Maataloustieteellinen Aikakauskirja Vol. 56: 309—323, 1984 Xylitol, polyol molasses and glucose in the diet of newborn calves 11. Content of antimicrobial factors in blood and saliva, bacteria in faeces and health status HANNU KORHONEN 1, EIJA ALASAARI and MATTI ANTILA Department of Dairy Science, University of Helsinki, SF-00710 HELSINKI 71, Finland MIKKO TUORI and ESKO POUTIAINEN Department of Animat Husbandry, University of Helsinki, SF-00710 HELSINKI 71, Finland Abstract. The concentrations of various antimicrobial factors in the saliva and plasma of newborn calves with special reference to possible effects of a diet supplemented with dif- ferent sugar alcohols were studied. Eighteen calves were assigned alternately at birth to three groups, each comprising six animals. All calves were fed a pooled colostrum diet for the first four days, thereafter whole milk plus milk replacer. Concentrates and hay were given ad libitum. The diets of the different groups were supplemented with the following test sub- strates: xylitol, polyol molasses or glucose (control). The daily doses of each substrate were progressively increased from 0.5 to 1.0 g/kg of live weight for the last four weeks. Plasma and saliva samples were taken from all calves before colostrum feeding (day 0) and on days 1,2, 4,7, 21 and 35 after birth. The following factors were determined quantitatively: lacto- ferrin (LF), lactoperoxidase (LP), lysozyme (LZM) and immunoglobulins IgG,, lgG 2 , IgM and IgA (only in plasma). Further determinations included weekly counts of total aerobic bacteria, aerobic haemo- lytic bacteria, coliforms, lactobacilli and clostridia in faeces. Salivary LF increased from the average 0 day level of 3.6—17.0 /rg/ml in the different groups to a maximum of 38.7—55.6 jig/ml within one week, and declining thereafter slowly in all groups. LF was not found in the saliva of all calves at birth, but was consistently present later on. No LF was detected in plasma. Salivary LP increased from the average 0 day level of 56.3—86.6 /rg/ml in the different groups to a maximum of 228—296 /j.g/ml within three weeks and declined markedly by day 35. LP was found in all saliva samples, but the plasma concentrations were very low and not always detectable. Salivary LZM was high at birth com- pared to the plasma level (average 0.8—2.1 /rg/ml vs. 0.3—0.5 ng/ml in the different groups) Present address: Ministry of Livestock Development, P.O. Box 68228, Hill Plaza, Nairobi, Kenya, Index words: antimicrobial factors, lactoperoxidase, lactoferrin, lysozyme, calves, blood, saliva, xylitol, polyols, faecal bacteria 309 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=Y0wkI_hR9BYS7-G2.gZAgDo-QZndDlbBi7g6nIA._1a1SgeVruW4rvXN_Vvyz8J803muC7j08sjAlIT8msQevFPrIJX9IakI9ujhnWxSSZVGZYPTBvhn-gKYnyHf2NX_YnzzgN7XpyH_yWKiaULY_g1RJWo6mR6PjEcMGtLGzKDjzFMv9hMQbbBuOCj_-ZZzQqx-VU_weXVeULzYNwKWwe20TbKIwQIuqgI3drgJfMtuYPz0z5HUg4SwtjO1YimTZa6FB4STWO2EzQyKhgKMyV87_gv6tdCWzw but dropped within one week in all groups, reaching the plasma level by day 21. No clear changes were observed in the LZM plasma concentration. Except for IgG 2 , there was a rapid but transient increase in the plasma levels of all Ig’s, in particular of IgG„ after the first colostrum feeding. From day 7 the levels of Ig’s with the exception of lgG2 , started to rise again. There were no statistically significant differences in any of the investigated antimicro- bial factors or bacterial groups between the feeding groups, except for Clostridia which exhib- ited the highest count (P <0.05) in the xylitol group. Also the average salivary LF, LZM and IgG, levels were highest, and the health status was best in the xylitol group. The results suggest that, besides antibodies, LF, LZM and LP, which are present in maternal colostrum and also in saliva of the newborn calf, may contribute considerably to the protection of the calf against pathogens during the first weeks of life. Introduction The importance of colostrum feeding for the survival and health of the newborn calf is well established (for recent literature, see Porter et al. 1977, Ann. Rech.Vet. 9 (2) 1978 and Roy 1980). The protection pro- vided by the colostrum is primarily attribut- able to antibodies which are absorbed from the calf’s intestine into circulation during the first 24—36 hours after birth, the absorption time depending on the class of immunoglob- ulin (Penhalecl al. 1973, Logan et al. 1978, Stott and Menefee 1978, Stott et al. 1979 a, 1979 b, Bush and Staley 1980). In addition to systemic immunity, colostral antibodies seem to confer local protection against microbial infections within the