Effects of Florfenicol, Chloramphenicol, and Thiamphenicol on Phagocytosis, Chemiluminescence, and Morphology of Bovine Polymorphonuclear Neutroph I I Leukocytesl

PHYSIOLOGY AND MANAGEMENT

Effects of Florfenicol, Chloramphenicol, and Thiamphenicol on Phagocytosis, Chemiluminescence, and Morphology of Bovine Polymorphonuclear Neutroph i I Leukocytesl

M. J. PAAPE and R. H. MILLER Milk Seaetkn and Masaitls Labambq USA, ARS Bdtsville. MD awo5

0. ZIV Kimon Veterinary institute Bet-DWan, Israel

ABSTRACT tron micmscopy showed that the percent- Florfenicol, chloramphenicol, and age of neutrophils without pseudopcxjia thiampheniwl were tested in vitro to de- averaged 67.94,32, and 1696, respective- ly. Results indicated that neither flor- termine their potential toxic effects on feniml thiamphenicol dted neutro- bovine neutrophils. Antibiotics were nor phil function, but they did alter neutrophil tested at 4O00, 2000, and 10 CLghnl of incubation mixture. Percentage phagocy- morphology, although to a lesser extent tosis was determined incubations with than did ChloramphenicoL by (Key words: antibiotics, neutrophils, neutrophils isolated from milk of five cows and %-labeled StaphyZococcus Phagocytosis) aurelrs and 5% skim milk. The effect of 4OOO pg of each antibiotic on chemilumi- INTRODUCTION nescence was determined neutrophils on previous studies have shown that chluram- isolated from mammary secretims of pheniwl had detrimental effects on bovine three nulliparous heifers. Morphological polymorphonuclear neutrophil phagocytosis evaluation by transmission and scanning (11, 25) and morphology (11). Because the electron microscopy was perfonnea on drug causes aplastic anemia in humans, its use neutrophils isolated firom two heifers at in the United States in food-producing animals antibiotic concentrations of 4OOO and 10 is prohibited, However, chloramphenicol is ap- pg/ml. momnphenim1 depresses proved for use in several European countries, phagocytosis at the high and medium and evidence indicates that vetezhirhs and and blocked chemiluminescence doses others in the United States are using the drug in activity at the high dose. No effects were food-producing animals (17). TWOanalogs Of observed for florfeaicol and thiam- Chloramphenicol fl~rfenicol and thiamphenicol. Transmission electron micro- phenicol, have been synthesized. The major scopic examination showed that at the difference between the analogs and chloram- high concentration of drugs, 99, 99, 97, phenicol is that the p-nitro group is replaced by and 76% of the neutfophils med with a methyl sulfonyl group (Figwe 1). Thep-nitro florfeniwl, chlorampheniool, thiam- group has been implicated with irreversible in- phenicol, and dimethyl sulfoxide were hibition of growth of bone mamxv precursor abnormal. Examination by scanning elec- cells (15). Widespread use of thiampheniwl in Europe has not been associated with aplastic anemia (17). Both analogs possess an antimi- dial spectrum similar to that of chluram- phenicol (10, 20). However, florfenicol has been mpmd (20) to possess supexior in vitro bactericidal activity compared with chloramphenicol and thiampWcoL This has been at-

1990 J Dairy Sci 73:1734-1744 1734 EFFlXTS OP CHLOmCOLON N@UTROFWILS 1735 Corp., Union, NJ), and florfenicol (Shering- Plough Corp.) were dissolved in dimethyl sulfoxide (DMSO)(Sigma Chemical Co.) and di- luted to desired concentrations in either balanced salt solution or .01 M phosphate buffered saline (PBS).

