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Plasma protein loss in growing pigs during the prepatent and early patent periods of infection with high doses of Hyostrongylus rubidus larvae
J. COMP.
PATH.
1974.
VOL.
84.
PLASMA PROTEIN THE PREPATENT INFECTION WITH
399
LOSS IN GROWING PIGS DURING AND EARLY PATENT PERIODS OF HIGH DOSES OF HYOSTRONGYLUS RUBIDUS LARVAE BY
R. N.
TITCHENER*,
I.
Deparlmmt
V.
of Applied
and A. J.
HERBERT,
U.C.N.
PROBERT
W., Banp
INTRODUCTION
Hyperplastic gastritis in swine is a close analogue to human disorders in which gastro-intestinal protein loss, particularly albumin, has been demonstrated (Nielsen, 1968). Studies in our laboratory have failed to demonstrate hypoproteinaemia in hyostrongylosis of growing pigs fed on adequate diets (Lean, 1969; Ford, 1970). Serum protein levels in the low normal range and low serum albumin levels have, however, been attributed to Hyostronpvltls rubidus infection in field cases where a severe gastritis was observed (Nielsen, 1966). The levels of infection employed by Lean (1969) and Ford ( 1970) were, however, below those normally necessary to cause clinical signs of hyostrongylosis in growing pigs, as described by various authors-viz. diarrhoea, vomiting, anorexia and weight loss (see Castelino, Herbert and Lean, 1970). These clinical signs occur during early prepatency and again at or shortly after patency. The present study was undertaken to determine whether hypoproteinaemia and hypoalbuminaemia accompany high levels of infection with H. rubidus larvae in growing pigs during prepatent and patent periods at levels which Castelino et al. (1970) have shown to precipitate the clinical signs described above. Following this period further weight loss does not occur in adequately fed animals. MATERIALS
AND
METHODS
Conventionally reared Large White x Landrace M.D. (minimal disease) pigs of mixed sexes (females and castrate males) were fed 635 g. of a commercial pig meal (Sow and Weaner Meal, B.O.C.M., Ltd.) twice daily. Water was unrestricted in experiment I, but restricted in experiment II to 1000 ml. and in experiments III and IV to 1500 ml. per feed. In experiments III and IV, to prevent radioactive iodine uptake by the thyroid, potassium iodide solution was added to this water to give a final concentration of 0.005 per cent. The pigs were infected orally with the infective larvae, as a single dose, at 12 weeks of age and approximately 25 kg. weight. Experiments I and II were designed to determine the approximate level of infection required to produce clinical signs of severe hyostrongylosis together with lowered plasma protein and albumin levels. In experiment I, a litter of 10 pigs was divided as follows: Group la, 3 pigs infected with 60 000 larvae, group lb, 3 pigs infected with 200 000 larvae and group lc, 4 * Present Auchincruive,
address: Ayr.
Department
of Zoology
and
Nematology.
West
of Scotland
Agricultural
College,
400
R. N. TITCHENER
f?t d.
uninfected controls. In experiment II, 9 litter mates were divided into group 2a, 5 pigs infected with 350 000 larvae and group 2b, 4 uninfected controls. All pigs used in these 2 experiments were bled from the anterior vena cava. Blood samples, each of 10 ml., were collected in heparinized tubes (Stayne Laboratories Ltd.). Total plasma protein and albumin levels were determined by the biuret method described by Wootton (1964). Experiments III and IV covered the period just before and during which low plasma total protein and albumin levels were observed and was designed to investigate hyostrongylosis as a protein-losing enteropathy by measuring the turnover of radio-iodinated albumin and the half life and faecal output of iodine labelled PVP (polyvinyl-pyrrolidone) in infected pigs. In experiment III a litter of 10 pigs was divided into, group 3a, 6 pigs and group 3c, 4 uninfected controls. The six pigs in group 3a were infected with 250 000 larvae since the results of experiments of I and II indicated that this level of infection would possibly be sufficient to produce clinical symptoms and likely to lead to an increased radioiodinated albumin turnover. Porcine albumin (Sigma Chemicals Ltd.), labelled with 1311 by the electrolytic method of Rosa, Scassellati, Pennsis, Riccioni, Griagnoni and Giordani, (1964), was diluted with porcine albumin and 1 ml. of the resulting solution (dose 30 &i) immediately injected into the ear vein of each pig in groups 1 and 2, 15 d. after infection. Heparinized blood samples, each of 5 ml., were taken from the anterior vena cava 15 min. after the injection of the radioactive albumin, after which further samples were taken daily for the next 10 days. Aliquots of plasma, each of 1 ml., obtained from the blood by centrifugation were counted in a well type scintillation counter with a twin channel scaler. Plasma radioactivity, corrected for isotope decay, was expressed as a percentage of the activity of the 15 min. sample. The fractional catabolic rate was calculated according to the method described by Matthews (1957). In experiment IV a litter of 8 pigs was divided into 2 equal groups. Group 4a, was infected whilst group 4b were uninfected controls. A higher larval dose of 325 000 was used since gross clinical signs of hyostrongylosis were only apparent in half of the pigs infected with 250 000 larvae in experiment III. It appeared probable that the higher dose rate would increase the likelihood of the development of overt clinical symptoms and thus, possibly, provide a greater contrast in the plasma half-life