gas- trointestinal tract (Logan et al. 1974 b). Besides antibodies, the colostrum and postcolostral milk contain varying amounts of non-specific antimicrobial factors such as lactoferrin (LF), lysozyme (LZM) and the lactoperoxidase (LP) system as well as living cells (for recent reviews, see Reiter 1978 a, 1978 b, Korhonen et ai. 1978). Their poten- tial role in the protection of neonatal calves against enteric or systemic infections is as yet little explored, but their concentrations in colostrum have been the subject of recent studies (Korhonen 1977, Meriläinen et ai. 1979). Recently, Reiter et ai. (1980) showed that the LP system can be activated in vivo in the abomasum of the calf, and this may contribute to its resistance to infections. It appeared, therefore, worth investigating the occurrence of the above antimicrobial fac- tors in the saliva and blood of newborn cal- ves and the effect of colostrum feeding on their concentrations. The present study was carried out as part of a trial on the effects of supplementing the colostrum-based diet with different sugar alcohols. Therefore their potential influence was evaluated, with special reference to studies of Mäkinen et al. (1975) according to which long-term xylitol diet enhanced the peroxi- dase activity in human salivaand reduced the frequency of caries. In the first part of the present study, the results of certain physio- logical parameters and growth of the calves are described (Tuori 1984). Materials and methods Arrangement of the feeding trial The details of the experimental procedures have been described in the first part of the present paper (Tuori 1984). In brief, the fee- ding was arranged as follows: 18 newborn, unsuckled calves were assigned randomly, within sex, to three groups, each comprising six animals. Each group was fed during the first week a basic colostrum- and whole milk based diet, then a commercial milk replacer diet, supplemented with one of the following test substrates; polyol molasses, xylitol and glucose (control group). The feeding scheme was as follows: 310 Day after Colostrum Whole milk Test substrate Total amount birth % of live % of live g/kg of live of liquid weight weight weight diet 1 % of live weight 1 7 0.5 2 7 0.5 3 7 0.5 4 8 0.5 5 43 0.75 9 6 0 6 0.75 10 7 4 1.0 11 8— 4 1.0 12 9—35 1.0 12 1 Liquid diet contained whole milk, milk replacer (140 g powder in one litre water) + 1 g test substrate per kg live weight. Concentrates and hay were given ad libi- tum. The composition of thepolyol molasses has been given in the first part of the present paper (Tuori 1984). All test substrates were mixed in a liquid form with colostrum or milk immediately before feeding. The calves were fed individually twice a day. The first feeding took place about 2—6 hours after birth. Colostrum and milk for feeding Colostrum was pooled from the first six milkings post partum from several cows and stored frozen until feeding. Whole milk was collected from milk tank at a time and frozen. The concentrations of various anti- microbial factors were examined, and the result is given in the following table: Sampling and analyses Blood and saliva were collected from all calves according to the following schedule: 0 (before colostrum feeding), 1,2, 4 and 7 days after birth and at three weeks (21 days) and five weeks (35 days) of age. Blood samples were taken from the jugular vein into heparinized tubes. The red cells were separated by centrifugation, and plasma was stored at —2O°C until analyzed. The saliva samples were also stored at —2O°C. The concentrations of the different immu- noglobulins (IgG!, IgG2, IgM and IgA) were measured in plasma samples by the radial immunodiffusion gel technique according to Fahey and McKelvey (1965). The specific antisera and lyophilized standard Ig’s were purchased from Miles Laboratories (India- na, USA). Sample Lysozyme Lacto- Lactoferrin Immunoglobulins peroxidase /rg/ml ng/ml /tg/ml IgG, IgG 2 IgM IgA Colostrum 0.8 61.5 0.50 4.2 0 0.4 2.9 Whole milk 0.4 46.0 0.07 0.6 0 0.3 0.3 The data on the compositional parameters analyzed from colostrum and milk have been presented in the first part of the present paper (Tuori 1984). Both plasma and saliva samples were as- sayed for concentrations of lysozyme (Par- ry et al. 1965), lactoferrin (Ahonen et ai. 1978) and lactoperoxidase (Korhonen et ai. 1977). ' Oil311 Rectal faeces samples were taken weekly from every calf and the following quantita- tions were made: Total count of aerobic bacteria: 10 % sheep blood agar (Blood Agar Base, Merck Ag, Darmstadt), incubation for 2—3 days at + 37°C. Haemolytic aerobic bacteria: as above, but