Neutrophils Mammary gland neutrophils were isolated from lactating cows 14 h after inmammary inoculafion of 50 pg of Escherichia coli endotoxin (E. coli 026:B6, Boivin TCA procedure, endotoxin code 3920, Difco Laboratories, De- troit, MI) m 50 ml of sterile PBS. To induce milk ejection, 7 Iu of oxytocin (Anpro Phama- ceutical. Arcadia, CA) were administered into the tail vein, and 250 ml of milk were collected tributed to the substitution of a fluorine atom (by hand milking) into a polypropylene flask for the hydroxyl on the 3' carbon. The Samples and reagents were kept at 5'C during group pIvmxke. Milk was filtered efficacy intramammary florfenicol the the isolation of for cloth (NSG Precision hc., treatment of mastitis in lactating cowsis cur- through silk Cells, rently under investigation Ziv, unpublished Hickwille, NY), paured into siliconid bottles (G. (Cares, Pant Co., Newtown. and cen- observations). Du 0, trifuged at x g for 15 min. The cream The study was determine loo0 purpose of this to layer was removed, the skim milk was in vitro effects of chloramphenicol, fldenicol, discard- and the pellet was gently suspended in and thiamphenicol phagocytosis, chemilumi- ed, cell on 80 ml PBS. After centrifugation at 200 x nescence, and morphology bovine neutro- of g, of neutrophils were washed again in The phils. PBS. suspension was centrifuged again, and the sedi- mented cells were suspended in PBS and then MATERIALS AND MmHODS adjusted to 25 x 106/ml. On the average, 97% of the cells recovered were neutrophils with Cattle 95% viability as determined by Trypan blue exclusion. Five lactating cows and three nulliparous Neutrophils from the mammary gland of heifers were used as sources of neutrophils. On nulliparous heifers were isolated after in- the basis of results of bacteriologic culture of tramammary inoculation of 5 p.g of E. coli mammary secretions on blood esculin agar, endatoxin in 5 ml of PBS. At 14 h after endo- these cattle were free of intmmmnmy patho- toxin administration, 20 ml of PBS were inm- gens. lated into the endotoxin-treated quarters, and Each of two separate trials consisted of mammary secretions were collected into a poly- phagocytosis measurements on mammary gland propylene flask. The neutrophils were isolated neutrophils from three and two lactating cows, and their concentration was adjusted, similarly and three separate trials consisted of chemilu- as for those from lactating cows. Typically, minescence (CL) and morphologic measure 94% of the isolated cells were neutrophils, of ments on mammary neutrophils collected alter- which %% were viable. natively from one of the three heifers. Phagocytosis Drugs Percentage of phagocytosis was measured Chloramphenicol (Sigma Chemical Co., St. after a 60-min incubation as described (7). Louis, MO), thiamphenid (Shering-Plough Drugs were tested at in vim concentrations of

