J. Vet. Med. B 39, 537-545 (1992) 0 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1793

International Food Institute of Queensland, Australia

In vitro Antibacterial Activity of the Lactoperoxidase System Towards Enterotoxigenic Strains of Escherichia coli P. A. GRIEVE, D. A. DIONYSIUS and A. C. Vos Address of authors: International Food Institute of Queensland, 19 Hercules Street, Hamilton, Q. 4007, Australia

With 3 figures and 3 tables (Received for publication July 19, 1991)

Summary The lactoperoxidase-thiocyanate-hydrogenperoxide (LP) system inhibited the growth of enterotoxigenic Eschrrzchia coli strains responsible for scouring in neonatal and post-weaning piglets. An enzymatic system for hydrogen peroxide generation (glucose oxidase, GO; 0.1 U/ml) and a chemical source (sodium carbonate peroxyhydrate, SCP; 90 mg/l) were used in the LP system to test 19 strains in a 6-h growth assay at 37°C. Only three strains were highly sensitive to the LP/GO system, while all exhibited significant growth inhibition with the LP/SCP system. Hydrogen peroxide alone had less effect than the complete system. The bactericidal activity of the LP/GO system towards a previously resistant strain was greatly increased by increasing the level of glucose oxidase in the system by three- or five-fold.

Introduction The inhibitory effect of the lactoperoxidase system on many bacterial species in vitro has been well documented [I I]. Lactoperoxidase (LP) catalyses the oxidation of the thiocyanate ion (SCN-) by hydrogen peroxide ( H 2 0 2 )to form hypothiocyanite (OSCN-), the putative antibacterial agent [l, 91. Higher oxyacids, including HOPSCN and HO,SCN, have also been implicated in the inhibitory mechanism [3, 8, 121. The oxidation of essential protein sulfhydryl groups by LP reaction products is considered the main cause of the inhibitory effect, as reducing compounds can reverse the inhibition [l 11. The in vitro antibacterial activity of the LP system towards enteric bacteria has resulted in the investigation and use of this system for the prevention of scours in neonatal animals. REITERet al. [15] showed a growth-promoting effect of the LP system on newborn calves, while STILLet al. [18] reported that a preparation based o n the LP system and lactoferrin was effective in the treatment of enteric colibacillosis in calves. GRUN[7] found that feeding calves and piglets milk supplemented with thiocyanate and peroxide to activate the LP system led to a lower incidence of diarrhoea and better weight gain. Piglet colibacillosis has a substantial commercial impact on the pig industry in Australia, with losses in production due to mortality, slower growth rates and treatment costs. There are two forms of colibacillosis: neonatal colibacillosis, caused by predominantly non-haemolytic enterotoxigenic Escherichia coli (ETEC), and post-weaning coliform enteritis, caused by predominantly haemolytic ETEC 161. In this investigation we have US.Copyright

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GRIEVE, DIONYSIUS and Vos

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examined the in d u o antibacterial activity of the LP system towards enterotoxigenic strains of porcine E. Cali isolated from cases of neonatal and post-weaning scours. The effects of two exogenous sources of hydrogen peroxide, a glucose/glucose oxidase H202generating system and a chemical system (sodium carbonate peroxyhydrate, SCP) on the antibacterial activity of the LP system were also studied.

Material and Methods Bacteria and Culture Conditions Enterotoxigenic Escherichia coli (ETEC) strains were isolated from cases of neonatal and postweaning scours in piglets. Eight isolates (PEC 1-8) were obtained from the Regional Veterinary Laboratory (Wagga Wagga, NSW, Australia) and were kindly provided by Dr. PAT BLACKALL (Animal Research Institute, Brisbane, Q., Australia). Eleven isolates (PEC 9- 19) were kindly FAHY(Regional Veterinary Laboratory, Bendigo, Vic., Australia). provided by Dr. TONY Each isolate, on receival at the laboratory, was allocated a laboratory reference number (PEC 1- 19), regenerated in Isosensitest broth (Oxoid Limited, Basingstoke, Hampshire, England), and inoculated and grown overnight on peptone-yeast extract (PYE) agar slants at 37°C. PYE agar contained 1 % peptone, 0.5% yeast extract, 0.5% NaCl and 1.0% Agar No. 1 (Oxoid Ltd.). All isolates were routinely maintained by subculturing onto PYE agar slants at three monthly intervals and stored at 4 “C. Working inocula for antibacterial assays were prepared by subculturing isolates from agar slants into Isosensitest broth and incubating at 37°C for 16h. The serotype, haemolytic activity and scouring type of each isolate is shown in Table 1.

