J. Cow.

PATH.

1974. VOL.

84.

381

A BIOCHEMICAL
I. PLASMA PROTEIN

STUDY

OF EXPERIMENTAL DISEASE
INTO BY THE INTESTINE

JOHNES
OF SHEEP

LEAKAGE

LV. M.

ALLEN*,

SYLVIA
Central

BERRETT,
Veterinary Laboratory,

and D. S. P. PATTERSON
Weybridge

INTRODUCTION

Enteric diseasesof animals and man are frequently associated with excessive loss of plasma protein into the intestinal tract and consequent hypoalbuminaemia (Jejeebehoy, 1964; Nielsen, 1966a,b). In cattle, specific enteric infections (including parasitic gastro-enteritis and Johnes disease) are characterized by hypoproteinaemia and protein loss by the gut (Nielsen, 1966b; Patterson, Allen and Lloyd, 1967). I n J oh nes disease of cattle, this loss progressively results in the development of the typical protein deficiency state (Patterson, Allen, Berrett, Ivins and Sweasey, 1968). Sheep infected with Mycobacterium johnei similarly frequently show gross and severe intestinal lesions, although severe wasting (Stamp and Watt, 1954) and the typical symptoms of protein deficiency do not always develop (Allen, 1971). This paper describes the importance of plasma protein loss into the gut and its contribution to the pathogenesis of clinical disease in the sheep.
MATERIALS AND METHODS

Plasma protein loss was measured in 5 healthy sheep and 10 sheep experimentally infected with M. johnei. Based on the method described by Gilmour and Brotherston (1966) the sheep were infected by oral administration of a saline suspension (2 ml.) of M. johnei at I-day intervals for a period of 10 weeks. The calculated dose over the 1 O-week period was equivalent to 15 mg. wet weight of organisms. From the calculation of Gilmour and Brotherston (1966), this was approximately 2-5 x lo9 viable units of M. johnei. Observations on the healthy controls and the infected group of animals were made at either 11 or 15 months after oral infection. The results from both series of observations were combined and the consequent variation in age and time after infection was considered during the statistical evaluation of results. The sheep were trained to stand in a metabolism cage to provide separate collection of urine and faeces. Food intake was measured daily. Plasma protein was labelled intravascularly with 51CrC1, by a method based on that described by Van Tongeren and Reichert (1966) and the rate of decreaseof activity in plasma and the loss of activity into the urine and faeces were determined. Approximately 1 mCi. 51CrC1, was diluted in isotonic saline and injected i.v. At intervals after dosing the radioactivity of plasma, urine and faeces was determined using a scintillation counter according to the method described in detail by Allen (1971). The total activity passed into the faeces and urine over a 100 h. period was calculated and the activity of the plasma was determined over a similar period of time.
* Present address: Compton, Newbury, Agricultural Berkshire. Research Council Institute for Research on Animal Diseascr,

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Zone electrophoresis of serum samples and subsequent autoradiography demonstrated that the Wr label was bound predominantly to the P-globulin. Initially there was a small proportion attached to albumin but this did not persist. Examination of washed cells confirmed that W2rCls did not attach to erythrocytes. After the protein loss had been measured, healthy and control sheep were examined at autopsy. Histological and bacteriological examinations were carried out on several portions of the small intestine.
RESULTS

The total loss of Wr into the faeces for each group of sheep in a 100 h. period is shown in Table 1. The loss of label in both the healthy controls (group 1) and the 7 sheep which were infected, but were without gross lesions at autopsy (group 2) was similar. However, in the 3 sheep with gross lesions (group 3) the percentage of dose lost into the faeces was significantly greater than those of the other groups. Plasma protein half-life, presumably a measure of I&globulin turnover, was calculated from each of the logarithmic decay curves. The mean value for the animals with gross lesions was considerably less than that for controls, but because of the wide spread of values it was not significantly different. The volume of plasma lost into the faeces was calculated according to the method described by Van Tongeren and Reichert (1966).
TABLE 1
GASTRO-INTESTINAL PROTEIN LEAKAGE DURING EXPERIMENTAL INFECTION OF SHEEP WITH

M.johnei

Mean

and range

of values % oj-T4r Cl3 dose in.faeces during 100 h. observation 0.762 (0.566-1.017) Ml plasma cleared into intestine per 100 h. per kg. $ body weight 1.90 ( 1.30-2.83)

Sheep group (no. qf animals) Controls (5)

Body weight (kg) 41.2 (33.849.8)

,4pParen t plasma half & 117.8 (102-125)

M. johnei infected, without gross lesions. (7, two of these animals were examined at both 11 and 15 months after infection) M. johnei infected lesions (3) 8 Residual * t $ 0 standard with gross
(R.s.D.)