Jodof Dairy Sciemx Vol. 73, No. 7, 1990 1736 PAAPE ET AL. 4000,2000, and 10 ~&IIof incubation medi- and numerous pseudopodia; 2) altered, charac- um. The final concentrations of DMSO in the terized by slight rounding of the cell and nu- incubation medium were 6.25, 3.12, and .02%. clear lobes with partial loss of pseudopodia; 3) The incubation mixture included .5 ml of drug swollen, gross rounding of nuclear lobes and or corresponding dilution of DMSO, .5 ml of complete rounding of the ce& and 4) lysed, 10% skimmed milk in PBS, .5 ml of the neu- charaderimxi by nuclear swelling, extrusion of trophil suspension, and .5 ml of 32P-labeled cell contents, and partial loss of the plasma Staphylococcus aureus (2 x 106). All determi- membrane. The number of cells with cytoplas- nations were performed in triplicate. mic vacuoles and with cytoplasmic blebs pro- mding from the cell surface were also record- Chemllumlnescence ed. Two hundred cells per trealment were charaderimxi. For scanning electron microsco- Balanced salt solution was prepared as pre py, fixed cells were processed (9) and 200 cells viously described (8), except that phenol red were examined using a scanning electron mi- was not added. The solutions and methodology croscope operating at 20 kV. The number of used were as previously described (6). cells with no pseudopodia (rounded) was re- Luminol-enhanced CL was measured in a liq- corded. uid scintillation counter (LS lOOC, Bechnan Instruments, Inc., Silver Spring, MD) at 21’C, Statlstlcal Analyses using the tritium channel and the incoinci- dence mode. Isolated mammary gland neutro- Oneway analysis of variance procedures phils (5 x 106/.2 ml) from a nulliparous heifer were used to make comparisons of the means were added to 7-ml scintillation vials caritain- and percentage change in phagocytosis from ing luminol(1.8 ml), 1 ml of zymosan (5 x le DMSO control. Groups were drug and concen- particles/ml), and 1 ml of drug or DMSO in tration combinations. Chemiluminescence data balanced salt solution. Final concentrations of were analyzed by general linear models proce- drug and DMSO in the incubation mixture were dum of SAS (SAS Institute, Cary, NC) with a 4000 pdml and 6.25%.Vials were counted for model including drug, incubation time, and in- 1 min at 16-min intervals for a maximum of teraction of drug x time. The tests of signifi- 128 min. All determinations were performed in cance were based on log (baseld of counts per duplicate. minute (mean of two counts). For morphologic results, comparisons were made separately for each concentration of each antibiotic versus Morphologic Study DMSO by each morphologic category, by two- Neutrophils (12.5 x 106) collected from way factorial chi-square analysis. mammary secretions of two nulliparous heifers were incubated for 2 h at 37’C at the high and RESULTS low concentrations of antibiotics and DMSO. Cells were then fixed in 2.5% glutaraldehyde in .1 M cacodylate buffer (pH 7)at room tempera- Phagocytosis ture, fixed as described (21), and dehydrated in A significant (R.05) inhibitory effect on a graded series of ethanol. For light mi- phagocytosis was observed for chloramphenicol py, neutrophils were embedded in Spun’s me at the high and intemxdiate drug concentra- dium (18) and sectioned. For transmission elec- tions (Table 1). Floxfenicol and thiamphenicol tron microscopy, ultrathin sections (60 to 90 had no effect (l5.05) at any of the concentra- nm) were stained with uranyl acetate and lead tions tested. citrate and examined, using an electron micro- scope operating at 60 kV. A morphologic anal- Chemllumlnescence ysis was performed on leukocytes incubated with each drug in culture medium, and values Activity of neutrophils was depressed were compared with those of DMSO controls. (P<.M) by chloramphenicol between 16 and Cells were classified as pviously described 112 min compared with DMSO (Figure 2). (11): 1) normal, cells with a multilobed nucleus Although CL activity compared with DMSO

Journal of Dairy Science Vol. 73. No. 7, 1990 EPPECIS OF CHUlRAMpHENICOL ON NEUTR0PHJI.S 1737

TABLE 1. JBWt of chloramphcnieol dm phagocytosis.' 12 CCma&ail'on, w 4ooo 2ooo 10 6) l: CblOnUIlphClliicol -16.6' -18.6' 4.3 Plorphenicol -7.8 -3.0 3.3 Thiamphenicol -5 2 .8 3.7 i6 .Different (Pc.05) from 625,3.125,and .M% dimethyl B 4. sulfoxide control vlllacs of 31.8. 36.4, and 47.6%. lmta expressed as pacentage of change relative to DMSO control. 5 2' ,- ol,L - e - e e4...... r ...... * 0 16 32 48 64 80 Q6 112 128 T=Wlh38) was reduced by florfenicol and thiamphenicol, the differences were not significant (h.05). Maximum CL activity occurred at 48 min of incubation.

Morphology Study Transmission electron microscopic eXamina- were observed with cytoplasmic vacuoles when tion of cells incubated in drugs and DMSO incubated in the lowest concentration of flor- indicated that most cells had altered morphol- fenicol (Table 3). However. more (R.01) neu- ogy (Table 2). At the high concentration of trophils were observed with cytoplasmic vacu- drug, fewer (R.01) normal neutrophils were oles at the high concentration of florfenicol and observed than with DMSO. More normal cells chloramphenicol. were observed at the lowest Concentration of A commonly observed abnormality was the drug. Interestingly, the percentage of normal blebbmg of cytoplasm from the surface of neu- cells for florfenicol (54%) and DMSO (58%) trophils incubated in the high concentration of were very similar, although not different florfenicol (Figure 50. Significantly more neu- (b.05) from chloramphenicol (37%) and trophils (26%) incubated in the high dose of thiamphenicol (33%). Cytoplasmic vacuoles florfenicol had blebbing than in the DMSO were commonly observed in neutrophils for all control (Table 3). Although a high percentage conmtrations of drugs and DMSO (Table 3, of neutcophils incubated in the low (2Wo) and Figures 3,4, and 5). Fewer (Pc.05) neutrophils high (18%) doses of thiamphenicol also had