Analyses Lactoperoxidase activity was determined spectrophotometrically using 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS; Sigma Chemical Co.) essentially by the method of PUTTER and BECKER[14]. Assay mixtures (2.38ml) contained 1.67mM ABTS and 0.18mM H202 in O.1M

Table 1. Enterotoxigenic Escherichia coli isolates from piglets with neonatal and post-weaning coli bacillosis Laboratory Reference Number

Serotype‘)

Haemolysis ~

PEC

1 2 3 4 - 5 6 7 8 9 10 11 12 13 14 15 16 17

: OX : 018ac : 08 :

18

: :

19

0157

0149 08 01

: : :

OX

: :

08 09 020 064 0101

OX 0138 0149 0141 0157 0141

: :

: : : : : :

K’V17’,K83,K12,K87, K88ac K91, KXXac KSNT,K12,KSNT, K88ab KSNT,KSNT,KL238, K88 KV142, K99 KSNT,K87, K88 K81,K91, K88 K85ab,KV17,K85ac,-

Colibacillosis Type7 ~

~~

: HNT : H11 : €320

-

N

-

: H19

-

: H2

-

: HNM

N N N N N

-

+

N

: H19

: H10

. . : :

-

. . -

. . . . . -

-

+

-

+ +

+ + + +

N N N N N

N PW PW PW PW PW

PW

,’ Serotype (0: capsular, fimbrial: flagellar); + N, neonatal colibacillosis; PW, post-weaning colibacillos19.

Antibacterial Activity of Lactoperoxidase Towards E. coli

539

citrate buffer, p H 5.5. The reaction was started by the addition of 20 pl enzyme and the rate of change of absorbance at 405 nm measured on a recording spectrophotometer at 25 "C. The hydrogen peroxide concentration of samples was determined by a modification of the LP assay using ABTS as the hydrogen donor. The reaction mixture (3.0ml) contained 1.34mM ABTS, 6.67 pg/ml horseradish peroxidase (Calbiochem-Behring Corporation, LaJolla, California, USA) and sample (containing up to 300 pM H202)in 0.1 M citrate buffer, p H 5.5. The reaction was started by the addition of enzyme and the rate recorded until a maximum absorbance value was obtained at 405 nm. A linear relationship was obtained between Hz02concentration and absorbance at 405 nm. Catalase activity was determined spectrophotometrically at 240 nm using the method of BEERS and SIZER[2]. Assays for LP activity and hydrogen peroxide were carried out in duplicate and the means reported. Antibacterial Assays

