l c cuf blood cells were immobile after two h o ~ . - were immobile before fixation. This could be beta[use such . . . incubation. After three hours incubation an incomplete cells are only attracted to immobile, and possibly dead, halo o f eosinophils (Fig 1) was observed surrounding larvae or because the eosinophils cause the larvae to become larvae. After four hours a mass of cells surrounded larvae immobile. However, since larvae incubated in the absence (Fig 2) and it was difficult to identify eosinophils amongst of leucocytes were invariably mobile before fixation, some them. facto r associated with the leucocytes m lust have remsulted in the la ck of larval movement. Analogy Hri th Nemarolspiroides Study 4 d u b iIS ~ (MacKenzie et al 1978) and Sc-histosoma mansoni (..~.a n m o u det a1 1975) suggests this f actor was Iprobably No mesoDrills survlveu 48 n o u n I I I L ~ in~ vitro ~ ~ but ~ , ~ . associated with t h e eosinophil cell p o p ulatlon. 45 per cent of eosinop'hils, 27 per cent o f lymphocytes and are an t is sugge equently, i 22 per ce nt of neljtrophils did survive. Eosinophil Cons' sted that eosinophils adherence to larvae \.sas seen in all cultures containing imp01rtant elemel tot the ho!st's defence against D v iviparus. .. . unheated serum. Where heated serum was used, adherence v i v i ~7rus. l was seen 01ily in cultu res contain ing hyperimmune seru Received forpublication April 1 5 , 1981 Cells from I he calf trea led with levamisole did Accepted July 13, 1981 behave diff erently frorn those fro!rn other sou 8
9 . .
,I% %'4DI,,
~
F..a",...--
- -
- 3 . .
..-L
.L
-A
.
Discussion
Eosinopb . , . 111-rlcn cell mlxtures prepared from periton' wasnlngs in laboratory animals have been used in the Stu of cell adherence in schistosomes (Mahmoud et a1 1975) nematode worms (MacKenzie et at. 1978). In the current work cell types found in the blood stream were used as far as Possible in the natural proportions existing in calves, SO that nn l*ndue domination in numbers o r stimulation (by .
L -
.
rences
:Em. E.E. - (-1973) Veterinlory Record 9 . t N L I t , L.1)..
PRESTON. P.M. & ( rure 276, 826.-828 .. - MAHMOUD, A.A.F., WAKREN, s.W. & PETEKS, P.A. (IY75) Journal of ErperirnenfolMedicine142.805-813 MICHEL. J.F. & CORNWELL, R.L. (1959') Veterinary Record 71, 912-913 OAKLEY. G:A: (1980) PhD thesis. CNM/L.IVCIPUUIUIniversity. -
'
CHRISTISON, G.1. & CURTIN, T.M. (1%9) Laboratory An'-'Care 19.259-262 DAVIES. A.J., FLEET. I.R.. HARRISON. F WALKER. F.M. (1979) Journal of Physiology 3 DOUGLAS. R.H. & GINTHER, O.J. (1976) Prostaglandrns 25 1-260 GLEESON. A.R.. T H ORBURN, G.D. & C(>X, R.I. (I 974) Prosta~la, ndins 5,521- 529 GURPIDE, E. (1975) Tr acer Method s in Hormonle Research. New . , York. S~nneer-vcrrag . DD 74-75 . RINO, S.P % H A N I N G ; R V . , ~ 1 LI'SZEK. F 7 SPEROFIF. L. (1977) I3rostanlandiri >gicalReview HORTON. E.W. &POYSER. N.L. (I
..
'
..
.. -"
595-651 KINDAHL, H.L.. EDQVIST, L.E., BANE, A. & GRANSTROM. ' (1976a) Acta Endocrinolonica 82, 134- ' '^ I A H L , H.L., EDQVIST, L.E., GRAl (1976b) Prostaglandins 111.871-878 CHELL, M.D., FLINT,, 4.D.F. & TLIRNBULL, I ostaglandins 11,319-329 PET1ERSON, A.J.. TERVIT, H.R., FAIRCLOUGH, R.1I., HAVIK, P..1. & SMITH, J.F. (1976) Prostaglandins 12, 551-551S SHE:MESH, M. & HANSEL, W. (1975) Proceedings of the Society Biology and Medicine 148. 123-126 ~ O Experintental I S H l l.LE. V.M.. KARLBOM, I., EINARSSON, S., LARaau1., -., NDAHL, H . & EDQV in. A 26, 169. terinarn~edizr
Research in Veterinary Science 1981,31,389-391
ktyocaulus viviparus in vitro
herence to larv: N. H. H. KNAPP-,
G . A. OAKLEY
Imperial Chemical Ind~triesLimited, Phurrrruccur Division, Alderley House. Alderley rarK, Mamesj~eru, Cheshire
Bovine eoslnopnlis survivea lor up l o 48 ho urs in vitro in a medium of undiluted bovine serum and bee9m e adherer~ t t o 1he surface of I>ict~~ocaulus viviparus larv ae earlier 1than was other cells found in peripheral blood. Cell adherence .. . associated with a hea I labile fact o r in normr11 bovine serum and a hea I stable fac lor in hypetrimmune slerum. A f a ctor associated with Ieuco,cytes in vitl.o appeared lo cause la immobilit:y. Cells fi.om calves treated wvith levamisole , .. .. to those from orner .. - sources. I : behaved iaen~ically suggested that eosino phils are an important element in defence aglainst I> viviiparus infec lion.