only colonies with haemolytic zones around them were observed. Coliform bacteria: Ergitol —7— Agar (Merck Ag, Darmstadt) aerobic incubation for 1 day at + 37°C. Lactobacilli: MRS Agar (Merck Ag, Darmstadt), anaerobic incubation in Gas Table 1. Means (x) and standard deviations (s) of lactoferrin concentrations 0»g/ml) in saliva of different feeding groups. Feeding Days postpartum group 0 1 2 4 7 21 35 All samples xs xs xs xs xs xs xs xs Xylitol 17.0 11.9 12.6 8.9 16.8 10.7 40.0 30.4 55.6 30.3 30.7 13.1 20.5 27.5 27.2 23.8 Polyol 12.7 9.4 13.2 7.3 16.8 3.7 29.2 12.6 46.3 29.4 32.2 16.8 28.8 29.1 25.4 20.4 Glucose 3.6 5.0 5.5 11.0 8.8 8.2 22.2 13.2 38.7 33.5 23.5 9.5 4.3 7.4 16.1 19.5 Fig. I. Mean concentrations of lactoferrin in saliva of different feeding groups. 312 Table 2. Means (x) and standard deviations (s) of lactoperoxidase concentrations Org/ml) in saliva and plasma of different feeding groups. Feeding Sample Days postpartum group 0 1 2 4 7 21 35 All samples X s X s X s X s X s Xs Xs Xs Xylitol Saliva 86.6 72.6 120.9 53.9 77.3 35.8 240.0 259.2 200.3 120.1 295.8 231.9 87.8 141.9 157.2 167.0 Plasma 0.2 0.3 0.7 1.0 1.1 1.7 0.9 0.7 1.6 2.0 1.6 1.3 0.5 0.4 1.0 1.2 Polyol Saliva 78.8 46.4 134.8 78.9 111.8 77.7 203.8 81.4 252.2 143.4 253.8 129.9 167.3 134.9 171.8 115.9 Plasma 0.5 0.8 0.3 0.4 1.2 1.7 1.2 0.7 0.5 0.5 2.3 2.1 0.7 0.7 1.0 1.3 Glucose Saliva 56.3 89.4 162.7 295.6 92.9 71.9 124.3 131.4 135.8 110.9 228.3 182.9 83.2 91.7 127.1 149.9 Plasma 2.1 1.1 0.4 0.7 0.5 0.6 0.4 0.3 1.7 2.4 1.6 2.2 1.1 1.0 1.1 1.5 Pak chamber under 5 % C0 2 for 3 days at + 37°C. Clostridia: RCM-broth (Merck Ag, Darmstadt), anaerobic incubation for 7 days at + 37°C. The health status of the calves was exam- ined by daily recordings of possible incidence of diarrhoea and general physiological con- dition. Results Lactoferrin (LF) LF was detected in no plasma samples. Neither was it found in the saliva ofall calves at birth, but it was present later on. In each feeding group, the LF concentration peaked within seven days and declined almost to the birth level by day 35 (Fig. 1). There were great interindividual variations in LF in each group, as seen from the standard deviations in Table 1. Throughout the trial the average level of LF was lower in the glucose group than in the other groups, but statistically sig- nificant (P < 0.05) differences could be confirmed only when the calves were 35 days old. Lactoperoxidase (LP) LP was present in all saliva samples, but the concentrations varied widely (range 1.0—690.0 jtg/ml), depending on the time of sampling and the calf (Table 2). In all groups, the LP saliva level rose clearly, though transiently, after the first colostrum feeding. It rose again from day 2 and peaked in all groups (3—4 times the birth level) on day 21 after which the level fell sharply al- most to the birth level (Fig. 2). Throughout the trial the average LP concentration was noticeably lower in the glucose group com- pared to the other groups, but this difference was not statistically significant at any sam- pling time. Compared to saliva, the LP plasma con- centration was very low and was not always 313 detectable in all calves (Table 2). Although the average LP plasma level parellelled quite well with that of saliva, no statistically signi- ficant correlation between these concentra- tions could be established. Lysozyme (LZM) At birth, LZM activity was present in the saliva and plasma of all calves. In subsequent samples it was, however, not always detect- able. During the first week of life, the sali- vary LZM concentration was about twice the plasma concentration, but thereafter the sali- vary LP dropped to the plasma level in all groups (Fig. 3, Table 3). In contrast to the wide interindividual and time-related fluc- tuations observed in the salivary LZM, the LZM plasma level remained relatively con- stant throughout the trial. No statistically significant differences in the level of this enzyme were observed between the different groups. However, theaverage level tended to be lowest in the glucose group. Fig. 2. Mean concentrations of lactoperoxidase in sali- va and plasma of different feeding groups. Fig. 3. Mean concentrations of lysozyme in saliva and plasma of different feeding groups. 