In vitro antibacterial activity of the LP /glucose oxidase / thiocyanate (LP/GO) and the LP / sodium carbonate peroxyhydrate / thiocyanate (LP/SCP) systems towards porcine ETEC was determined in Isosensitest broth at 37°C. Assays were modifications of the method of REITERet al. [16]. The LP/GO system contained 2.24 mM KSCN, 1.5 ABTS U/ml LP (Sigma Chemical Company, St. Louis, Missouri, USA), 0.3 %, ( w h ) glucose and 0.1 U/ml glucose oxidase (Sigma Chemical Co.) in Isosensitest broth. All liquid components added to the broth were sterilised by passage through a 0.2 pm membrane filter (Flow Laboratories Australasia, Sydney, Australia). The LP/SCP system had the same composition as the LP/GO system, except that glucose oxidase was replaced by 90mg/l sodium carbonate peroxyhydrate (SCP, 2Na2C03. 3 H202; Kali-Chemie, Hannover, FRG) as the H202-generating source. Assays were inoculated with a 16-h culture of the test organism, grown in Isosensitest broth at 3 7 T , to obtain an initial bacterial population of approximately lo4 colony forming units (CFU) per ml. Control assays contained all components except glucose oxidase or SCP, while the effect of the H202-generating system was determined in assays which contained all components except LP. The antibacterial effect of the LP systems on the growth rate of each organism was measured by determining the viable count at selected intervals using a pour plate method. Samples were serially diluted into sterile 0.1 %, Bacto-Peptone (Difco Laboratories, Detroit, Michigan, USA) at p H 7.0, and 1.0-ml aliquots of appropriate dilutions were then pipetted onto Petri dishes and mixed with lOml Tryptone Glucose Extract Agar (Oxoid Ltd.) a t 45°C. After the agar solidified lates were incubated ' p. at 37°C for 48h and bacterial colonies counted. Plate counts were performed in duplicate and the means are reported. Assays for hydrogen peroxide and LP activity were carried out at regular intervals during the incubation period.

Results

Effect of the LP System on Bacterial Growth The in vitro antibacterial effect of the LP/GO and LP/SCP systems on the growth of porcine ETEC strain PEC 3 is shown in Fig. 1. The LP/GO system (Fig. 1 A) had no effect on growth, while glucose oxidase in the absence of LP slowed growth for at least 6h. When LP/SCP was used as the inhibitory agent, substantial differences in growth were observed (Fig. 1 B). The complete system (LP/SCP) showed bactericidal activity against PEC 3, while SCP alone was bacteriostatic for 4 h. However by 24 h both cultures had again regrown to control (stationary phase) levels (lo9 CFU/ml). The inhibitory effect of the LP system on the growth of a second strain, PEC 18, is shown in Fig. 2. In contrast to PEC 3, this strain was extremely sensitive to the LP/GO system, and was also inhibited by glucose oxidase in the absence of LP (Fig. 2 A). LP/SCP, and SCP alone, both exhibited bactericidal activity against PEC 18 (Fig. 2 B). Despite the initial effectiveness of SCP in inhibiting growth, bacterial numbers had recovered to control levels after 24h. In comparison, no recovery was observed with the LP/SCP system, or with glucose oxidase in the presence or absence of LP. Hydrogen peroxide concentrations were measured during the incubation of PEC 3 with both LP systems (Fig. 3 A). For both LP/GO and LP/SCP, hydrogen peroxide was not detectable during the entire incubation period. In the absence of LP, glucose oxidase generated 220 pM H 2 0 2 over 4 h, but the concentration then decreased to zero by 8 h.

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Fig. 1. Effect of the lactoperoxidase system on the growth of enterotoxigenic E . coli, strain PEC 3, in Isosensitest broth at 37 "C.a, lactoperoxidase / glucose oxidase system: W control; 0 control with glucose oxidase; A test. b, lactoperoxidase / sodium carbonate peroxyhydrate system: control; 0 control with sodium carbonate peroxyhydrate; A test

