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        Àn²O¤Ú²Ó­M¤§¤ÀÂ÷»P¯Â¤Æ¡G±Ä¥Î¤§¤èªk«Y¨ÌHiggins(9)©ÒµoªíªÌ¤©¥H­×§ï¡C¥ý¦ÛÀnÁl¤UÀR¯ß±Ä¦å¡A¨Ã¥HEDTA (0.2¢H)§Ü¾®¡C¸Ó¦å²G¡A¸g4¢J¡A1300¡Ñg¤UÂ÷¤ß30¤ÀÄÁ¡A¦¬¶°buffy coat¡A»P§t0.2¢H EDTA¤§µ¥¶q1¡ÑPBS (pH7.2¡A41¢J)²V¦X¡C¦A±N¤§¸m©óµ¥¶qFicoll-Paque (density¡G1.077¡FSigma, St Louis, Mo., USA)¤W©ó4¢J¡A200¡Ñg¤UÂ÷¤ß25¤ÀÄÁ¡A±N²Ó­M¼h§l¥X¡A¥ý¥H1¡ÑPBS²M¬~¤@¦¸¡A©ó4¢J¡A200¡Ñg¤UÂ÷¤ß10¤ÀÄÁ«á¦A¥H§tEDTA(0.2¢H)¤§RPMI-1640²M¬~¨â¦¸¡A¨ú±o°ª¯Â«×¤§²O¤Ú²Ó­M¡C°£¥Htrypan blue (0.2%)¦ævital stain´ú©w²Ó­M¦s¬¡²v¥~¡A¨Ã¨ú¾A¶q²Ó­M²G¥H²Ó­M©Ù¤ùÂ÷¤ß¾÷(Shandon cytospin, Shandon Southern Instruments, Sewelicky, PA, USA)»s¦¨©Ù¤ù¡A¸gDiff-Quick¬V¦â(International Reagents Corp, Japan)«á©óÅã·LÃè¤U­p¼Æ200­Ó²Ó­M¡A´ú©w²Ó­M¤§¤ÀÃþ¡C¨Ì¤W­zµ²ªG¡A±N²Ó­MÄa¯B²G¥H§t20¢H­L¤û¦å²M¡B«CÅð¯À(100U¡þmL)¡ÏÃìÅð¯À(100£gg¡þmL)¤Î1¢HL-glutamine¤§RPMI-1640½Õ¾ã³Ì²×²Ó­M¿@ÁY¬°1¡Ñ107²O¤Ú²Ó­M¡þmL¡C

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Table l.   The    experimental    design    of     the    effect    of     various    mitogens    on     duck    lymphocyte    transformation
Treatment
¡@ Lym1)¡Ï mitogen 0£gg/mL Lym¡Ï mitogen 5£gg/mL Lym¡Ï mitogen 10£gg/mL Lym¡Ï mitogen 20£gg/mL Lym¡Ï mitogen 40£gg/mL Lym¡Ï mitogen 80£gg/mL
RPMI

Lymphocytes

(1¡Ñ107 cells¡þmL)

Mitogen*

(200£gg¡þml)

Total

120£gL

80£gL

 

0£gL

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200£gL

115£gL

80£gL

 

5£gL

¡@

200£gL

110£gL

80£gL

 

10£gL

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200£gL

100£gL

80£gL

 

20£gL

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200£gL

80£gL

80£gL

 

40£gL

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200£gL

40£gL

80£gL

 

80£gL

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200£gL

*Mitogens included BSS¡BPHA¡BPNA¡BPWM.

1) Lym¡×Lymphocytes.

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Table 2.   The    experimental    design   of   the    effects   of    AFB1   on    PHA-induced   duck   lymphocytes    transformation
Treatment *
¡@ CTRL- CTRL+ MIC A4+MIC A4 A1+MIC A1 A-2+MIC A-2
RPMI

Lymphocytes

(1¡Ñ107¡þmL)

PHA

(100£gg¡þmL)

AFB1

Microsome

(5mg¡þmL)

MADPH

(0.25mM)