43.4 (35.5-50.5) 30.1* (25.0-37.0) 6.17

112.1 (87-155) 91.7 (73-107.5) 21.6

0.903 (0.671-1.044) 3.31t (2.654.15) 0.165

1.77 ( 1.55-2.03) 749t (6.27-9.45) 0.406

deviation

Significantly less than groups 1 and 2, P<O.O5. Significantly greater than groups 1 and 2, Pt0.001. Calculated according to Van Tongeren and Reichert ( 1966). The R.S.D. is defined as the pooled standard deviation (square root of the error variance) representing the variation within groups after allowing for differences between groups due to age, time and status of infection.
DISCUSSION

The importance of protein plasma loss into the faeces has been demonstrated in cattle (Patterson et al., 1967). Cows affected with Johnes disease lost, on average, 39 g. of protein/d. more than controls and this may be an important factor in the pathogenesis of the wasting process. In this experiment the sheep with gross lesions lost an average of 3.96 g. (calculated from data in Column 5, Table 1 for a sheep of 30 kg. with a plasma protein concentration of

EXPERIMENTAL

JOHNES

DISEASE

: PLASMA

PROTEIN

LEAKAGE

383

7.06 g./lOO ml.) of plasma protein/day into the faeces, which was 2.9 g. more than the controls and equivalent to 43 ml. of plasma. This was similar on a body weight basis to the loss in infected cattle. Holmes and McLean (1971) used 1311labelled albumin to measure the catabolism of plasma albumin, and 51CrC13to measure faecal clearance of labelled protein in sheep. They measured the lossesin sheep infected with 900 000 Ostertagia ostertagi larvae. Plasma loss into the intestine increased up to 64 ml./d. more than healthy controls during the clinical stages of the disease which was associated with diarrhoea and inappetance. Compensatory mechanisms, however, apparently maintained the albumin concentration in plasma at normal levels. Alexander and Kiesel (1968) had also demonstrated that sheep could compensate for the loss of up to 50 ml. plasma. Plasma protein concentration could be maintained at normal concentrations provided the animals received adequate dietary protein. In the present experiment the approximate daily food intake for the control sheep (group 1) and all the sheep infected with M. johnei (groups 2 and 3) was 300 g. of concentrates and 700 g. of hay. This was equivalent to a daily intake of about 43.5 g. of protein, assuming that the digestible crude protein content of hay is 3 per cent. and of concentrates is 7.5 per cent. Thus the extra lossof 2.9 g. plasma protein represents 6.7 per cent. approximately of the daily protein intake. If this loss continued without a compensatory increase in appetite or absorption it could account for the muscular wasting observed in the severely affected animals. However, in the sheep with severe lesions due to ,21.johnei infection, adaptation must have occurred because, although protein loss occurred into the gut, almost normal concentrations of plasma protein were maintained. Electrophoresis and autoradiography had confirmed that the 51Cr label was bound only by the P-globulin and plasma protein losseswere estimated on the basis of 51Cr excretion. The lossesof other protein fractions e.g. albumin were not estimated separately and because the molecular weight of albumin is several times smaller, the actual loss of the albumin fraction may well have been greater than that implied by our calculation for plasma proteins. The relationship between malabsorption and excessive protein loss into the gut and protein metabolism in the whole body will be discussed in subsequent papers.
SUMMARY

Sheep were infected by repeated oral dosing with Mycobacterium johnei. The rate of plasma protein loss into the intestine was measured by an intravascular labelling technique using 51CrC13. The label attached predominantly to the P-globulin fraction. Sheep which exhibited gross lesions lost on average 4-O times the volume of plasma into the intestinal tract than either healthy sheep or infected sheep without lesions. The importance of this lossin the pathogenesis of the severe clinical disease in the sheep is discussed.
REFERENCES

Allen,
H

W. M. (1971). and mice. Ph.D.

Biochemical aspects of Myocobacterium johnei infection Thesis, University of London.

in sheep

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Alexander, H. D., and Kiesel, G. K. (1968). The effect of blood loss on weight gain, haemoglobin and haematocrit in lambs fed different levels of protein. Auburn Veterinarian, 21, 114-l 17, 129. Gilmour, N. J. L., and Brotherston, J. G. (1966). Further studies on immunity to Mycobacterium johnei in sheep. Relationship between hypersensitivity and host response to infection. Journal of Comparative Pathology, 76, 341-349. Holmes, P. H., and MacLean, J. M. (1971). The pathophysiology of ovine ostertagiasis: a study of the changes in plasma protein metabolism following single infections. Research in Veterinary Science, 12, 265-27 1. Jejeebehoy, K. N. (1964). In The Role of the Gastro-intestinal Tract in Protein Metabolism. Ed. H. N. Munro, pp. 357-383. Blackwell, Oxford. Nielsen, K. (1966a). Metabolism and distribution of 1311 labelled albumin in pigs with gastro intestinal disease. Acta Veterinaria Scandinavica, 7, 32 l-329. Nielsen, K. (196613). Gusfrointestinal Protein Loss in Cattle. Ed. C. R. Mortensen, Copenhagen. Patterson, D. S. P., Allen, W. M., and Lloyd, M. K. (1967). Clinical Johnes disease as a protein losing enteropathy. Veterinary Record, 81, 717-718. Patterson, D. S. P., Allen, W. M., Berrett, S., Ivins, L. N., and Sweasey, D. (1968). Some biochemical aspects of clinical Johnes disease in cattle. Research in Veterinary Science, 9, 117-l 29. Stamp, J., and Watt, J. A. (1954). J o h nes disease in sheep. Journal of Comparative Pathology, 64, 26-40. Van Tongeren, J. H. M., and Reichert, W. J. (1966). Demonstration of protein losing gastroenteropathy. The quantitative estimation of gastrointestinal protein loss, using Cr labelled plasma proteins. Clinica Chimica Actu, 14, 42-48. [Received.for publication, December 3rd, 19731