and faecal output of i311 PVP between infected and control animals. A larval dose in excess of 325 000 was not considered advisable as severe clinical signs developed in 2 pigs infected with 350 000 larvae in experiment II. Whilst these extreme signs were exceptional and had not been observed in previous studies in our laboratory using high levels of infective larvae (Castelino et al., 1970) they were associated with a degree of haemoconcentration which if it occurred in the 1311 PVP test would result in a misleadingly high plasma concentration and increased plasma half life of 1311 PVP. One millilitre of 13iI labelled PVP (dose 200 l&i) was injected into the ear-vein of each pig in groups 4a and 4b 12 days after infection. Plasma samples were taken and counted as previously described at 24 h. after injection, after which samples were taken daily for the next 8 days. Plasma. radioactivity corrected for isotope decay, was expressed as a percentage of the sample 24 h. after injection. The method of “least squares” was used to draw the best straight line through the points on the plasma disappearance curve of 1311 PVP so that the plasma half-life could be calculated. From 14 to 24 days after infection the 24 h. faecal output of each pig in experiment IV was collected and 4 g. aliquots taken and assessed for radioactivity. Faecal activity was expressed as a percentage of the injected dose found in each 24 h. faecal sample. In all 4 experiments daily faecal egg counts were made on each 24 h. faecal sample during the period 14 to 30 days after infection using the Clayton-Lane centrifuge flotation method (M.A.F.F. 1971) using saturated magnesium sulphate (specific gravity l-25).
Ii. rubidus
INFECTION:
PLASMA
PROTEIN
401
LOSS
RESULTS
No clinical signs of diarrhoea, anorexia or weight loss were observed in animals infected with 60 000 or 200 000 larvae. In experiment II, in which animals were infected with 350 000 larvae, vomiting and anorexia were observed in all the infected pigs sometime during the period 15 to 27 days and were most severe 18 to 20 days after infection. Two pigs (B65 and B70) were most severely affected, B70 dying on the 20th day after infection. In addition to the clinical signs already described, both animals became lethargic, showed cyanosis of the nose, ears and ventral abdomen and scoured slightly, the faeces being of a sticky yellowish consistency. Nematode eggs were first detected in the faeces 17 days after infection in experiments I and II. In experiment I the mean peak egg counts were: group la, 52 eggs per gram (e.p.g.) 21 days and group lb, 159 e.p.g. 23 days after infection. Egg counts were considerably higher in experiment II and a mean peak egg count of 2902 e.p.g. 20 days after infection was observed. Particularly high individual egg counts of 3612 e.p.g. and 12 540 e.p.g. were recorded from the faeces of pigs B65 and B70 respectively. No difference in mean concentration of total plasma protein or plasma albumin was observed in experiment I between the pigs in group la, infected I
I
I
I
I
I
I
I
I
I
I
I
I
I
I A
7.5 -
tlllllllllll 0
4
6
12 Days
I6 after
20
l 24
III 28
i
3;
mfectton
Fig. 1. Mean total plasma protein and plasma albumin levels in pigs infected with 200 000 H. rubidas larva (Exp. I, group lb). A. Group mean total plasma protein. B. Group mean plasma albumin. (e-0) Mean value group lb infected with 200 000 larvae; (0-O) Mean value uninfected group.
402
R.
N.
TITCHENER
et Ul.
with 60 000 larvae, and the controls. Mean total plasma protein and plasma albumin levels of the pig; in group lb, infected with 20 000 larvae, showed a sharp decline 20 days after infection (Fig. 1). Whilst these levels were not statistically different from the control levels, 2 out of the 3 infected pigs had plasma albumin levels below the limit of the normal range for pigs. II
IIIII
IIIIIIII
1. A
_
IO -
2-
I 0
I 4
I
I 8
I
I 12 Days
,
, I6 after
20
24
28
32
mfectlon
Fig. 2. Mean total plasma protein and plasma albumin levels of 2 pigs (B65 and B70) in Exp. II infected with 350 000 H. rubidus larvae together with these values expressed against a constant urea value. A. Total plasma protein. B. Plasma albumin. (o-0) Mean value without basis of constant urea value; (0-n) Mean value with basis of constant urea value (40 mg. urea/ 100 ml. plasma). Broken line indicates death of one of the infected animals, (B70).
In experiment II no significant difference was observed in mean total plasma protein and plasma albumin levels between the infected and control pigs and only one infected pig, B71, had a plasma albumin level 20 days after infection below the limit of the normal range for pigs. Some degree of haemoconcentration appears to have occurred in pigs B65 and B70 at this time and for this reason total plasma protein and plasma albumin levels have been expressed on the basis of a constant urea value since, in our opinion, in the absence of renal malfunction, one might expect elevated urea levels during haemoconcentration. The urea levels were in fact elevated in both of these animals. While it is appreciated that corroborative evidence of haemoconcentration from haematocrit studies would ideally have been obtained, the evidence from our radiation studies does confirm lowered plasma protein during the same period. Mean total protein and plasma albumin levels of pigs B65 and B70, expressed on the basis of a constant urea value, are shown in Fig. 2. There was a decline in total plasma protein and plasma albumin levels from 16 days which reached
ff.