Total

150£gL

100£gL

¡@

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¡@

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250£gL

125£gL

100£gL

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        ¦³Ãö¤£¦Pµõ´Þ­ì¹ïÀn²O¤Ú²Ó­MÂ૬§@¥Î¤§´ú¸Õ¡A¨äµ²ªG¥Ü©óªí3¡Cªí¤¤Åã¥ÜBSS¹ïÀn²O¤Ú²Ó­MÂ૬¤ÏÀ³³Ì¨Î¿@«×¬°40£gg¡þmL¡APHAl¬°5£gg¡þmL¡APNA¬°40£gg¡þmL¡A¦ÓPWM¬°5£gg¡þmL¡C¸ÕÅç¨Ï¥Î¤§µõ´Þ­ì¡A°£PHA¤ÎPHA¤§CPM¶W¹L40000¥H¤W¡A¨ä¾lBSS¤ÎPWM¤§CPM­È§¡§C©ó10000¡C¦¹»PHiggins.(9)¤§³ø§i«ü¥X³Ì¨Î¤ÏÀ³¿@«×¤ÎCPM­È¤À§O¬°BSS 40£gg¡þmL¡A23415¡FPHA 20£gg¡þmL¡A4754¡F PNA 20£gg¡þmL¡A6962¡F¤ÎPWM 40£gg¡þmL¡A11297¡A¦³©Ò®t²§¡CÂk¯Ç¨ä¥i¯à­ì¦]¦³¡G¦å²M¨Ó·½¤£¦P¡A¥»¸ÕÅç¨Ï¥Î­L¤û¦å²M¡A¦ÓHiggins(9)¨Ï¥Î¦¨¼ôÀn¦å²M¡F¨ä¦¸¥i¯à¬O¦å®ê²yªº¦s¦b¿@«×¡A¦å®ê²y¤@¯ë»{¬°¨ã¦³§í¨î²O¤Ú²Ó­M¹ïµõ´Þ­ìÂ૬§@¥Îªº¤ÏÀ³(23)¡C¦¹¥~¡AÀnºØ¤£¦P¥i¯à¹ïµõ´Þ­ìªº¤ÏÀ³¥ç¤£¦P¡A¦Ó¦¹ÂI¥i¯à«Y¦]¯S²§¨ü¾¹ªº¦s¦b»P§_¤Î¦s¦bªº¶q¦³Ãö¡Cµõ´Þ­ì¥i¯S²§©Êªº¦ì©ó°Êª«²Ó­Mªí­±¿}³J¥Õ¤Î¿}¯×½è¤º¤§³æ¿}©Î¹è¿}¡A¦Ó²O¤Ú²Ó­MªºÂ૬§@¥Î¤£¶È¦³¿à¾A·íªº¨ü¾¹¦s¦b¡A¥t¥~¸Óµ¥¨ü¾¹¤À¥¬ªº±K«×¨Ï¨ä¯à»Pµõ´Þ­ì§Î¦¨¥æ¤eÁäµ²(cross-linking)¡A¥ç¬O¨M©w¦]¤l¤§¤@¡C¬G¦³¨Çµõ´Þ­ìÁö»P¨ü¾¹µ²¦X¡A¦ý¦p¨ü¾¹±K«×§C®É¡A¥çµLÂà§Î¤ÏÀ³²£¥Í(18)¡C¥Ñ©óµæÀn²O¤Ú²Ó­M­M¹ï¢â²Ó­M¨ë¿E­ìPHA¤ÎB²Ó­M¨ë¿E­ìPNA¦³¸û¨ÎªºÂ૬¤ÏÀ³¡A¦ý¹ï©óB¤ÎT²Ó­M§¡¦³¨ë¿E§@¥ÎªºBSS¤ÎPWMªºÂ૬¤ÏÀ³«o¤£¨Î¡C¥Ñ©óPHA¤ÎPNA¯à»P²Ó­M¿}³J¥Õ¤Î¿}¯×½è¤§N-acetylgalactosamineµ²¦X¡A¦Ó¦¹µ¥¨ü¾¹¦bÀn²O¤Ú²y¤£¶È¦s¦b¥B¨ä¤À§Gªº±K«×·¥¾A¦XPHA¤ÎPHA»P¤§§Î¦¨¥æ¤eÃìµ²¡A¦]¦Ó¾É­PÀn²O¤Ú²Ó­M©úÅ㪺¦³µ·¤Àµõªº¶i¦æ(8,9)¡C


Table 3. Effects   of  various  mitogens  at  different  concentrations   on  duck blood  lymphocyte  transformation
µõ´Þ·½ Cpm values at various concentraction1)
mitogen* 0 5 10 20 40 80
BSS

PHA

PNA

PWM

828±230

853±218

736±244

763±210

3705±828

44565±2627

3916±1257

7260±1527

4603±787

39926±4579

6730±1662

6390±1630

3444±769

31020±4691

14213±2980

1586±344

5197±643

24748±5935

41068±6992

2269±1342

3585±1313

2845±972

2586±1011

765±322

1) CPM:(count per minute).