314 Table 3. Means (x) and standard deviations (s) of lysozyme concentrations o*g/ml) in saliva and plasma of different feeding groups. Feeding Sample Days postpartum group 0 1 2 4 7 21 35 All samples X S X S X S X S X S X s X S X s Xylitol Saliva 2.0 1.0 2.0 2.1 0.8 1.1 2.0 1.8 1.4 1.4 0.6 0.4 0.5 0.4 1.3 1.3 Plasma 0.5 0.1 0.5 0.5 0.7 1.5 0.4 0.3 0.3 0.2 0.7 0.8 0.5 0.6 0.5 0.7 Polyol Saliva 2.1 2.2 1.1 0.9 3.2 2.6 0.9 0.2 1.2 1.4 0.4 0.1 0.3 0.2 1.3 1.6 Plasma 0.5 0.2 0.4 0.5 0.4 0.2 0.4 0.3 0.2 0.2 0.6 0.2 0.4 0.2 0.4 0.3 Glucose Saliva 0.8 0.6 1.7 2.2 1.1 1.7 0.7 0.5 1.2 1.3 0.6 0.2 0.5 0.4 0.9 1.1 Plasma 0.3 0.0 0.8 0.7 0.5 0.3 0.6 0.5 0.4 0.3 0.4 0.2 0.3 0.2 0.5 0.4 Immunoglobulins (Ig) Before the first colostrum feeding, the concentrations in all Ig classes were low or undetectable in the plasma of all calves (Ta- ble 4). Following colostrum ingestion the levels of all Ig’s increased rapidly and peaked within one or two days (Fig. 4). In particu- Fig. 4. Mean concentrations of various immunoglob- ulins in plasma of different feeding groups. 315 316 Table 4. Means and rangesof immunoglobulin concentrations in plasma of all feeding groups. Ig class Days postpartum 0 1 2 4 7 21 35 x range x range x range x range x range x range x range IgG, 0.17 0.0— 0.5 1.50 0.7—4.0 2.06 0.9— 6.0 2.10 0.7—7.6 1.43 0.0— 3.2 2.20 0.0— 6.0 4.40 0.0—25.0 IgG, 0.37 0.0— 0.5 0.40 0.3—0.4 0.23 0.0—0.5 0.37 0.0—0.6 0.33 0.0— 0.4 0.40 0.4— 0.5 0.40 0.4—0.5 IgM 0.20 0.0— 0.4 0.43 0.2—0.6 0.33 0.0—0.8 0.33 0.0—0.7 0.50 0.2— 0.9 1.27 0.6— 2.6 1.00 0.3— 2.6 IgA 0.37 0.0— 2.2 0.83 0.0— 1.9 0.30 0.0— 1.1 0.10 0.0— 1.1 0.57 0.0— 2.0 1.30 0.0— 1.9 1.23 0.0— 2.4lar, the IgG, level increased by manyfold, but declined after a few days, remaining, however, above the birth level in most calves. In the polyol group, no such decline was ob- served. The rise of IgM and IgA was relatively small and transient, whereas the lgG2 level remained almost unchanged. Towards the end of the trial period (at 21 and 35 days) the concentrations of all Ig’s, except for IgG 2 , began to rise sharply. There was no statistically significant dif- ference in any Ig class between the different groups. As seen from Table 4, there was great interindividual variation in the Ig classes. Fig. 5. Geometric means and ranges of variation of total aerobic bacteria and conforms in the faeces of different feeding groups. E 3 = xylitol group, □ = polyol group, Q = 8 ,u* cose group. Bacterial content Total aerobic bacteria The total number of aerobic bacteria in faeces of individual calves varied between 106—lOVg during the first three weeks, the mean count for all feeding groups being IOVg (Fig. 5). Thereafter, the counts de- creased in all groups to an average of IOVg, but the interindividual range of variation re- mained large (104—lOVg). Aerobic haemolytic bacteria These bacteria were found irregularly in faeces of all calves, and their occurrence in high numbers coincided with the diarrhoea. The average counts in positive samples of different groups varied between 107—lOVg during the first and second week, and de- creasing thereafter to 103 —lOVg by the fifth week. Also the frequency of positive findings diminished in all groups in the course of the trial. The total number of positive samples was lowest in the xylitol group (9 positive, n = 30) and highest in the polyol group (17 positive, n = 30). In the glucose group, 12 out of 30 samples were positive. Coliforms The average number of coliform bacteria varied in different groups between 106 and 10Vg (mean lOVg) during the first two weeks. Thereafter, the counts of all groups de- creased slowly to an average of IOVg, but the range of variation between different calves still remained large (10 4—l08/g) (Fig. 5). Lactobacilli The number of lactobacilli increased slightly in every group during the first three weeks. Mean count 10Vg, thus represented the predominant bacterial group. The count then decreased to an average level of IOVg (Fig. 6). Clostridia The number of Clostridia in faeces varied greatly between individual calves (102 lOVg), but an average level of 104—lOVg was maintained in all groups throughout the trial (Fig. 6). The counts were constantly higher in the xylitol group than in the other groups, the difference being significant at P < 0.05. In the case of other investigated bacterial groups, no significant differences were established between the feeding groups. Relationships between antimicrobial factors and bacterial content A correlation analysis was carried out in order to determine the possible relationships Fig. 6. Geometric means and ranges of variation of lactobacilli and Clostridia in the faeces of dif- ferent feeding groups. m = xylitol group, □ = polyol group, E 3 = glucose group. 