?dkiXS
INFECTION:
PLASMA
PROTEIN
403
LOSS
their lowest values 20 days after infection after which a gradual recovery occurred. The infection in the parasitized pigs of experiment III became patent 17 days after infection. Significantly higher egg counts (P <0*05) were recorded from the 3 pigs, R83, R84 and R86 which showed clinical signs of hyostrongylosis, than from the other 3 infected pigs, R81, R82 and R85 which showed no clinical TABLE PARASITOLOGICAL
AND
1
Ia11 ALBUMIN TURNOVER PERIOD 15-25 d. AFTER
DATA IN EXPERIMENT INFECTION
III FOR THE
Clinical signs Group
Group R83 R84 R86
3a
~hfean
+ S.D.
Group R81 R82 R85 Mean
3b
anorexia, diarrhoea and vomiting
-
t +
+
-
-
* s.o.
Standard
2194 1436 1573+ 565 P
Croup 3c (Control) R87 R88 R89 R90 Mean+s.D. SD.
+
Peak egg count e.p.g.
-
15
-
Plasma half 1+/h.
76 120 109k28.7 P
Fractional catabolic rate ~_____ ~- --.. -.- -.
40.00 26.74 3 1.99 fr 7.05 P
Deviation.
signs (Table 1). The low egg counts and lack of clinical signs possibly suggest a lower level of parasitic infestation in these pigs and for this reason group 3a was split into two groups: group 3a, pigs R83, R84 and R86, group 3b, pigs R81, R82 and R85, for analysis of the albumin turnover data. Albumin turnover data for the period 15 to 25 days after infection is given in Table 1. The pigs of group 3a, when compared with the pigs in group 3b and the control group 3c had shorter plasma half-lifes and higher fractional turnover rates. Compared with the control group, mean plasma half-life was significantly shorter (PC 0.002) and mean fractional catabolic rate significantly higher (P
404
R. xv. TITCHENER I
I
I
I
I4
I5
I6
I7
et al. I
I
1
I
I6
19
20
21
d i
i d N 8 E is 0 kz J
I.46 -
I .26 -
I.10 -
0.92
-
0.74 -
056-
13
Doys
Fig. 3. Plasma half-life in Experiment I
I
ofter
of 1311 PVP in infected and control groups of pigs, IV. (e-0) Mean value infected group; (0-O) I
I
I
I
13 to 18 days after infection, Mean value control group.
I
11 I6
Doys
after
22
infection
I9
I
LA 20
21
I
I
22
23
infectcon
Fig. 4. Faecal output of Is11 PVP in infected and control group of pigs, 14 to 23 days after infection, Experiment IV. (m) Mean value infected group; (IJ) Mean value control group.
in
H. rubidus
INFECTION:
PLASMA
PROTEIN
LOSS
405
In experiment IV patency occurred 16 days after infection and a mean peak egg count of 2430 e.p.g. was recorded on the 22nd day. Severe anorexia was observed in all the infected pigs 15 days after infection and continued until 23 days. One infected pig vomited and had slight diarrhoea during this period. Fourteen to 18 days after infection the mean plasma half-life of i311 PVP in the infected group of pigs (19 h. fS.D. 3.5) was much shorter than in the uninfected controls (60 h. *S.D. 7.9, Fig. 3), and this result was highly significant (P
In experiments I and II low levels of plasma total protein and albumin were detected shortly after patency at a time when the worms were at or approaching their peak egg production. The results of experiments III and IV show that these low levels were indicative of an earlier increased plasma protein loss in the period just before (experiment IV) and just after (experiment III) patency. Previous studies to detect gastro-intestinal plasma protein loss have been carried out in natural infections by Nielsen (1966), and in experimental infections by Jacobs (1970) and Dey-Hazra, Kolm, Enigk and Giese (1972). Only in the study of Dey-Hazra et al. (1972) was there a demonstrable Ioss of plasma protein in the infected pigs, but these workers did not commence their studies until 25 days after infection of 3 week old animals. It is difficult to compare the results detailed by Dey-Hazra et al. (1972) and those described here since they involve studies of different periods of infection. Using our strain of parasite, day 25 corresponds to the period after which the majority of adult worms have been lost, when egg counts are low and when a long lived “residual” infection aggregates in the fundic region (bordering the pyloric region) of the stomach. Such aggregations of worms are associated with erosions of the stomach wall similar to those described by Dey-Hazra et al. (1972) and these persist for varying periods of time. It is also difficult to compare the results in view of the fact that the two studies have dealt with different infection levels in pigs of’ different ages and when the prepatent periods are different. The prepatent period described in our study corresponds with that described by Davidson, Murray and Sutherland, (1967). Dey-Hazra et al. (1972) considered that the parasite used the epithelial cells of the gastric mucosa for nourishment and that loss of these cells, particularly where this was fairly extensive and small ulcers formed, resulted in increased mucosal permeability which was responsible for the increased gastro-intestinal protein loss. Studies in this laboratory agree with those of Davidson et al. (1967) that epithelial cytolysis occurs at the time
406
R. N. TITCHENER
et d.