*BBS:bandeiraea simplicifola; PHA:phytohaemagglutinin; PNA:peanut agglutinin; PWN:pokeweed mitogen.

Higgins.(9)«ü¥X¦b§C¿@«×¤§PHA¦³¸û¨Î¤§Â૬¤ÏÀ³¥i¯à©M¨ü¾¹ªº²§½è©Ê(heterogenecity)¦³Ãö¡C§Y·í¤ÏÀ³ªºµõ´Þ­ì¿@«×§ïÅܮɻP¿}¯×½è©Î¿}³J¥Õ½è¤§¿Ë©M¤O©Îµ²¦X¤O(association)§Y§ïÅÜ¡C¦¹¥~¡A¦b¤ÏÀ³ªº²Ó­M¸s¤¤¤À¤Æ²Ó­MÄâ±a¦³¤£¦Pªº±µ¨ü¾¹¡A¬G¹ïµõ´Þ­ì¦³¤£¦Pªº¤ÏÀ³¡C

        ¦³ÃöAFB1¹ïÀn²O¤Ú²Ó­MÂ૬§@¥Î¤§¼vÅT¡A¨äµ²ªG¥Ü©ó¹Ï1¡C¹Ï¤¤Åã¥ÜAFB1¦b§C¿@«×®É(101¤Î10-2ng¡þmL)¹ï¥ÑPHA©Ò¤ÞµoªºÀn²O¤Ú²Ó­MÂ૬§@¥Î¨ã¦³¨ë¿Eªº®ÄªG¡A¥BÅãµÛ¸ûPHA¶§©Ê³B²z²Õ¬°°ª(P¡Õ0.05)¡A¤ÏÆ[·íAFB1ªº¿@«×´£¤É¦Ü104ng¡þmL®É¡A§Y¶}©l¹ïPHA©Ò¤ÞµoªºÀn²O¤Ú²yÂ૬§@¥Î¤S¦³§í¨îªºÁͶաAÁöµM¦¹§í¨î®ÄªG¦b²Î­p¾Ç¤W¨Ã¤£ÅãµÛ¡CµM¦Ó·í¦UAFB1²Õ¤¤¥[¤J¨xŦ·L²ÉÅé«á¡AµL½×¬O101©Î¬O10-2§¡¹ïPHA©Ò¤ÞµoªºÀn²O¤Ú²yÂ૬§@¥Î²£¥Í·¥ÅãµÛªº§í¨î§@¥Î¡C¥Ñ¥H¤Wªºµ²ªGÅã¥Ü¨xŦ·L²ÉÅé¦bAFB1¹ïÀn²O¤Ú²yÂ૬§@¥Î§í¨î©Ê¤W§êºt­«­nªº¥[­¼®ÄÀ³¡A¦¹ÂI©MAFB1¹ï¤õÂû¸¡µÄ¥¨¾½²Ó­Mªº¬r©Ê¬ÛÃþ¦ü(17)¡CAFB1¦p¥¼¸g¨xŦ·L²ÉÅ骺¬¡¤Æ¡A§Y¨Ï¦b°ª¿@«×¤U40£gg¡A¹ï¤õÂû¸¡µÄ¥¨¾½²Ó­MªºªþµÛ¤O¡B§ÎºA¾Ç¡A¤Î§]¾½¯à¤O§¡µLÅãµÛªº¼vÅT¡F¤Ï¤§¡A¸g¨xŦ·L²ÉÅé¥NÁ«á¡AAFB1¦b·¥§Cªº¿@«×¤U(1¡ã5£gg)§Y¥i³y¦¨¤õÂû¸¡µÄ¥¨¾½²Ó­MªþµÛ¤O©M§]¾½¯à¤Oªº©úÅã¤U­°¡A¥B²Ó­Mªí­±§e²yª¬¬ð°_¦Ó®Ö¥ç§e¤À¸Ñª¬(17)¡CAFB1¦b®ñ©MNADPHªº¦s¦b¤U¡A¥i³Q¥õ¿àcytochrome P450ªº¨xŦ·L²ÉÅémixed function oxidase (MFO)¨t²Î©Ò¬¡¤Æ(12)¡A¨ä¤¤¤@­Ó¥NÁ²£ª«¡A2,3-oxide¥i©MDNA¤WN7-guanineµ²¦X¦Ó²£¥Í²Ó­M¬r©Ê¡B­PÀù©Ê¤Î·î­F©Êµ¥¼vÅT(11)¡CBodine et al. (4)¥ç´¿«ü¥X¨üµõ´Þ­ìPHA­P¬¡¤§¤û²O¤Ú²y¯à±NAFB1¶i¤@¨B¥NÁ¬°¥i©MDNAµ²¦Xªº²Ó­M¬ðÅܪ«½è¡C¥B§í¨î¬¡Åé¥~²O¤Ú²Ó­M¹ï¦UºØ§Ü­ì©Î²Ó­Mµõ´Þ­ìªº¨ë¿E¤ÏÀ³(21,24)¡C