TABLE 2. Effect of antibiotics on oeatropbil morphology as detuminui by transrmsm* 'on electron microscopy. C- C- 'on. w Normal Altered SWOllUl Lysad mg 10 4Ooo 10 4ooo 10 4ooo 10 4Ooo

Chloramphenicol 37 1' 52 83 a 9 3 7 morphenicol 54 1' 34 69 3 19 9 11 Thiamphmicol 33 3' 58 72 7 16 2 9 DMSO' (58) (24) (33) (63 (2) (6) (7) (7) %hcs within each dmg concentration am diffaent from DMSO control (P<.Ol). of dimthy1 sulfoxide wae .02 aud 625%.

Jodof Dairy Sacnce Vol. 73. No. 7. 1990 1738 PAApe ET AL.

’ * Hgure 3.l.mmm.W’ ‘on elactron micrographs of ntatrophils incutmtcd in media A) .U2% dimethyl dfoxide (DMSO), B) 10 crglml of chloramphdcol, C) 10 clg/ml of 5orfenic04 and D) 10- Whdwl.NmmpM8 incubated m chloramphniml (B), florfenicol (C), and thiampheniml (D) sppcatcd morphologicaUy similac to those mcubatcd in DMSO (A). ~3900.

Journal of Dairy science Vol. 73, No. 7, 1990 EFPECIS OP CHLoRAMPHwlCOL ON NEUTROPHILS 1739

Figure 4. Scanning electron micmpplu of nmtmpldb ioabntd in media contaimlog A) .OZ% dimthy1 sulfoxide (DMSO), B) 10 c1B/ml of chlomqkbl, C) 10 pghd of fhfdml, md D) 10 clghnl of mW-1. Pseadopodia WQC not aa distinct on nmtmphils incubated m chi-. 1 (6). mdcells can be seen at the bottom for Uds chlstaof cells. '2bccell sarf.Cesfor~~iacabated m florfhl (C) and thiamphrmicol@) wcre similar to those for ncatmphilr incubated m DMSO (A). x4ooo. 1740 PAAeE ET AL..

Jopmal of Dairy Scicace Vol. 73, No. 7, 1990 EPPECFS OF CHLORAMpHENlCOL ON "TROPHILS 1741

figure 6. Scanning electroo micrographs of ncutmphils incubated in media Containing A) 6.25% dimethyl sulfoxide, B) 4OOO p@ml of chloramphdcol, C) 4OOO p@ml of florfdl, and D) 4ooo pghl of thiamphcniml . More rounded cells can be observed for neutrophils exposad to chloramphenicol (B). Bleb of cyloplasm arc seen Coming from the surface of neutrophils incabated in florfenicol (c)end tbiamp~l@).x3900.

Journal of Dairy Science Vol. 73, No. 7, 1990 1742 PAAPE ET AL.