when the adult worm migrates onto the stomach surface. This lesion is different from that caused by residual aggegations of worms described above. Loss of plasma protein occurs at the same time as this epithelial cytolysis occurs. In bovine and ovine ostertagiasis, structural changes in the mucosa and increased gastro-intestinal protein loss similar to those described here in hyostrongylosis also occur at the time when the parasites are emerging from the gastric glands (Jennings, Armour, Kirkpatrick and Murray, 1967). Murray, Jennings and Armour (1970) in a study of the gastric mucosa at this time have shown that the functional gastric gland mass is replaced by undifferentiated cells and have found by electron microscopy, that the junctional complexes between many of these undifferentiated cells are open, which they consider accounts for the enhanced permeability of the gastric mucosa to macromolecules. It seems probable that such structural changes in the gastric mucosa associated with the emergence of H. rubidus adult worms as well as the described epithelial cytolysis could account for the gastro-intestinal loss of plasma protein. SUMMARY
levels of total plasma protein and albumin have been detected in pigs infected with 200 000 and 350 000 larvae of Hyostronpylus rubidus. Further studies using 1311labelled albumin and 1311polyvinyl pyrrolidone have shown that this depression in total plasma protein and albumin results from plasma protein leakage into the gastro-intestinal tract at a time when the worms were migrating, or, had just finished migrating from the gastric glands on to the mucosal surface. LOW growing
ACKNOWLEDGMENTS
The authors wish to thank the ance. We are indebted to Dr W. A. labelling of the porcine albumin Titchener thanks the Sir William
Agricultural Research Council for financial assistHemmings, U.C.N.W., Bangor, for the radioactive and his advice on radioactive techniques. R. N. Roberts trust (U.C.N.W.) for financial support. REFERENCES
-197
1. Manual of Veterinary Parasitological Laboratory Techniques. Ministry of Agriculture, Fisheries and Food. Technical Bulletin No. 18. H.M.S.O. London. Castelino, J. B., Herbert, I. V., and Lean, I. J. (1970). The live-weight gain of growing pigs experimentally infected with massive doses of Hyostrongylusrubidus (Nematoda) larvae. British Veterinary Journal, 126, 579-582. Davidson, J. B., Murray, M., and Sutherland, I. H. (1967). Observations on the Clinical Pathology of Natural Strongyle Infestations in the Pig and their Control, with Special Reference to Hyostrongylus rubidus. Proceedings of the 3rd International Conference of the World Association for the Advancement of Veterinary Parasitology, Lyon, pp. 9-23, Elwert, Marburg, Lahn. Dey-Hazra, A., Kolm, H. P., Enigk, K., and Giese, W. (1972). Zum Gastrointestinalen Plasmaproteinverlust beim Hyostronpylus- befall des Schweines.
H. rubidus
INFECTION:
Gordon, R. S. (1959). Exudative intestinal tract demonstrable,
PLASMA
PROTEIN
407
LOSS
enteropathy. Abnormal permeability with labelled polyvinylpyrrolidone.
of gastroLancet, i,
325-327. JacobsiafJ)$,t
(1970).
Studies
on helminthiasis
in the pig. Ph.D. Thesis,
University
of
Jennings, F. W., Armour, J., Kirkpatrick, K. S., and Murray, M. (1967). Biochemical Consequences of Ostertagia Infections in Ruminants. Proceedings of the 3rd Conference of the World Association for the Advancement of Veterinary Parasitolqp, I,yons, pp. 38-42. Elwert, Marburg, Lahn. Lean, I. J. (1969). Studies on the pathogenesis of infection with Hvostrongylus rubidus (Nematoda) in pigs with particular reference to haematological and nitrogen metabolism changes. Ph.D. Thesis, University of Wales. Matthews, C. M. E. (1957). Theory of tracer experiments with iodine-131-labelled plasma proteins. Physics in Medicine and Biology, 2, 36-53. Murray, M., Jennings, F. W., and Armour, J. (1970). Bovine ostertagiasis; Structure, function and mode of differentiation of the bovine gastric mucosa and kinetics of the worm loss. Research in Veterinary Science, 11, 417-427. Neilsen, K. (1966). Metabolism and distribution of I 131 labelled albumin in pigs with gastrointestinal disease. Acta Veterinaria S’candinauica, 7, 321-329. Neilsen, K. (1968). Hypoproteinaemia associated with Helminthic Infections with Special Reference to Gastrointestinal Parasites. Isotopes and Radiation in Parasitology, p. 125. International Atomic Energy Agency, Vienna. Rosa, V., Scassellati, G. A., Pennsis, F., Riccioni, N., Griagnoni, P., and Giordani, R. ( 1964). Labelling of human fibrinogen with I 131 by electrolytic iodination. Biochimica et biophysics acta, 86, 5 19-526. Wootton, I. D. P. (1964). Micro-Analysis in Medical Biochemistry, J. & A. Churchill Ltd., London. [Received-for publication,
December 6th, 19731
PATH.