317 between the concentrations of LF, LP and LZM in saliva and plasma and also between these factors and the content of different bacterial groups in faeces. These correlations were determined separately for each feeding group and for the total of all samples. The analysis indicated that with regard to all samples, salivary LF and LP correlated high- ly significantly (r = 0.441, P < 0.001) with each other. The correlation was highly signi- ficant (r = 0.484, P < 0.001) also in the polyol group and significant (r = 0.441, P < 0.01) in the xylitol group, whereas in the glucose group it showed no significance. No significant correlation was established between LZM and LF or LP, respectively. None of the above antimicrobial factors cor- related significantly with any of the investi- gated bacterial groups. No statistical analysis was made on the relationships between the different Ig classes in plasma and the bac- terial content of faeces. It is, however, inter- esting to note that the occurrence of aerobic haemolytic bacteria was most frequent (17 positive samples) in the polyol group where the average concentrations of all Ig classes were lowest during the first week of life. Health status In general, the calves of the xylitol group were healthiest. Only two of the six calves in this group showed any signs of illness during the trial. These calves exhibited mild scouring for a few days at two or three weeks of age, respectively, but no haemolytic bacteria were found in their faeces at the time of illness. All the calves of the polyol group showed signs of scouring during the trial. Four calves showed diminished appetite on week two with haemolytic bacteria detected in their faeces. At one week of age, one of these calves had a transient increase in body tem- perature associated with a simultaneous high count of coliform in faeces. The remaining two calves had mild diarrhoea for a few days on the second and third week, respectively. It coincided with the appearance of haemolytic bacteria in the faeces. In the glucose group, three calves exhib- ited mild diarrhoea on the second week. The other three calves showed dimished appetite during most of the time. An elevated tem- perature was recorded twice in one of these calves. During the duration of diarrhoea, haemolytic bacteria were found in faeces of all but one of the calves in this group. A sele- nium injection was administered intramuscu- larly to two and three calves of the glucose and polyol groups, respectively, during the first week. None of the calves in the xylitol group needed the injection. Discussion Recent studies have shown that bovine colostrum contains substantial quantities of non-specific antimicrobial factors. It appears therefore attractive to extend the immunol- ogical concept of colostrum for protection of the newborn to include also these non- antibody factors. However, in vivo evidence to support this is yet very limited and even the knowledge about the occurrence of anti- microbial factors, other than immunoglobu- lins, in the fluids of newborn and colostrum- fed calves is scanty. The principal aim of the present study was, therefore, to obtain a pic- ture of the levels of these factors in the saliva and plasma of newborn calves before and after colostrum feeding. For comparison the immunoglobulin concentrations were deter- mined also in plasma, but no in saliva due to difficulties in obtaining quantitatively ade- quate samples. In general, the results of this study reveal that there are enormous interindividual varia- tions in the concentrations of all investigated antimicrobial factors in saliva and plasma, irrespective of the same age and sex and sim- ilar environmental condition. The reasons for this variability cannot be explained, but some influence of hereditary factors may be suggested. With regard to the results on LF, LP and 318 S 319 LZM concentrations no direct comparisons can be made with other studies due to lack of relevant data. It seems, however, justified to compare the present results with concentra- tions of the respective substances in colos- trum and milk to obtain some indication of the importance of different antimicrobial factors to the calf. The average level of salivary LF increased maximally by 3- to 10-fold within one week after birth. Nevertheless, even at its peak the LF concentration remained about ten times lower than that in colostrum and was also slightly lower than in the milk used for feed- ing. Since the LF level rose sharply during the time (day 4) the diet was converted from colostrum to whole milk, the observed in- crease is not attributable to colostral LF. In- stead, it is probably due to a more pro- nounced synthesis of this protein in the buc- cal epithelium of the calf. Physiologically this is supported by the fact that the LF con- centration of colostrum drops drastically within two days after parturition (Korhonen 