TABLE 3. Effect of antibiotics on neutrophil morphology TABLE 4. Effect of antibiotics on rounding of neutrophils as determined by translllssl' 'on electron microscopy. as detcrmiraed by Scanning eleclron micrOscopy. Concentration, p@ml 96 Rounded neufxo- CytoPlasmiC PhilS, vacuoles Blebs concentration, pgfml Dw 10 4ooo 10 4Ooo mg 10 4ooo chloramphenicol 5 94'L PlOrphmicOl 4 67' Chloramphenicol 44 54' 5 9 Thiamphenicol 4 32 FlOrpheniCOl 42' 1 26' 16' DMSO~ (2) Thiamphenicol 36 26 20 18 (10) DMSO in F'BS' 135) (21) (11) (8) %ignificantly different from DMSO control (Pe.01). koncentrations of dimethyl sulfoxide and within each concentration different were .02 'Values drug are 6.25%. fium DMSO control (Pe,Ol). koncentrations of dimethyl sulfoxide were .UZ and 6.25% in phosphatbbuffed saline. low dose of antibiotics or in the low and high doses of DMSO. cytoplasmic blebs, the percentages were not different from the DMSO controls. DISCUSSION Another commonly observed abnormality was the loss of pseudopodia from the surface of The drug concentrations in the present neutrophils incubated in the high concentration study represent what is currently found in & of chloramphenicol and florphenicol (Table 4, during the first 12 h after intramammaq treatment at recommended doses (24, 26). Figure 6). Counts made on 200 cells by scan- In- ning electron microscopy revealed that for cells tramammq chloramphenicol therapy at doses ranging between .5 and 5 g/quarter is used in incubated in 4000 pdd Of chloramphenicol many and other countries (22) for the and florfenicol94 and 67% were rounded (Ta- European treatment of coliform mastitis. Thus, con- ble 41, which was greater (P<.Ol) than what centrations of the drug are in the & was Observed for the conml* No effect soon after treatment. In the present study, 4ooo was observed for incubated in thiam- and 2000 pg/d concentrations of chloramphenicol* The distribution Of pseudopodia On phenicol resulted in 16.4 and 18.6% reductions for in phagocytosis. kvious studies (11,25) using in low doses of drugs and Dm0 Crable 4, in vitro concentrations of 4000 pg/d repfied Figure 4)- However, pseudopodia for Chloram- greater decreases in phagocytosis by phenicol-treated cells did not ~PF to mammary neutrophils, on the order of 47.9 to out as far from the cell as did pwuhpodia on 38%. Variation among cows in sensitivity of cells incubated in other drugs or DMSO. Addi- neumphils to CMrnamfiedcol, or the Some of tied abnormalities included a greater rounding the nmtro@ls, could explain the less &tic of the nuclear lobes and WOP~SmiCgrades effects on phagocytosis observed in the present in neUtrOphils incubated in the high concentra- study. variation exists among COWS in the ab& tions of florfenicol (Figure 5c) compared with ity of neutrophils to phagocytose (2, 14). In the other drugs at this concentration and with vitro concentrations of 1000 p&d or less are DMSO. not inhibitory to phagocytosis (4). Importantly, Transmission electron microscopic examina- parenterally administered chloramphenicol does tion of neutrophils revealed a condensing of the not result in milk drug concentrations found to cytoplasmic granules in neutrophils incubated impede phagocytosis (23). in the high concentration of all three antibiotics A significant observation was the lack of an (Figures 5B, C, and D). The movement of inhibitoryeffect on phagocytosis by florfenicol granules away from the penphev of the cells and thiamphenicol at all concentrations tested. was readily discernible. This phenomenon was However, in a recent study (4), incubation of not observed in neutrophils incubated in the neutrophils in 5,125, or 1000 pg/rnl concentra-