1974.
VOL.
84.
PLASMA PROTEIN THE PREPATENT INFECTION WITH
399
LOSS IN GROWING PIGS DURING AND EARLY PATENT PERIODS OF HIGH DOSES OF HYOSTRONGYLUS RUBIDUS LARVAE BY
R. N.
TITCHENER*,
I.
Deparlmmt
V.
of Applied
and A. J.
HERBERT,
U.C.N.
PROBERT
W., Banp
INTRODUCTION
Hyperplastic gastritis in swine is a close analogue to human disorders in which gastro-intestinal protein loss, particularly albumin, has been demonstrated (Nielsen, 1968). Studies in our laboratory have failed to demonstrate hypoproteinaemia in hyostrongylosis of growing pigs fed on adequate diets (Lean, 1969; Ford, 1970). Serum protein levels in the low normal range and low serum albumin levels have, however, been attributed to Hyostronpvltls rubidus infection in field cases where a severe gastritis was observed (Nielsen, 1966). The levels of infection employed by Lean (1969) and Ford ( 1970) were, however, below those normally necessary to cause clinical signs of hyostrongylosis in growing pigs, as described by various authors-viz. diarrhoea, vomiting, anorexia and weight loss (see Castelino, Herbert and Lean, 1970). These clinical signs occur during early prepatency and again at or shortly after patency. The present study was undertaken to determine whether hypoproteinaemia and hypoalbuminaemia accompany high levels of infection with H. rubidus larvae in growing pigs during prepatent and patent periods at levels which Castelino et al. (1970) have shown to precipitate the clinical signs described above. Following this period further weight loss does not occur in adequately fed animals. MATERIALS
AND
METHODS
Conventionally reared Large White x Landrace M.D. (minimal disease) pigs of mixed sexes (females and castrate males) were fed 635 g. of a commercial pig meal (Sow and Weaner Meal, B.O.C.M., Ltd.) twice daily. Water was unrestricted in experiment I, but restricted in experiment II to 1000 ml. and in experiments III and IV to 1500 ml. per feed. In experiments III and IV, to prevent radioactive iodine uptake by the thyroid, potassium iodide solution was added to this water to give a final concentration of 0.005 per cent. The pigs were infected orally with the infective larvae, as a single dose, at 12 weeks of age and approximately 25 kg. weight. Experiments I and II were designed to determine the approximate level of infection required to produce clinical signs of severe hyostrongylosis together with lowered plasma protein and albumin levels. In experiment I, a litter of 10 pigs was divided as follows: Group la, 3 pigs infected with 60 000 larvae, group lb, 3 pigs infected with 200 000 larvae and group lc, 4 * Present Auchincruive,
address: Ayr.
Department
of Zoology
and
Nematology.
West
of Scotland
Agricultural
College,
400
R. N. TITCHENER
f?t d.
uninfected controls. In experiment II, 9 litter mates were divided into group 2a, 5 pigs infected with 350 000 larvae and group 2b, 4 uninfected controls. All pigs used in these 2 experiments were bled from the anterior vena cava. Blood samples, each of 10 ml., were collected in heparinized tubes (Stayne Laboratories Ltd.). Total plasma protein and albumin levels were determined by the biuret method described by Wootton (1964). Experiments III and IV covered the period just before and during which low plasma total protein and albumin levels were observed and was designed to investigate hyostrongylosis as a protein-losing enteropathy by measuring the turnover of radio-iodinated albumin and the half life and faecal output of iodine labelled PVP (polyvinyl-pyrrolidone) in infected pigs. In experiment III a litter of 10 pigs was divided into, group 3a, 6 pigs and group 3c, 4 uninfected controls. The six pigs in group 3a were infected with 250 000 larvae since the results of experiments of I and II indicated that this level of infection would possibly be sufficient to produce clinical symptoms and likely to lead to an increased radioiodinated albumin turnover. Porcine albumin (Sigma Chemicals Ltd.), labelled with 1311 by the electrolytic method of Rosa, Scassellati, Pennsis, Riccioni, Griagnoni and Giordani, (1964), was diluted with porcine albumin and 1 ml. of the resulting solution (dose 30 &i) immediately injected into the ear vein of each pig in groups 1 and 2, 15 d. after infection. Heparinized blood samples, each of 5 ml., were taken from the anterior vena cava 15 min. after the injection of the radioactive albumin, after which further samples were taken daily for the next 10 days. Aliquots of plasma, each of 1 ml., obtained from the blood by centrifugation were counted in a well type scintillation counter with a twin channel scaler. Plasma radioactivity, corrected for isotope decay, was expressed as a percentage of the activity of the 15 min. sample. The fractional catabolic rate was calculated according to the method described by Matthews (1957). In experiment IV a litter of 8 pigs was divided into 2 equal groups. Group 4a, was infected whilst group 4b were uninfected controls. A higher larval dose of 325 000 was used since gross clinical signs of hyostrongylosis were only apparent in half of the pigs infected with 250 000 larvae in experiment III. It appeared probable that the higher dose rate would increase the likelihood of the development of overt clinical symptoms and thus, possibly, provide a greater contrast in the plasma half-life and faecal output of i311 PVP between infected and control animals. A larval dose in excess of 325 000 