.
~
betvYeen protect~on ana clrculatrng eoslnopnlls ana, or, corn plement fixing antibodies has been noted (Michel and Cor nwell 1959, Oakley 1980). Possibly these factors repr esent parts of a complex system which can only P.._. lunc:tion when all components are present and working in Coolperation. It was therefc)re decided t o investiga .te the in vit ro activity d or serumI containinr eosinoph!ils against o f \t hole- - bl001 . . nnrtlr lor"
-
erials and n 1 were withdrawn in heparinised lood sampl nges trom tour I W ~g calves, two of which had been ntained wor.m free and two infected twice at a four week .us per kg rval with 210 third stat;e larvae o ueight.
1 1 has tLL,, JUBBcb,cU [.,(111
n
.
FIG 1: Larva after three hours incubation
-
The coffin bottles and contents were incubated at 37°C' for 48 hours. The base of each coffin bottle was the]n examined through an inverted microscope, but it was on1Y possible t o view larvae under low power because of th ' ickness of the plastic and poor transmission of liehI 1 rough the medium. d The fluid was discarcled and tht: coffin bo th methyl alcohol to fix larvae or cells whicl1 aanerea [I Q rhn I.-..,, , n o r h m " out of th-* ,I1 e base of the bottle. '.,,L bcjttle and us;:d as a micr oscope slid(e. Larvae a]nd cells wer e Stiained with (jiemsa and examined u~ndera micl.oscope.
..%.% A range of incubation times were used :one, two, wrr*l*sed. three, f'our, five, six. 12. 24 and 48 hours. UQ
: study4I
..,
,..I
udy 2
-. . ..
ntooa cells In v.> ml wnole o ~ o o awere wasnea tnree tirnes by centrifugation and resuspension in serum and made up to a final volume of 5 ml. This was transferred by Stl:rile Pasteur pipette to a 34 by 45 mm perspex box containing a 20 by 40 mm coverslip (replicates with bot glass and plastic coverslips were prepared). Othe constituents were then added so that the final culture war 0.5 ml whole blood taken, the cellular fraction washed i rum and re)suspended in a final 1rolume of 5 ml; 5.0 a rum (duplilcate samplts were pre*pared with either fet: df serum ()r hyperim mune seru m); 0 . I ml antibiotic - t ~ u u uunits per ml); 10 thir lutamine; (1.2 ml hepar~n st;age larvae of D vivipams; and one piece of !rtrand nylo th read. Control samples in which 1cells from whole bloo WIere omitted were also prepared. Cultures were incubated for thre:e hours at 37°C. Fluid .. wer as then removed by pipette and discarum. I ne.ooxes ~ o d e dwith methyi kc; )hol to fix aIny materiall adherent t e cover s lips. ~ Cover!dips were removed, !stained wit iemsa and etxamined urider the mic:roscope. ,.A,.,.
..--2..J
VL
L.
'udy3 Cultures w=,, p,Lp,.
Cult'ures were preparea as ~n stuay J except rnar one s l y worm free calf was infected with D viviparus (20 previo~ third stage larvae, per kg) and the infection treated with 24 hours later. Blood samples were taken ...- .. 14 1~vnmlsoIe .-.-.... days a fter levamisole treatment. Also replicates I~f each rl were serum unheated and heated (56°C for one hou~ includebd in the cultures prepared. To check survival of eosinophils a difrctcrtuat LCucocyte .e use and iafter 48 was made o~nblood saliples befo~ incubation. on culturt: fluid fro1n control (:ultures of D vivipa'ms. ~
'
rae incubated in the absence of blood were mobile ~cubationamd before fixing. Those incubated in the ce of blood were immobile (irrespective of the source ~ - - > .. Im usea) and on stainine found to be surrounded bv a halo ()f densely packed m lononuclear, and poly onuclear cells. F...-l..
.. .
.., .