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°Ñ¦Ò¤åÄm

        1.§dÄ~ªÚ¡B¾G²M´Ë¡B´å¸q¼w¡BÄY®a²M¡B³¢©¾¬F¡C1988¡C¹}®Æ¤¤§t§C¿@«×¶ÀÄT¯À¹ï¥Íªø½Þ¥Íªø©Ê¯à»P²Õ´´Ý¯d¶q¤§¼vÅT¡C¤¤µØ¹A¾Ç·|³ø. 144:85-93

        2.Ãe­¸¡C1994¡C¶ÀÄT¬r¯À¹ï½Þ°¦§K¬Ì¤O¼vÅT¤§¬ã¨s¡C¥Í©R¬ì¾Ç²°T¡C8(3):1-9.

        3.Blount, W. P. 1961. Turkey X disease. J. Brit. Turkey Federation. 9:52.

        4.Bodine, A. B., S. F. Fisher and S. Gangjee. 1984. Effect of aflatoxin B1 and major metabolites on phytohemagglutinin stimulated lymphoblastogenesis of bovine lymphocytes. J. Dairy Sci. 67:110-114.

        5.Boyum, A. 1968. Isolation of leucocytes from human blood. A two phase system for removal of red cells with methyl cellulose as erythrocyte-aggregatting agent. Scand. J. Clin. Lab. Invest. 21 (suppl. 9),4.

        6.Chang, C. F. and P. B. Hamilton. 1979. Impaired phagocytosis by heterophils from chickens during aflatoxicosis. Toxicol. Appl. Pharmacol. 48:459-466.

        7.Ciegler, A. and E. B. Lillehoj. 1968. Mycotoxins. Advan. Appl. Microbiol. 10:155-219.

        8.Higgins, D. A. and S. H. Chung. 1986. Duck Lymphocytes: I. Purification and preliminary observations on surface markers. J. Immunol. Methods. 86:231-238.

        9.Higgins, D. A. 1990. Duck Lymphocytes: III. Transformation responses to some common mitogens. Comp. Immunol. Microbiol. Infect. Dis. 13:12-23.

        10.Kadian, S. K., D. P. Monga and M. C. Goel. 1988. Effect of aflatoxin B1 on the delayed type hypersensitivity and phagocytic activity of reticuloendothlial system in chickens. Mycopathologia 104:33-36.

        11.Karenlampi, S. O. 1987. Mechanism of cytotoxicity of aflatoxin B1: Role of cytochrome P1-450. Biochem. Biophys. Res. Commun. 145:854-860.

        12.Koser, P. L., M. B. Faletto, A. E. Maccubbin, and H. L. Gurtto. 1988. The genetics of aflatoxin B1 metabolism, Association of the induction of aflatoxin B1-4-hydroxylase with the transcriptional activation of cytochrome P3-450 gene. J. Biol. Chem. 263:12584-12595.

        13.Lowry, O. H., N. J. Rosebrough., A. L. Far and R. J. Randall. 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193:265-275.

        14.Lucas, A. M. and C. Jamroz, 1961. Atlas of Avian Hematology, Agriculture Monograph 25 (U.S. Department of Agriculture, Washington, DC).

        15.Michael, G. Y., P. Thaxton and P. B. Hamilton. 1973. Impairment of the reticuloendothelial system of chickens during aflatoxicosis. Poult. Sci. 52:1206-1207.

        16.Muller, R. D., C. W. Carlson, G. Semeniuk and G. S. Harshfiled. 1970. The response of chicks, ducklings, goslings, pheasants and poults to graded levels of aflatoxins. Poult. Sci. 49:1346-1350.