Journal of Dairy Science Vol. 73, No. 7. 1990 EFFIKTS OF CHLO-COL ON NEUTROPHILS 1743 tions of florfenicol resulted in significant inhi- highest concentration of drug the percentage of bition of phagocytosis by bovine neutrophils normal cells was only 1 to 3%. Almost all of compared with 5% bovine serum in PBS. How- the neutrophils (94%) exposed to the highest ever, the florfenicol used in that study was in concentration of chloramphenicol had no pseu- an organic solvent. The authors did not spec@ dopodia. This fmding suggests that disruption if appropriate dilutions were made of the sol- of the microtubule assembly occurred. Microtu- vent used to dissolve the florfenicol in the 5% bules play a role in degranulation (5) and main- serum control medium. Thus, the organic sol- tenance of pseudopodia (19). A classic example vent used with the florfenicol and not flor- of the importance of microtubules in degranula- fenicol itself may have been responsible for the tion comes from results of studies on the dis- observed inhibitory effects on phagocytosis. ease, Chediak-Higashi Syndrome. Neutrophils A saiking finding was the complete block- from patients with this disease are unable to ing of CL activity of neutrophils by the 4O00 undergo noddegranulation (16). In vitro pg/ml concentration of chloramphenicol. As a treatment of Chediak-Higashi Syndrome cells result of respiratory burst activation, neutro- with Concanavalin A produces “capping” (12). phils produce highly unstable oxygen metabo- which is construed as evidence of defective lites that are involved in the killing of bacteria microtubule assembly. Furthermore, the signifi- (3). Chemiluminescence, the accompanying cant inhibition of chloramphenicol on phagocy- light emission, can be meadspectrophoto- tosis reported in this and other studies (1 1, 25) metrically and has been used to study oxygen- suggests inhibition of the assembly of actin dependent microbicidal systems of neutrophils monomers into filaments, a critical step for (1). Results from this investigation indicated phagocytosis to occur (19). that although neutrophils may still have been capable of microbial ingestion in medium con- CONCLUSIONS taining 4O00 pgml of Chloramphenicol, oxida- tive killing mechanisms may be totally sup- Chloramphenicol suppressed phagocytosis pressed. Fortunately, although high and completely blocked respiratory burst activ- concentrations of the drug are present in the ity of neutrophils, effects not observed in neu- milk soon after treatment, the drug is rapidly trophil~ exp~sedto florfenicol and thiam- absorbed from the udder (27). Intramammary phenicol. All three drugs altered the injection of 5 g of chloramphenicol into a morphology of neutrophils with effects being lactating mammary quarter will result in a milk slightly greater in neutrophils treated with concentration of 115.9 pgml 2 h after injection chloramphenicol. Because both florfenicol and (13). Thus, suppression of intracellular oxida- thiamphenicol possess an antimicrobial spec- tive microbicidal systems may be of short dura- trum similar to that of chloramphenicol, use of tion. In a previous study (13), neutrophils ex- these two drugs as therapeutic agents appears posed in vivo to chloramphenicol concentration appropriate. of 414 pgml of milk showed no diminishment of CL activity. A favorable fmding from the REFERENCES present study was the detection of CL activity 1 AUen, R. C., and L. D. Loose. 1976. F%agocytic activa- in neutrophils after exposure to florfenicol and tion of a IuminoIdependent chemiluminescencein rabbit thiamphenicol. Depending on results from effi- alveolar and peritonial macrophages. Biochem. Res. cacy trials, results of these functional studies conrmun. 69:u5. 2 Anduson, K. L. 1984. Infkmmation, phagocytosis and seem to indicate that both florfenicol and effect of flunixin megluminc during endotoxin-induced thiamphenicol should be considered as suitable mastitis. ph9. Diss., Univ. Illinois, Urbana therapeutic replacements for chloramphenicol. 3Badwey. J. A., and M. L. Karwvdcy. 1980. Active Results from the morphologic study pro- oxygen species and the functions of phagocytic leako- cytcs. Ann. Rev. Biochem. 49:695. duced some unexpected &dings. From the 4BreW, K. N., C. A. Neff-Davis, R. S. 0% G. D. functional studies, we anticipated that flor- Koritz, B. K. Gustafmn, and L. E. Davis. 1987. fenicol and thiamphenicol would be less dam- Florfenicol in non-lactating dairy cows: pharmacokinet- aging to neutrophils than would chlorampheni- ics, binding to plasma proteins, and effects an phagocytosis by blood neutrophils. J. Vet. F%mmacol. Ther. 10: col. However, all three drugs altered the 233. morphologic features of neutrophils. At the 5 Deporter, D. A. 1978. Microtabules, cyclic AMP and

Journal of Dairy Science Vol. 73, No. 7, 1990 1744 PAApe ET AL.