1
L a r{ae ~ incubat ea In tne aasence or a ~ o o acells were mobile: before fix ing. Those incubated in the presence of blood cells were i mmobite and partially surrounded by an ' ' ~ncoml prere naro of cells. After fixation it was fouina tnat larvae adhered to plastic coverslips but not t() glass. Examination after staining showed that cells adhl:rent to larvae were eosinophils. No cells adhered to nylon.
.. . .
cell aanerence ro D vivil 3d of prepa ration) wou Id be accorcied toany c:ell type. was. ..found, .despite . . a. 1(oss. in. all. cc211 types, thlat many. eoslnioph~lssurvlvect In vltro In aovlne serum anaI ala not -ently require special additives t o prolong viability. Adhe rence to larvae of D viviparus was not thle special prerogative of any cell type, but eosinophils we!re found -AL...ing earlier than other cells. Adherence was mca.. a-r;1.r :d when a plastic micrc,scope slide was used, as larvae viviparus did not adher,e to glass alFter fixationI. No cell .ence to nylon was noted. >.-. - method of quantifying rne uegrer V I cru atdherence was etvolved. Estimation of percentage of parasite surtace covered (MacKenzie et al 1978) did not prove practicable as larval: were invariably coiled and the surface partially o b s c!red ~ in places. Eosinophils were always involved in .ed the p i c tIre. ~ adhe1,ence beforc:other cell t ypes obscu~ At least two f actors associated with bovine ser um were invollved, one be ing heat la1~ i l eand the other a he,at stable facto r found on1y in hyperir nmune seru~ m It. was 1:resumed -. --> >~ that t he former was complemenr anu rne latter anti1oouy. probable that eosinophils in a non imm .eems une host It s FIG 2: Larva after four hcJurs incubatic may c:ombat invading D viviparus larvae in the prc:sence of compilement and that this killer cell activity urould be ^^I.^. t Iced by circulating antibody in an immune ho!>,. Leitamisole used 24 hours after infection did ncbt appear Larvae i~ncuoatea ~n rne aosence of blood cells wc're t o exc:rt any influence on eosinophil adherence to larvae in mobile afte r 48 hours incubation. Those incubated in I.he vitro. Where eosinophils were adherent to larvae, t he larvae ,rr v l c ~ c l l c cuf blood cells were immobile after two h o ~ . - were immobile before fixation. This could be beta[use such . . . incubation. After three hours incubation an incomplete cells are only attracted to immobile, and possibly dead, halo o f eosinophils (Fig 1) was observed surrounding larvae or because the eosinophils cause the larvae to become larvae. After four hours a mass of cells surrounded larvae immobile. However, since larvae incubated in the absence (Fig 2) and it was difficult to identify eosinophils amongst of leucocytes were invariably mobile before fixation, some them. facto r associated with the leucocytes m lust have remsulted in the la ck of larval movement. Analogy Hri th Nemarolspiroides Study 4 d u b iIS ~ (MacKenzie et al 1978) and Sc-histosoma mansoni (..~.a n m o u det a1 1975) suggests this f actor was Iprobably No mesoDrills survlveu 48 n o u n I I I L ~ in~ vitro ~ ~ but ~ , ~ . associated with t h e eosinophil cell p o p ulatlon. 45 per cent of eosinop'hils, 27 per cent o f lymphocytes and are an t is sugge equently, i 22 per ce nt of neljtrophils did survive. Eosinophil Cons' sted that eosinophils adherence to larvae \.sas seen in all cultures containing imp01rtant elemel tot the ho!st's defence against D v iviparus. .. . unheated serum. Where heated serum was used, adherence v i v i ~7rus. l was seen 01ily in cultu res contain ing hyperimmune seru Received forpublication April 1 5 , 1981 Cells from I he calf trea led with levamisole did Accepted July 13, 1981 behave diff erently frorn those fro!rn other sou 8
9 . .
,I% %'4DI,,
~
F..a",...--
- -
- 3 . .
..-L
.L
-A
.
Discussion
Eosinopb . , . 111-rlcn cell mlxtures prepared from periton' wasnlngs in laboratory animals have been used in the Stu of cell adherence in schistosomes (Mahmoud et a1 1975) nematode worms (MacKenzie et at. 1978). In the current work cell types found in the blood stream were used as far as Possible in the natural proportions existing in calves, SO that nn l*ndue domination in numbers o r stimulation (by .
L -
.
rences
:Em. E.E. - (-1973) Veterinlory Record 9 . t N L I t , L.1)..
PRESTON. P.M. & ( rure 276, 826.-828 .. - MAHMOUD, A.A.F., WAKREN, s.W. & PETEKS, P.A. (IY75) Journal of ErperirnenfolMedicine142.805-813 MICHEL. J.F. & CORNWELL, R.L. (1959') Veterinary Record 71, 912-913 OAKLEY. G:A: (1980) PhD thesis. CNM/L.IVCIPUUIUIniversity. -