1977). Thus, the reduced intake of LF from colostrum is probably compensated by the commencement of the calf’s own LF produc- tion. This seems to coincide with the time when the passive immunity provided by co- lostral antibodies is decreased (Fig. 4), and the calf’s own Ig production has not yet reached the full capacity (Logan et al. 1974 b, Husband and Lascelles 1975). Therefore, at this particular period, the sali- vary LF could contribute to a greater extent to the defence mechanisms of the calf. LF re- mains active in the intestines, and the condi- tions there appear favourable for LF to be- come inhibitory (Reiter 1978 a, 1978 b). It is of particular interest in this respect that no LF was found in the plasma of colostrum- fed calves. This suggests that LF is not ab- sorbed from the intestines into the circula- tion, even from the first colostrum feeding, though immunoglobulins having a higher molecular weight, are transferred unchanged in high amounts (Roy 1980). There are probably no such receptors for LF in the in- testinal epithelial surfaces, thus making it functional only within the gastrointestinal tract. Hence, further studies on the physio- logical significance of the colostral and sali- vary LF to the calf and the possible synthesis of thisprotein in the intestines would be war- ranted. The level of salivary LP followed roughly the same pattern as that of salivary LF, with the exception of two peaks being observed for LP. At birth, the concentration was on the average at the same level as in the in- gested colostrum but 3—4 times the milk concentration. The small temporary increase on the first day may be attributable to colos- trum intake, but the sharp increase found al- ready on day 4 is probably due to the com- mencement of active LP synthesis in the sali- vary glands. Within three weeks, the con- centration rose maximally by five tenfold compared to that in colostrum and milk, declining thereafter to the original level by the end of the trial. The present results are contradictory to other studies (Morrison and Steel 1968, Gothefors and Marklund 1975 and Reiter et al. 1980) in which the saliva of newborn calves was found to be poor in LP or devoid of it. Regarding the subsequent increase, our results are, how- ever, in a line with the above studies. The biological significance of LP is asso- ciated with its antimicrobial action in the presence of thiocyanate ions and hydrogen peroxide (Reiter et al. 1964). This system has been previously shown to kill in vitro pathogens in human saliva (KtEBANOFFet al. 1966) and in bovine milk (Reiter et al. 1976) and was recently found to be activated also in vivo in the abomasum of the calf (Reiter et al. 1980). The results of the latter study suggest that salivary LP does not contribute to the bactericidal activity exerted by the LP system in the abomasal fluid. Thus, this ac- tivity is catalyzed principally by the LP con- tained in the ingested colostrum or milk. The above concept is supported by the fact that the colostral LP activity peaks within two— three days postpartum (Korhonen 1977). The results of the present study, however, show that in addition to colostrum and milk, also saliva is an important source of LP in the newborn calf. However, it is not known whether the LP system is active in the calf’s saliva, since it seems to contain only little SCN~ (0.06 mM) (Reiter et al. 1980), and the source of H 2 G2 is uncertain. Therefore, further studies would be necessary to clarify this point. According to Reiter et al. (1980) the LP system is likely to be most active in the upper intestinal tract where there is enough 02 present to permit formation of H 2 02 . It may be provided either by a H 2G2 generating system (e.g. glucose oxidase and glucose) with the diet or by H 2G2 producing lactobacilli which occur naturally in large quantities in the abomasum of the calf. Suf- ficient SCN- is partly provided by colos- trum and milk and partly by the active secre- tion in the abomasum. It appears, therefore, probable that this non-specific system has considerable significance in vivo in the pre- vention of enteric infections in preruminant calves. It would be interesting to investigate, whether the LP system is active in the case of feeding milk replacers instead of raw milk upon initial colostrum diet. In principle, the system should be active, since all its compo- nents are present, being provided by the calf itself. The negligible peroxidase activities found in plasma throughout the trial suggest that the biological function of LP, like in the case of LF, is confined to the gastrointestinal tract. At birth, the