Small intestinal microbiological and morphological observations in young calves fed milk replacers enriched with a combination of lactoperoxidase system and lactoferrin

A study has been made of the effects of a combination of lactoperoxidase system (LP-s) and lactoferrin (LF), supplemented to milk replacer for 2 weeks, on the morphology of the small intestinal mucosa, microbiology of the intestinal tract and the incidence and severity of diarrhoea in young calves. The experiment was conducted in two groups of 15 young calves, aged 7 to 21 days. The results showed a lower incidence and severity of diarrhoea (P<0.05) in the calves of the LP-s/LF group compared to the control group. CFU (Colony forming units) of E. coli in colonic digesta (P=0.10) and in faeces (P<0.05) were significantly lower in the LP-s/LF group compared to the control group. Dissecting microscopy of the small intestinal mucosa indicated more finger-shaped villi in the distal jejunum of the calves of the LP-s/LF group compared to the control group (P=0.01). Histometrical measurements showed that these villi were significantly (P=0.002) higher. The mean contents of immunoglobulins (IgG) increased (P=0.1) during the test period whereas those of the control group remained unchanged.


INTRODUCTION
A combination of two bioactive proteins, lactoperoxidase system (LP-s) and lactoferrin (LF), was added to a milk replacer.The chemical and biological properties of the LP-s have been reviewed by De Wit and Hooydonk (1996) and Reiter LACTOPEROXIDASE AND LACTOFERRIN FOR CALVES and Perraudin (1991) while the structure and functions of lactoferrin (LF) were reviewed by Lonnerdal and Iyer (1995).
The present study was carried out to investigate the effect of adding LP-s and LF to milk replacer on the incidence and severity of diarrhoea, the intestinal microbiology and the morphology of the small intestinal mucosa.

MATERIAL AND METHODS
Thirty Fresian-Dutch calves aged approximately 7-d were assigned to one of two treatment groups (n=T5) based on body weight.The calves were fed a commercial milk replacer diet without antibiotics.The animals of the test group (LP-s/LF) received the diet with a supplement of bovine LP-s/bovine LF concentrate.Bovine LP-s and bovine LF were supplied by DMV International, Veghel, The Netherlands.The milk replacer had a final concentration of 200 mg/kg LP and 1000 mg/kg of bovine LF (20% Fe saturated).The LP activity was 1000 U/g using the ABTS method (De Wit and van Hooydonk, 1996).The control group (C) received the same diet with placebo supplementation.
Both groups were fed twice a day according to a standard feeding schedule.During the experiment feed intake and faecal score, with 0 for normal faeces and 3 for severe diarrhoea, were recorded.The experiment was concluded on day 13 after arrival.Faecal samples were taken daily from the rectum of each animal from day 6 to day 10 for determination of bacterial counts.Three h post-feeding blood samples were taken on days 2 and 13.Haematological parameters were determined in blood taken on day 13, and IgG contents were determined in blood sera taken on days 2 and 13.On day 14 (one day after the end of the experiment) dissection was conducted at 0.5 h after feeding.
The shape of the villi was studied with a dissecting microscope and characterized according to Mouwen (1972).Villus height, crypt depth and the numbers of goblet cells (no. per 100 mm crypt) were determined according to previously described procedures (Kik et al., 1990).
Analysis of variance (using treatment as a factor) were carried out with the software package of SPSS/PC+ V5.0 (SPSS, 1992).

RESULTS AND DISCUSSION
Animals arrived at the Institute in good health.On day 6, one animal from the control group was euthanized because of very severe diarrhoea.Feed intake was similar (P>0.1) between groups.Average faecal consistency over the 13 d experimental period was 0.80 for the control and 0.61 (PO.05) for the LP-s/LF group.For the major part of the experimental period the LP-s/LF group had a lower faecal consistency score than the control group.The differences were significant (PO.05) at days 4, 8 and 9.
Results of the haematological examination determined at the end of the experiment showed that the numbers of basophils (0.64%) were higher (P<0.05) in the control group compared to the LP-s/LF group (0.07%).The numbers of eosinophils were increased (P<0.1) in the LP-s/LF (0.00%>) compared to the control group (0.20%).There were no significant differences in haemoglobin, erythrocytes, haematocrit, leucocytes, neutrophils, lymphocytes and monocytes between the control and the LP-s/LF group.
The results of immunoglobins in blood serum are summarised in Table 1.The IgG level of the LP-s/LF group was lower (P<0.1)than those of the control group on day 2. Overall, the IgG content of LP-s/LF group increased between days 2 and 13 whereas the content slightly decreased in the control group.The change in the IgG content (DIgG/Dt (g/1 per day)) over the experimental period was different (P=0.1) between the groups.The lower IgG content at the start of the experiment suggests that the calves from the LP-s/LF group had a lower passive immunization than the control group.The increase in the IgG content of the LP-s/LF group during the experiment was probably related to an improved immune status.In the present experiment, no significant (P>0.1)correlation was found between IgG contents and the mean faeces score (not presented).The relation between IgG concentration and the occurrence of diarrhoea was studied by Rajala and Castren (1995).They concluded that IgG concentrations did not explain diarrhoea occur- -goblet cells, n/100 mm crypt 6.1 (0.65) 6.9 (0.62) NS 1 NS= difference is not significant (P>0.05)* = difference is significant (P<0.05) 2 scale: 0 (ideal) to 3 (highly affected villi) rence.In addition, the lower CFU of E. coli in the distal GI tract is a factor which can, secondarily, affect the immune status.
The CFU of E. coli in the colonic digesta and in the faeces of the control calves were higher than those of the LP-s/LF group (P = 0.10 and P<0.05, respectively) (Table 2).There were no significant differences (P>0-.1) in the CFU of E. coli in the digesta from the jejunum.These results are in accordance with Still et al. (1989) who showed an effect of a combination of LP-s and LF on the severity of diarrhoea in calves over the period 0 to 6 days after an experimental E. coli infection.They concluded that LP-s/LF had preventive and curative effects after the E. coli infection.
Counts of lactic acid producing bacteria (LAB) in the jejunum and colon were not significantly different (P>0.1) between the groups.The colonic contents of one animal of the control group were positive for salmonella whereas no salmonella positive animals were found in the LP-s/LF group.
Dissecting under the microscope, no differences in morphology of villus shape were found in the proximal or mid jejunum.However, in the distal jejunum, the villus shape scores were higher (P<0.05) in the LP-s/LF fed calves compared to those of the control group (Table 3).Histometrically, the mean villus height in the distal jejunum of the LP-s/LF group was also significantly higher (P<0.05)than that of the control group.There were, however, no differences in crypt depth or goblet cell density (P>0.1) between the groups.
In summary, in the present experiment LP-s/LF addition to milk replacer decreased the severity of diarrhoea and decreased the numbers of E. coli in faeces and colon of young calves.Moreover, a beneficial effect on the shape and height of the villi in the distal jejunum was observed in young calves fed a milk replacer diet supplemented with LP-s/LF.