was not considered advisable as severe clinical signs developed in 2 pigs infected with 350 000 larvae in experiment II. Whilst these extreme signs were exceptional and had not been observed in previous studies in our laboratory using high levels of infective larvae (Castelino et al., 1970) they were associated with a degree of haemoconcentration which if it occurred in the 1311 PVP test would result in a misleadingly high plasma concentration and increased plasma half life of 1311 PVP. One millilitre of 13iI labelled PVP (dose 200 l&i) was injected into the ear-vein of each pig in groups 4a and 4b 12 days after infection. Plasma samples were taken and counted as previously described at 24 h. after injection, after which samples were taken daily for the next 8 days. Plasma. radioactivity corrected for isotope decay, was expressed as a percentage of the sample 24 h. after injection. The method of “least squares” was used to draw the best straight line through the points on the plasma disappearance curve of 1311 PVP so that the plasma half-life could be calculated. From 14 to 24 days after infection the 24 h. faecal output of each pig in experiment IV was collected and 4 g. aliquots taken and assessed for radioactivity. Faecal activity was expressed as a percentage of the injected dose found in each 24 h. faecal sample. In all 4 experiments daily faecal egg counts were made on each 24 h. faecal sample during the period 14 to 30 days after infection using the Clayton-Lane centrifuge flotation method (M.A.F.F. 1971) using saturated magnesium sulphate (specific gravity l-25).
Ii. rubidus
INFECTION:
PLASMA
PROTEIN
401
LOSS
RESULTS
No clinical signs of diarrhoea, anorexia or weight loss were observed in animals infected with 60 000 or 200 000 larvae. In experiment II, in which animals were infected with 350 000 larvae, vomiting and anorexia were observed in all the infected pigs sometime during the period 15 to 27 days and were most severe 18 to 20 days after infection. Two pigs (B65 and B70) were most severely affected, B70 dying on the 20th day after infection. In addition to the clinical signs already described, both animals became lethargic, showed cyanosis of the nose, ears and ventral abdomen and scoured slightly, the faeces being of a sticky yellowish consistency. Nematode eggs were first detected in the faeces 17 days after infection in experiments I and II. In experiment I the mean peak egg counts were: group la, 52 eggs per gram (e.p.g.) 21 days and group lb, 159 e.p.g. 23 days after infection. Egg counts were considerably higher in experiment II and a mean peak egg count of 2902 e.p.g. 20 days after infection was observed. Particularly high individual egg counts of 3612 e.p.g. and 12 540 e.p.g. were recorded from the faeces of pigs B65 and B70 respectively. No difference in mean concentration of total plasma protein or plasma albumin was observed in experiment I between the pigs in group la, infected I
I
I
I
I
I
I
I
I
I
I
I
I
I
I A
7.5 -
tlllllllllll 0
4
6
12 Days
I6 after
20
l 24
III 28
i
3;
mfectton
Fig. 1. Mean total plasma protein and plasma albumin levels in pigs infected with 200 000 H. rubidas larva (Exp. I, group lb). A. Group mean total plasma protein. B. Group mean plasma albumin. (e-0) Mean value group lb infected with 200 000 larvae; (0-O) Mean value uninfected group.
402
R.
N.
TITCHENER
et Ul.
with 60 000 larvae, and the controls. Mean total plasma protein and plasma albumin levels of the pig; in group lb, infected with 20 000 larvae, showed a sharp decline 20 days after infection (Fig. 1). Whilst these levels were not statistically different from the control levels, 2 out of the 3 infected pigs had plasma albumin levels below the limit of the normal range for pigs. II
IIIII
IIIIIIII
1. A
_
IO -
2-
I 0
I 4
I
I 8
I
I 12 Days
,
, I6 after
20
24
28
32
mfectlon
Fig. 2. Mean total plasma protein and plasma albumin levels of 2 pigs (B65 and B70) in Exp. II infected with 350 000 H. rubidus larvae together with these values expressed against a constant urea value. A. Total plasma protein. B. Plasma albumin. (o-0) Mean value without basis of constant urea value; (0-n) Mean value with basis of constant urea value (40 mg. urea/ 100 ml. plasma). Broken line indicates death of one of the infected animals, (B70).
In experiment II no significant difference was observed in mean total plasma protein and plasma albumin levels between the infected and control pigs and only one infected pig, B71, had a plasma albumin level 20 days after infection below the limit of the normal range for pigs. Some degree of haemoconcentration appears to have occurred in pigs B65 and B70 at this time and for this reason total plasma protein and plasma albumin levels have been expressed on the basis of a constant urea value since, in our opinion, in the absence of renal malfunction, one might expect elevated urea levels during haemoconcentration. The urea levels were in fact elevated in both of these animals. While it is appreciated that corroborative evidence of haemoconcentration from haematocrit studies would ideally have been obtained, the evidence from our radiation studies does confirm lowered plasma protein during the same period. Mean total protein and plasma albumin levels of pigs B65 and B70, expressed on the basis of a constant urea value, are shown in Fig. 2. There was a decline in total plasma protein and plasma albumin levels from 16 days which reached
ff.