lysomal enzyme release. A review. Biomedicine 285. Abnormal bactericidal, mtabolic, and lysosomal h- 6 Dulin, A. M., M. J. Paape, and S. C. Nickerson. 1988. tions of Chedialr-HigaShi Syndrome leukocytes. J. Clin. Comparison of phagocytosis and chemiluminescence by hveat. 51549. blood and msmmary gland neutrophils from multiparous 17 Settepani, J. A. 1984. Tbe hazard of using chlorampheni- and nullipamus cows. Am. J. Vet. Res. 49172. col in food producing animals. J. Am. Vet. Assoc. 184 7Dnlin,A.M.,M.J.Paepe,andB.T.Weinland.1984. 930. Detetmination of phagocytosis of ~ZP-~~CMStphylo- 18 Spurr, A. R. 1%9. A low-viscosity epoxy resin embed- coccus oureus by bovine polymorphonuclear leukocytes. ding medium for electron microscopy. J. Ultrastruct Am. J. Vet. Res. 45:786. Res. 2631. 8 Mishell, R. 1.. and R W.Dutton. 1967. Immunimtion of 19Stosse1, T. P. 1977. Phagocytosb. Page 87 in The dissociated spleen cell cnlhves from normal mice. I. +-e: fimction and clinical utilization. Alan R Exp. Med. 126423. Lis. Inc., New York, NY. 9Nemanic. M. K., and D. R. Pitelka. 1971. A scam@ 20 Syriopoulou, V. P., A. L. Harding, D. A. Goldmann, and electron microscopy study of the lactating mammary A. L. Smith 1981. In vim antiiterial activity of gland. J. Cell. Biol. 48:410. flUorinatCd analogs of Chloramphenicol and this- lONeu, H. C., and K. P. Fo. 1980. In vitro activity of phenicol. Antimicrob. Ag. Chemother. 19:294. chloramphenicol and thimpheuicol dogs. An- 21 Wmhol, I. B., D. C. Herbe~t,and E. G.Rennels. 1974. timicrob. Ag. chemother. 18311. An improved fixation procedure for microtubles and 11 Nickerson, S. C.. M.J. Paape, and A. M. Dulin, 1985. microhcnts in cells of the anterior pituitary. Am. J. Effect of antibiotics and vehicles on bovine mamrrrmy Anat. 141:427. polymorplnmuclear ldocyte morphologic features, vi- 22 Ziv, G. 1980. Availability and usage of new antibacterial abiity, and phagocytic activity in vitro. Am. J. Vet Res. dmgs in Europe. J. Am. Vet. Med. Assoc. 1761122. 46:2259. 23Ziv, G.. E. Bogin. J. Shani (Mishlciosky), and F. G. 120liver, J. M., R. B. Zmia, and R. D. Berlin. 1975. Sulman. 1973. Distribution and blood-to-milk trausfer of Concauavdin A cap leulrocytes of normal and beige labeled antibiotics. Antimicrob. Ag. chemother. 3607. (ChCdiaL-Higashi) mice. Nature (LOMI).253:471. WZiv, G., S. Gordin, G. Bachm, and S. Soback 1973. 13 Paape, M.J., S. C. Nickerson, and G. Ziv. 1990. In vivo conctntration and persistence of antibiotics in milk effects of chloramphenicol, tetracycline and gentamicin following . . nry infusion in cows. Rdu. Vet. on bovine polymorphonuclearneutrophil lenkocyte mor- 30%. phology. Am. J. Vet. Res. (in press). 25 Ziv, G., M.J. Paape, and A. M. Dulin. 1983. Influence of 14Paape, M. J., R. E. Pearson, and W.D. Schulm. 1978. antibiotics and in1-y antibiotic products on Variation among cows in the ability of milk to support phagocytosisof Staphyylococcus aweus by bovine 1- phagocytosis and in the ability of polymorphonuclear cytes. Am. J. Vet Res. 44:385. leukocytes to phagocytose Staphylococcus aweus. Am. 26Zv, G., A. Saran-Rosenzoaig, and R. Risenberg. 1973. J. Vet. Res. 391907. Retenton of antiiiotics in dry-udder secretions after the 15 pazdemik, T. L., and M. D. Corbett. 1979. Effects of iufusion of several "dry cow" antibiotic products. Zen- chloram-phenicol duction products on hemopoietic tralbl Vetainaernaed @3] 24415. precursor cells in vitro. pharmacology 19191. 27 Ziv, G., and F. G. slllman 1975. Absorption of antibiot- 16Root, R. K.,A. S. Rosenthal, and D. J. Balestra. 1972. ics by the bovine udder. J. Dairy Sci. 58:1637.

Journal of Dairy Science Vol. 73, No. 7, 1990