Detectable amounts of catalase (2.3 U/ml) were present after 8 h in this system. When SCP was used to generate H202, 350pM H202 was present immediately after the granules dissolved, and the level slowly decreased to zero after 12 h incubation. When incubations of PEC 18 with the LP systems were assayed for H202,similar findings were observed with LP/GO and LP/SCP (Fig.3B). However when glucose oxidase generated H202in the absence of LP, the level of H202continued to rise over 12 h, reaching 740 pM and remaining stable for a further 12 h. The inability of this strain to remove the H202 results in total destruction of the bacteria after 12 h. No catalase was detected during the 24-h incubation. With SCP as the inhibitory agent, 130 pM H202was still present after I2 h. The slower rate of H202removal by this strain resulted in a delayed growth recovery compared with the more resistant PEC 3. In control cultures, catalase activity of PEC3 was 5.6 and 5.2U/ml at 8 and 24h respectively, while the levels for PEC 18 at corresponding sample times were 0.8 and 1.5 U/ml, respectively. Measurements of LP activity during the incubation of PEC 3 at 37 "C showed that the enzyme in control samples containing all components except glucose oxidase o r SCP retained full activity (1.4U/ml) for at least 8 h, and more than 80 % of maximal activity after 24 h. Activity in the LP/GO sample gradually decreased with time, to 0.9 U/ml after 24h. In the presence of SCP, LP activity immediately decreased to 0.9U/ml, but then remained stable for the duration of the experiment. Similar results were obtained when PEC 18 was tested for LP activity.

Antibacterial Activity of Lactoperoxidase Towards E . coli

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Time ( h ) Fig. 2. Effect of the lactoperoxidasesystem on the growth of enterotoxigenic E . colz, strain PEC 18, in Isosensitest broth at 37°C. a, lactoperoxidase/glucose oxidase system: control; 0 control with glucose oxidase; A test. b, lactoperoxidase/ sodium carbonate peroxyhydrate system: W control; 0 control with sodium carbonate peroxyhydrate; A test

Bacterial Strain Sensitivity to the LP System The growth curves for P E C 3 and 18 in the presence of the antibacterial systems indicated that any substantial inhibition was observable after 6 h incubation at 37°C (Figs. 1 and 2). This 6-h incubation time was consequently chosen to measure the relative sensitivities of the bacterial strains to the LP system. The log,, reduction in CFU/ml of test assays compared with controls for 19 ETEC strains is shown in Table2. Only three strains were substantially inhibited by the L P / G O system. Strains in this LP/GO-sensitive category, with reductions in log,, CFU/ml of 3 o r greater, include PEC 9, 18 and 19. When the LP/SCP system was tested, all 19 strains were sensitive, with the 6 h reduction in log count ranging from 3.6 to 7.3. Incubation of bacteria with Hz02generated from SCP also proved highly inhibitory for all strains, with reductions in log count ranging from 2.6 to 6.2, but in most cases was not as effective as the combined LP/SCP system. Table 3 shows the effect of three- and five-fold increases in glucose oxidase concentration on the growth of LP/GO-resistant and sensitive strains, P E C 3 and 18, respectively. The antibacterial effect of the LP/GO system towards the previously resistant strain P E C 3 was dramatically enhanced by increasing the concentration of glucose oxidase in the system. Levels of H202remained very low (approx. 6pM with 0.5U/ml G O after 6h), indicating that virtually all the H202 generated was utilised by the L P system. In the

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absence of LP, increases in glucose oxidase concentration did not have such a large effect on bacterial growth: the reduction in bacterial numbers for resistant and sensitive strains did not reach that obtained with the complete system, despite the presence of up to 730 pM H 2 0 2after 6 h incubation.

Discussion Bacteriostatic and bactericidal effects of the LP system on Gram-negative bacteria have been documented previously [ll]. The source of H202for activation of the system may be enzymatic (glucose / glucose oxidase, xanthine / xanthine oxidase), chemical (H202 or a H202-generating species), or bacterial (lactobacilli, streptococci). In these studies, a system with continuous generation of H 2 0 2(LP/GO) was compared with one producing a single, initial burst of H202(LP/SCP). We have shown previously [5] that the production of hypothiocyanite (OSCN-), the active agent, differs greatly with these two systems: LP/ GO generates and maintains low levels (< 100 pM) for at least 10 h, while LP/SCP produces a burst of OSCN- (- 400 pM), but the level falls appreciably over 6 h. In both systems H202

Antibacterial Activity of Lactoperoxidase Towards E . colz

543

Table 2. Sensitivity of enterotoxigenic Eschevichia coli strains to the lactoperoxidase system PEC Number 1 2 3 4 5 6 7 8 93 10 11 12 13 14 15 16 17 18a 19