concentrations of salivary LZM were equal to or two—three times higher than the level found in the ingested colostrum. In spite of a large fluctuation, LZM remained on the average quite high during the first week, but dropped thereafter to the plasma level. The latter showed no clear changes throughout the trial and cor- responded to the LZM level of the ingested milk. These results indicate that the calf is born with high levels of salivary LZM and suggest that the synthesis of this enzyme is impaired during the first week of life. The physiological behaviour of salivary LZM is thus contradictory to that of salivary LF and LP. Since the colostral LZM level is relative- ly low in the first milkings and reach the peak only 3—4 days postpartum (Korhonen 1977), it appears logical to assume that this deficiency is compensated by high salivary LZM levels at birth. It remains, however, to be resolved whether the salivary or colostral LZM has any protective effect against enter- ic infections in the calf and whether the calf’s intestines are capable of synthesizing this en- zyme. It is interesting to note that in accord- ance with the results of LF and LP, the LZM plasma concentration was not affected sub- stantially by colostrum or milk ingestion. This may be due either to the low LZM con- tent which makes any change undetectable or to absorption being inhibited. The plasma of most pre-colostral calves contained detectable amounts of immuno- globulins. The average concentrations ob- served for each class are in accordance with recent studies (McGuire et al. 1976, Baumwart et al. 1977, Naylor and Kron- feldt 1977 and Jensen 1978) and indicate that the bovine foetus is capable of synthe- sizing Ig. However, due to lack of antigenic stimulus, their quantities in body fluids are considered too low at birth to provide ade- quate immune protection against invading pathogens (Roy 1980). Colostrum ingestion resulted in a rapid but transient increase of plasma IgGj being associated with a less pronounced rise of IgM and IgA levels. The average peak concentrations of all classes at 24 to 48 hours after birth are, however, 5 to 10 times lower than those found in previous studies (Porter 1972, Logan et al. 1974 b, McGuire et al. 1976, Naylor and Kronfeldt 1977, Jensen 1978). This difference would rather be ascribed to a relatively low Ig con- tent of ingested colostrum than to any inhibi- tion of Ig absorption except for IgG, in the polyol group. The decrease in all Ig levels during a few days, is attributable to the ter- minationof Ig absorption from the intestines 320 and to their distribution to different body fluids and gradual degradation (Porter 1972, Penhale et al. 1973, Logan et al. 1978, Stott et al. 1979 a, 1979 b). The sub- sequent significant increase in the levels of all Ig classes, except for IgG2, can be ex- plained by the activation of the calf’s own immune system. Depending on the duration of feeding of colostrum and amount of anti- bodies in it, the active intestinal synthesis of Ig may commence already within the first week of life (Logan et al. 1974 b, Husband and Lascelles 1975, Porter et al. 1977). Also in the saliva, Ig (mainly secretory IgA) is present at one week of age but not at birth (Mach and Pahudl97l, Butler et al. 1972). Noteworthy in this context is the behav- iour of plasma IgG, in the polyol group. In comparison to the other groups its level in- creased considerably less during the first 24 hours, but this was followed by a continuous increase resulting in peak IgG, level within 35 days. The apparent inhibition of absorp- tion of this particular Ig class, which remains unexplained, was thus compensated by a more vigorous autogenous production. It is interesting to note in this connection that al- though no statistical differences could be as- certained, haemolytic bacteria associated with diarrhoea were found most frequently in the polyol group. On the other hand, their incidence was lower in the xylitol group which during the colostrum feeding period showed the highest average IgG, plasma level. It must be noted, however, that the latter group exhibited also the highest aver- age levels of salivary LF and LZM. Also the level of LP was clearly higher in this group than in the glucose group. These results support the generally ac- cepted opinion of the importance of colos- tra! antibodies to the neonatal calf (Roy 1980). On the other hand, the present results suggest that also the non-specific antimicro- bial factors provided both passively from colostrum and actively through an endoge- nous production may contribute to the pro- tection of the calf during