?dkiXS
INFECTION:
PLASMA
PROTEIN
403
LOSS
their lowest values 20 days after infection after which a gradual recovery occurred. The infection in the parasitized pigs of experiment III became patent 17 days after infection. Significantly higher egg counts (P <0*05) were recorded from the 3 pigs, R83, R84 and R86 which showed clinical signs of hyostrongylosis, than from the other 3 infected pigs, R81, R82 and R85 which showed no clinical TABLE PARASITOLOGICAL
AND
1
Ia11 ALBUMIN TURNOVER PERIOD 15-25 d. AFTER
DATA IN EXPERIMENT INFECTION
III FOR THE
Clinical signs Group
Group R83 R84 R86
3a
~hfean
+ S.D.
Group R81 R82 R85 Mean
3b
anorexia, diarrhoea and vomiting
-
t +
+
-
-
* s.o.
Standard
2194 1436 1573+ 565 P
Croup 3c (Control) R87 R88 R89 R90 Mean+s.D. SD.
+
Peak egg count e.p.g.
-
15
-
Plasma half 1+/h.
76 120 109k28.7 P
Fractional catabolic rate ~_____ ~- --.. -.- -.
40.00 26.74 3 1.99 fr 7.05 P
Deviation.
signs (Table 1). The low egg counts and lack of clinical signs possibly suggest a lower level of parasitic infestation in these pigs and for this reason group 3a was split into two groups: group 3a, pigs R83, R84 and R86, group 3b, pigs R81, R82 and R85, for analysis of the albumin turnover data. Albumin turnover data for the period 15 to 25 days after infection is given in Table 1. The pigs of group 3a, when compared with the pigs in group 3b and the control group 3c had shorter plasma half-lifes and higher fractional turnover rates. Compared with the control group, mean plasma half-life was significantly shorter (PC 0.002) and mean fractional catabolic rate significantly higher (P
404
R. xv. TITCHENER I
I
I
I
I4
I5
I6
I7
et al. I
I
1
I
I6
19
20
21
d i
i d N 8 E is 0 kz J
I.46 -
I .26 -
I.10 -
0.92
-
0.74 -
056-
13
Doys
Fig. 3. Plasma half-life in Experiment I
I
ofter
of 1311 PVP in infected and control groups of pigs, IV. (e-0) Mean value infected group; (0-O) I
I
I
I
13 to 18 days after infection, Mean value control group.
I
11 I6
Doys
after
22
infection
I9
I
LA 20
21
I
I
22
23
infectcon
Fig. 4. Faecal output of Is11 PVP in infected and control group of pigs, 14 to 23 days after infection, Experiment IV. (m) Mean value infected group; (IJ) Mean value control group.
in
H. rubidus
INFECTION:
PLASMA
PROTEIN
LOSS
405
In experiment IV patency occurred 16 days after infection and a mean peak egg count of 2430 e.p.g. was recorded on the 22nd day. Severe anorexia was observed in all the infected pigs 15 days after infection and continued until 23 days. One infected pig vomited and had slight diarrhoea during this period. Fourteen to 18 days after infection the mean plasma half-life of i311 PVP in the infected group of pigs (19 h. fS.D. 3.5) was much shorter than in the uninfected controls (60 h. *S.D. 7.9, Fig. 3), and this result was highly significant (P
In experiments I and II low levels of plasma total protein and albumin were detected shortly after patency at a time when the worms were at or approaching their peak egg production. The results of experiments III and IV show that these low levels were indicative of an earlier increased plasma protein loss in the period just before (experiment IV) and just after (experiment III) patency. Previous studies to detect gastro-intestinal plasma protein loss have been carried out in natural infections by Nielsen (1966), and in experimental infections by Jacobs (1970) and Dey-Hazra, Kolm, Enigk and Giese (1972). Only in the study of Dey-Hazra et al. (1972) was there a demonstrable Ioss of plasma protein in the infected pigs, but these workers did not commence their studies until 25 days after infection of 3 week old animals. It is difficult to compare the results detailed by Dey-Hazra et al. (1972) and those described here since they involve studies of different periods of infection. Using our strain of parasite, day 25 corresponds to the period after which the majority of adult worms have been lost, when egg counts are low and when a long lived “residual” infection aggregates in the fundic region (bordering the pyloric region) of the stomach. Such aggregations of worms are associated with erosions of the stomach wall similar to those described by Dey-Hazra et al. (1972) and these persist for varying periods of time. It is also difficult to compare the results in view of the fact that the two studies have dealt with different infection levels in pigs of’ different ages and when the prepatent periods are different. The prepatent period described in our study corresponds with that described by Davidson, Murray and Sutherland, (1967). Dey-Hazra et al. (1972) considered that the parasite used the epithelial cells of the gastric mucosa for nourishment and that loss of these cells, particularly where this was fairly extensive and small ulcers formed, resulted in increased mucosal permeability which was responsible for the increased gastro-intestinal protein loss. Studies in this laboratory agree with those of Davidson et al. (1967) that epithelial cytolysis occurs at the time
406
R. N. TITCHENER
et d.