Mean

LP/GOt SE

0.9 0.5 0.3 0.4 0.3 1.9 2.5 1 .o 4.3 0.4 1.1 0.9 0.9 1.0 1.3 0.6 0.3 7.4 6.9

0.4 0.2 0.1 0.6 0.1 1.4 0.9 0.7 1.6 0.1 0.3 0.5 0.5 0.2 0.9 0.1 0.1 0.1 0.2

n 4 4

8 2 3 3 5 3 3 8 3 3 3 6 3 2 5 7 6

A Loglo (CFU/ml) at 6 h LP/SCP* Mean SE n 6.1 6.2 6.9 4.8 6.1 6.2 5.8 5.1 5.9 7.0 7.2 6.8 3.6 6.9 6.1 6.7 7.3 7.2 6.9

0.9 0.6 1.1 1.4 0.8 0.2 0.6 0.3 0.3 0.3 0.7 1.6 0.3 1.o 0.9 0.5 0.5 0.3 0.3

4 4 3 2 2 2 2 3 3 6 2 2 2 2 2 2 2 3 2

Mean

SCPS SE

4.1 4.9 4.3 4.3 4.0 4.3 5.0 5.7 4.8 5.0 6.2 4.5 4.0 3.7 2.6 3.8 5.5 4.9 4.2

n

0.5 1.2 0.6

3 3 2 1 1 2 1 1 1 1 1 2 1 2 2 2 2 3 2

1.0

0.7 0.5 0.4 0.1 0.1 1.2 0.1

'>Defined as the reduction in log bacterial numbers of the test compared with a control after 6 h incubation at 37 "C; t Lactoperoxidase / glucose oxidase; Lactoperoxidase / sodium carbonate peroxyhydrate; § Sodium carbonate peroxyhydrate; a L P / G O - sensitive strains.

is not detectable during the incubation, supporting the argument that the active species is a product of the LP reaction. PURDYet al. [13] have noted similar findings in studies on Sahonella typh irnurium. The LP reaction product had a greater antibacterial effect than H202in all assays except when LP/GO was tested against the resistant strain PEC3. In this experiment, OSCN- generated by LP/GO (< 100pM) did not affect growth, whereas H2O2generated by glucose oxidase (> 200 pM after 4 h) slowed growth for 6 h. The low level of OSCNmay be insufficient to affect bacterial metabolism in this resistant strain, while the higher

Table 3. Sensitivity of enterotoxigenic Escherzchza coli to the lactoperoxidase / glucose oxidase system

PEC Number 3

18

Glucose Oxidase (U/ml) 0.1 0.3 0.5 0.1 0.3 0.5

A Loglo (CFU/ml) at 6 h +

Mean

LP/GOt SE

n

Mean

GO* SE

n

0.34 4.79 6.88 7.39 7.55 7.55

0.12 1.38 0.76 0.11 0.09 0.09

8 5 6 7 3 3

1.91 2.69 3.57 2.88 3.67 5.42

0.28 0.50 0.63 0.29 0.85 1.18

5 3 4 5 3 4

Defined as the reduction in log bacterial numbers of the test compared with a control after 6 h incubation at 37 "C; t Lactoperoxidase / glucose oxidase antibacterial assay; Glucose oxidase antibacterial assay.