its first weeks of life. It appears that the physiological behav- iour of these factors in colostrum and the calf’s saliva differ from each other, but are complementary in a logical way, as is the case with immunoglobulins. Thus, the relatively low concentration of LZM in the first colostrum postpartum seems to be compensated by a high level of this enzyme in the calf’s saliva at birth. Con- versely, the decreasing levels of colostral LP, upon a short initial increase postpartum, and LF, in particular, seem to be compensated by increasing levels of these proteins in saliva. These changes appear to coincide with the period when the immunological protection provided by antibodies is lowest. At this par- ticular time, the. non-specific factors may, therefore, augment the effect of antibodies to a significant extent, not only by exerting synergistic action with them but also by ex- panding the range of the calf’s antimicrobial defence capacity, especially in the mouth and intestinal tract. It may be assumed that the presence of LF, LZM and LP in the calf’s sa- liva right from the birth is important in con- trolling the invasion of pathogens, especially between feeding times. Therefore, the con- centration of these factors at any time may be of special importance. The results of this study suggest a poten- tial contributory effect of xylitol intake on the health of the newborn calf but since a statistical confirmation could not be ob- tained, the validity of this hypothesis re- mains to be shown in further studies. 321 References Ahonen, T., Korhonen, H. & Antila, M. 1978. The characteristics and concentration of lactoferrin in Finnish bovine milk. Meijeritiet. Aikakausk. 36: 68—89. Baumwart, A.L., Bush, L.J., Mungle, M. & Corley, L.D. 1977. Effect of potassium isobutyrate on ab- sorption of immunoglobulins from colostrum by calves. J. Dairy Sci. 60: 759 —762. Bush, L.J. & Staley, T.E. 1980. Absorption of colos- tral immunoglobulins in newborn calves. J. 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Ternimaidon koostumus ja merkitys vastasyntyneelle vasikalle. (Significance of colostrum to the health and nutrition of the newborn calf). Suomen Eläinlääk,leh- ti 84: 375—391. —, Rintamäki, O. & Antila, M, 1977. A polyol mix- ture or molasses treated beet pulp in the silage based diet of dairy cows. 11. The effect on the lacto- peroxidase and thiocyanate content of milk and the udder health. J. Sci. Agric. Soc. Finl. 49: 330—345. Logan, E.F., Mcßeath, D.G. & Lowman, B.G. 1974 a. Quantitative studies on serum immunoglobulin level in suckled calves from birth to five weeks. Vet. Rec. 94: 367—370. —, McMurray, C.H., O’Neill, D.G., McFarland, P.J. & Mcßory, F.J. 1978, Absorption of colostral immunoglobulins by the neonatal calf. Br. Vet. J. 134: 258—262. —, Stenhouse, A., Ormrod, D.J. & Penhale, W.J. 1974 b. The role of colostra! immunoglobulins in in- testinal immunity to enteric colibacillosis in the calf. Res. Vet. Sci. 17: 290—301. Mach, J.-P. & Pahud, J.-J. 1971. 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Antimikrobiset tekijät veressä ja syljessä, sonnan bakteerisisältö ja eläinten terveys Hannu Korhonen 1, Eija Alasaari ja Matti Antila Helsingin yliopisto, maitotaloustieteen laitos, 00710 Helsinki 71 Mikko Tuori ja Esko Poutiainen Helsingin yliopisto, kotieläinlieteen laitos, 00710 Helsinki 71 Kokeessa tutkittiin eräiden antimikrobisten tekijöiden pitoisuutta vastasyntyneiden vasikoiden veressä ja syl- jessä. Lisäksi tutkittiin ksylitoli- ja polyolimelassilisän vaikutusta näihin tekijöihin vertailuryhmän saadessa vastaavan määrän glukoosia (n. 1 g/elopainokilo). Kaikkiaan 18 vasikalta otettiin veri- ja sylkinäytteitä syntymän jälkeenennen ensimmäistä ruokintaa ja sitten 1,2, ja 4 päivän sekä 1, 3 ja 5 viikon iässä. Seuraavat tekijät tutkittiin kvantitatiivisesti: laktoferriini (LF), laktoperoksidi (LP), lysotsyymi (LZM) sekä immuno- globuliinit IgG,, IgG 2 , IgM ja IgA (vain plasmasta). Kerran viikossa otetuista sontanäytteistä määritettiin 1 Nykyinen osoite: Ministry of Livestock Development, P.O. Box 68228, Hill Plaza, Nairobi, Kenia. bakteerien kokonaismäärä sekä anaerobisten hemolyyt- tien, koliformien, laktobasillien ja klostridien määrät. Ainoa tilastollisesti merkitsevä ero bakteerimäärissä oli ksylitoliryhmän korkein klostridipitoisuus (P < 0.05). Hemolyyttisten bakteerien runsas esiintyminen oli yh- teydessä vasikoiden ripuliin. Tulokset osoittavat, että immunoglobuliinien lisäksi myös ternimaidossa esiintyvät LF, LZM jaLP vaikutta- vat huomattavasti vasikan vastustuskyvyn muodostumi- seen patogeenisiä mikrobeja vastaan ensimmäisinä elin- viikkoina. 323