        17.Neldon-Ortiz, D. L. and M. A. Qureshi. 1991. Direct and microsomal activated aflatoxin B1 exposure and its effects on turkey peritoneal macorphage functions in vitro. Toxicol. Appl. Pharmacol. 109:432-442.

        18.Pearson, T. W., G. E. Roelants, L. B. Lundin and K. S. Mayor-Withey. 1979. The bovine lymphoid system:binding and stimulation of peripheral blood lymphocytes by lectins. J. Immunol. Methods 26:271-275.

        19.Richard, J. L. and J. R. Thurston. 1975. Effect of aflatoxin on phagocytosis of Aspergillus fumigatus spores by rabbit alveolar macrophages. Appl. Microbiol 30:40-47.

        20.Pier, A. C., S. J. Cysewski. J. L. Richard, and J. R. Thurston. 1977a. Mycotoxins in human and animal health. Edited by J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman. Park Forest South, Illinois, Pathotox Publ. pp. 745-750.

        21.Singh, J., R. P. Tiwari, G. Singh, S. Singh and D. V. Vadehra. 1987. Biochemical and immunological effects of aflatoxins in rabbits. Toxicol. Letters 35:225-230.

        22.Thurston, J. R. M., B. L. Royoe, A. L. Baetz, J. L. Rchard and G. D. Booth. 1974. Effect of aflatoxin of serum protein, complement activity, and the antiboby response to Brucellla abortus in guinea pigs. Am. J. Vet. Res. 35: 1097-1100.

        23.Traill, K. N. G. Bock, R. Boyd, G. Wick. 1983. Chicken thrombocytes. Isolation, serological and functional characterisation using the fluorescense activated cell sorter. Dev. Comp. Immunol. 7:112-115.

        24.Yang, W. C., 1983. Effects of aflatoxin B1 on the development of procine cellar and humoral immune responses. Ph. D. dissertation, Auburn University, Alabama, USA.                    

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Effects of Aflatoxin B1 (AFB1) on Duck Lymphocyte Transformation

Yeong-Hsiang Cheng(1) and Victor-Fei Pang(2)

Summary

        The purpose of this experiment was to isolate and purify duck lymphocytes from peripherial blood. Lymphocytes were then used to study transformation response to various mitogens. These results were used to evaluate the effects of aflatoxin on duck lymphocyte transformation.

        The buffy coat collected from the whole blood was anticoagulated with EDTA andcentrifuged at 1300¡Ñg for 30min at 4¢J, then resuspended with an equal wolume of PBS. The cell suspension was layered over a Ficoll-Paque gradient and centrifuged at 200¡Ñg for 25min at 4¢J. The band formed at the interface of the RBC layer contained cells of which greater than 95¢H were lymphocytes. The averaged numbers of lymphocytes obtained from 10 mL of whole blood wasapproximately 2 ¡Ñ107. Various mitogens (PWM, PHA, PNA, and BSS) at a final concentration of 0,5,10,20,40and 80£gg¡þmL were added to the lymphocyte suspension (8¡Ñ105 cells¡þmL). The Cpm value of the lymphocyte transformation response by 3H-thymidine integration after 72hrs incubation was measured. The results showed that PHA at 5 and 10£gg ¡þmL gave higher values than the others.

        The effects of aflatoxin on duck lymphocyte transformation response were evaluated, by addition of final concentration of 104,101, or 10-2 ng¡þmL aflatoxin B1 (AFB1) to lymphocyte suspension. Meanwhile, lymphocyte suspension with or without duck liver micorosome plus NADPH was also evaluated. The results showed AFB1 101 and 10-2ng/mL had significantly higher CPM values than the others (P¡Õ0.05). This indicated that AFB1 had a stimulative effect on duck lymphocyte transformation. AFB1 only at 104ng/mL had a slight inhibition. When liver microsome was present, AFB1 had an adverse effect on duck lymphocyte transformation even though at a low concentraction of 10-2ng/mL. This detrimental effect became more obvious as AFB1 concentration increased. It could thus be concluded that PHA-induced duck lymphocyte transformation was inhibited significantly when AFB1 was metabolized by duck liver micorsome, Key words:Aflatoxins, Lymphocyte Transformation, Duck

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1.Department of Animal Science, National I-Lan Institute of Agriculture and Technology, I-Lan Taiwan, R.O.C.

2.Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, R.O.C.

  (Received November 22, 1994; Accepted February 7,1995)

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