'h

*

544

GRIEVE, DIONYSIUS and Vos

concentration of H202 is sufficient to initiate oxidative damage. KLEBANOFF [lo] has speculated that the relatively low reactivity of H202 allows it to pass intact through bacterial cell membranes and consequently to be toxic under conditions in which more reactive oxidation products, e.g. OSCN- are readily scavenged. LP/GO assays employed concentrations of components shown by REITERet al. [16] to inhibit E. coli in a synthetic growth medium. In a richer growth medium, we observed only slight reductions in growth for most strains tested. However there were several exceptions, where significant bactericidal effects were observed. Further activation of the system by provision of additional H202-generating capacity led to enhanced bactericidal activity against a strain previously resistant. The greater efficacy of the oxidative species over H202was also demonstrated. In comparison, the LP/SCP system was consistently effective, and longer lasting than the effect of H202generated by SCP. Under these assay conditions bacteria are exposed to high initial concentrations of OSCN-, and oxidative damage to cell components occurs rapidly. The differences in sensitivity of P E C 3 and 18 to the LP/GO system may indicate metabolic differences, or variations in cell wall structure. Catalase levels in P E C 18 were much lower than PEC 3, and this probably accounts for the inability of P E C 18 to remove Hz02 before toxic levels accumulated. The strain’s sensitivity to OSCN- generated in a suboptimal LP/GO system suggests the lack of an efficient control and/or repair mechanism for oxidative damage. PURDYet al. [13] found that S.typhirnurium mutants with greater cell envelope permeability were more susceptible to the LP system, possibly through easier access of OSCN- to cellular sulfhydryl groups. The lack of an OSCN--scavenging enzyme, NAD(P)H-OSCN oxidoreductase, has been implicated in slower recovery of some streptococcal strains to inhibition by the LP system [4]. Another possible difference lies in the recovery mechanism postulated by THOMAS and AUNE[19]. With this scheme, intracellular sulfhydryls, e.g. glutathione, reduce the oxidised sulfhydryl groups (sulfenyl derivatives), and NAD(P)H is oxidised in the regeneration of glutathione by a reductase. A bacterial cell with lower reserves of oxidisable compounds, e.g. NAD(P)H o r sulfhydryls, may be irreversibly inhibited as sulfenyl derivatives are further oxidised by excess OSCN-. While our studies have not extended to cell structure characteristics, our results suggest that metabolic differences, e.g. catalase and other protective agents, may account for differences in strain sensitivity. The in vivo application of the LP sysrem requires an exogenous supply of H202, REITERet al. [17] showed that colonised lactobacilli can provide sufficient H202to kill E. coli in the calf abornasum; however most workers have used an enzymatic source, with glucose / glucose oxidase being used in studies to show bactericidal and growth-promoting effects [15, 16, 181. In conditions of limited oxygen availability, such as the small intestine of neonates where colonisation of enterotoxigenic E. coli occurs, an alternative source of H202may be more effective for activation of the LP system. Chemical species with H202releasing activity provide another option. The disadvantages of such systems are the discontinuity of OSCN- supply, which may necessitate repeated additions, and the need for a reliable delivery system for effective supply of OSCN- at the required site of action. However, these studies have shown that the SCP system is comparable with an enzymatic system for in vitro inhibition of E. coli responsible for neonatal enteric infections. Acknowledgements The authors wish to thank Dr. TONYFAHYand Dr. HUBERT ROGINSKIfor helpful discussions and Mr. STEPHENNOTTINGHAM for statistical analyses. This work was supported by the Australian Dairy Research and Development Corporation.

References 1. AUNE,T.M., and E.L. THOMAS,1977: Accumulation of hypothiocyanite ion during peroxidase - catalyzed oxidation of thiocyanate ion. Eur. J. Biochem. 80, 209-214.