when the adult worm migrates onto the stomach surface. This lesion is different from that caused by residual aggegations of worms described above. Loss of plasma protein occurs at the same time as this epithelial cytolysis occurs. In bovine and ovine ostertagiasis, structural changes in the mucosa and increased gastro-intestinal protein loss similar to those described here in hyostrongylosis also occur at the time when the parasites are emerging from the gastric glands (Jennings, Armour, Kirkpatrick and Murray, 1967). Murray, Jennings and Armour (1970) in a study of the gastric mucosa at this time have shown that the functional gastric gland mass is replaced by undifferentiated cells and have found by electron microscopy, that the junctional complexes between many of these undifferentiated cells are open, which they consider accounts for the enhanced permeability of the gastric mucosa to macromolecules. It seems probable that such structural changes in the gastric mucosa associated with the emergence of H. rubidus adult worms as well as the described epithelial cytolysis could account for the gastro-intestinal loss of plasma protein. SUMMARY
levels of total plasma protein and albumin have been detected in pigs infected with 200 000 and 350 000 larvae of Hyostronpylus rubidus. Further studies using 1311labelled albumin and 1311polyvinyl pyrrolidone have shown that this depression in total plasma protein and albumin results from plasma protein leakage into the gastro-intestinal tract at a time when the worms were migrating, or, had just finished migrating from the gastric glands on to the mucosal surface. LOW growing
ACKNOWLEDGMENTS
The authors wish to thank the ance. We are indebted to Dr W. A. labelling of the porcine albumin Titchener thanks the Sir William
Agricultural Research Council for financial assistHemmings, U.C.N.W., Bangor, for the radioactive and his advice on radioactive techniques. R. N. Roberts trust (U.C.N.W.) for financial support. REFERENCES
-197
1. Manual of Veterinary Parasitological Laboratory Techniques. Ministry of Agriculture, Fisheries and Food. Technical Bulletin No. 18. H.M.S.O. London. Castelino, J. B., Herbert, I. V., and Lean, I. J. (1970). The live-weight gain of growing pigs experimentally infected with massive doses of Hyostrongylusrubidus (Nematoda) larvae. British Veterinary Journal, 126, 579-582. Davidson, J. B., Murray, M., and Sutherland, I. H. (1967). Observations on the Clinical Pathology of Natural Strongyle Infestations in the Pig and their Control, with Special Reference to Hyostrongylus rubidus. Proceedings of the 3rd International Conference of the World Association for the Advancement of Veterinary Parasitology, Lyon, pp. 9-23, Elwert, Marburg, Lahn. Dey-Hazra, A., Kolm, H. P., Enigk, K., and Giese, W. (1972). Zum Gastrointestinalen Plasmaproteinverlust beim Hyostronpylus- befall des Schweines.
H. rubidus
INFECTION:
Gordon, R. S. (1959). Exudative intestinal tract demonstrable,
PLASMA
PROTEIN
407
LOSS
enteropathy. Abnormal permeability with labelled polyvinylpyrrolidone.
of gastroLancet, i,
325-327. JacobsiafJ)$,t
(1970).
Studies
on helminthiasis
in the pig. Ph.D. Thesis,
University
of
Jennings, F. W., Armour, J., Kirkpatrick, K. S., and Murray, M. (1967). Biochemical Consequences of Ostertagia Infections in Ruminants. Proceedings of the 3rd Conference of the World Association for the Advancement of Veterinary Parasitolqp, I,yons, pp. 38-42. Elwert, Marburg, Lahn. Lean, I. J. (1969). Studies on the pathogenesis of infection with Hvostrongylus rubidus (Nematoda) in pigs with particular reference to haematological and nitrogen metabolism changes. Ph.D. Thesis, University of Wales. Matthews, C. M. E. (1957). Theory of tracer experiments with iodine-131-labelled plasma proteins. Physics in Medicine and Biology, 2, 36-53. Murray, M., Jennings, F. W., and Armour, J. (1970). Bovine ostertagiasis; Structure, function and mode of differentiation of the bovine gastric mucosa and kinetics of the worm loss. Research in Veterinary Science, 11, 417-427. Neilsen, K. (1966). Metabolism and distribution of I 131 labelled albumin in pigs with gastrointestinal disease. Acta Veterinaria S’candinauica, 7, 321-329. Neilsen, K. (1968). Hypoproteinaemia associated with Helminthic Infections with Special Reference to Gastrointestinal Parasites. Isotopes and Radiation in Parasitology, p. 125. International Atomic Energy Agency, Vienna. Rosa, V., Scassellati, G. A., Pennsis, F., Riccioni, N., Griagnoni, P., and Giordani, R. ( 1964). Labelling of human fibrinogen with I 131 by electrolytic iodination. Biochimica et biophysics acta, 86, 5 19-526. Wootton, I. D. P. (1964). Micro-Analysis in Medical Biochemistry, J. & A. Churchill Ltd., London. [Received-for publication,
December 6th, 19731