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2. BEERS,R. J, Jr., and I. W. SIZER,1952: A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140. 1980: Correlation between concentration of hypothiocyanate and 3. BJORCK,L., and 0.CLAESSON, antibacterial effect of the lactoperoxidase system against Escherichia coli. J. Dairy Sci. 63, 919-922. 4. CARLSSON, J., Y. IWAMI,and T. YAMADA, 1983: Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogenperoxide. Infect. Immun. 40, 70-80. 5. DIONYSIUS, D. A,, P. A. GRIEVE,and A. C. Vos, 1992: Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation. J. Appl. Bact. 72, 146-153. S. J. DRIESEN,and E. M. SPICER,1987: A symposium: The 6. FAHY,V. A., I. D. CONNAUGHTON, control of pre and postweaning diarrhoea in the pig. In: Manipulating pig production, C.HANSEN, P . H . HEMSpp. 177-201 (Eds. BARNETT, J.L., B.S. BATTERHAM, G.M. CRONIN, WORTH,D.P. HENNESSY, P.E. HUGHES,N.E. JOHNSTON,and R . H . KING) Australasian Pig Science Association, Werrihee, Australia. 7. GRUN,E., 1984: Die Bedeutung des Laktoperoxidase-Thiozyanat-Peroxid-Systems fur die Erhaltung der Gesundheit des Euters und des Kalbes. Mh. Vet.-Med. 39, 698-700. 8. HOGG,D. Mc., and G. R. JAGO,1970: The antibacterial action of lactoperoxidase. The nature of the bacterial inhibitor. Biochem. J. 117, 779-790. W. SCHOLTES,and L. A. STODDARD, 1977: Hypothiocyanite 9. HOOGENDOORN, H., J. P. PIESSENS, ion; the inhibitor formed by the system lactoperoxidase-thiocyanate-hydrogenperoxide. Caries Res. 11, 77-84. 10. KLEBANOFF,S. J., 1988: Phagocytic cells: products of oxygen metabolism. In: Inflammation: Basic and Principles and Clinical Correlates, pp. 391 -444 (Eds. GALLIN,J. I., I. M. GOLDSTEIN, R. SNYDERMAN) Raven Press Ltd., New York. 11. PRUITT,K. M., and B. REITER,1985: Biochemistry of peroxidase system: antimicrobial effects. In: The Lactoperoxidase System: chemistry and biological significance, pp. 143-178. (Eds. PRUITT, K., and J. TENOVUO) Marcel Dekker, New York. R. W. ANDREWS, and T. MCKANE,1982: Lactoperoxidase-catalyzed 12. PRUITT,K. M., J. TENOVUO, oxidation of thiocyanate: polarographic study of the oxidation products. Biochemistry 21, 562 -567. 13. PURDY,M. A,, J. TENOVUO, K. M. PRUITT,and W. E. WHITE,Jr., 1983: Effect of growth phase and cell envelope structure on susceptibility of Salmonella typhimurium to the lactoperoxidasethiocyanate-hydrogen peroxide system. Infect. Immun. 39, 1187- 1195. 14. PUTTER, J., and R.BECKER,1983: Peroxidases. In: Methods of Enzymatic Analysis, Vol.3, pp. 286-293 (Ed. BERGMEYER, H. U.) Verlag Chemie, Weinheim. N.YARROW,M. J. DUCKER,and M.KNUTSSON, 15. REITER,B., R. J. FULFORD,V.M. MARSHALL, 1981: An evaluation of the growth promoting effect of the lactoperoxidase system in newborn calves. Anim. Prod. 32, 297-306. L. BJORCK,and C.-G. ROSEN,1976: Nonspecific bactericidal 16. REITER,B., V. M. E. MARSHALL, activity of the lactoperoxidase-thiocyanate-hydrogenperoxide system of milk against Escherichia coli and some Gram-negative pathogens. Infect. Immun. 13, 800-807. 17. REITER,B., V. M. MARSHALL, and S. M. PHILIPS,1980: The antibiotic activity of the lactoperoxidase-thiocyanate-hydrogenperoxide system in the calf abomasum. Res. vet. Sci. 28, 116-122. 18. STILL,J., P. DELAHAUT, P. COPPE,A. KAECKENBEECK, and J. P. PERRAUDIN, 1990: Treatment of induced enterotoxigenic colibacillosis (scours) in calves by the lactoperoxidase system and lactoferrin. Ann. Rech. Vet. 21, 143-152. 19. THOMAS,E. L., and T. M. AUNE, 1978: Lactoperoxidase, ,peroxide, thiocyanate antimicrobial system: correlation of sulfhydryl oxidation with antimicrobial action. Infect. Immun. 20, 456-463.