Effects of dietary molybdenum on nematode and host during Trichostrongylus vitrinus infection in lambs

Research in Veterinary Science 1992, 52, 224-229

Effects of dietary molybdenum on nematode and host during Trichostrongylus vitrinus infection in lambs N. F. SUTTLE, D. P. KNOX, F. JACKSON, R. L. COOP, K. W. ANGUS, Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH

The addition of molybdenum (0"05 mmol kg -1 dry matter) to the diet of lambs exposed for four weeks to a trickle (2500 third stage larvae per day) infection with Trichostrongylus vitrinus reduced the number and length of adult worms retrieved from the small intestine 11 days later: both effects were particularly marked in female worms from female lambs (P < 0.01). Worms from lambs given molybdenum contained less proteinase enzyme activity and secreted less proteinases in culture irrespective of the sex of the host. Pathogenicity was not attenuated by molybdenum. Damage to the intestinal mucosa was severe in both dietary groups but infected females given molybdenum developed lower plasma albumin concentrations and lighter dressed carcases than those not given molybdenum. Neither the effects on the parasite nor those on the host could be attributed simply to molybdenum-induced copper depletion, using conventional measures of copper status. Molybdenum may be toxic to T vitrinus but may also facilitate or enhance the inflammatory process limiting larval establishment or increasing parasite rejection.

THE exposure of lambs to improved hill pastures high in molybdenum can induce a severe hypocuprosis, one consequence of which is increased susceptibility to microbial infections (Woolliams et al 1986). Susceptibility to nematode infection, as judged by faecal egg counts, tended to be reduced, those breeds and lines of the lowest copper status occasionally having low worm burdens (Jones et al 1985). The only specific study of the effects of molybdenum-induced copper deficiency on the outcome of a parasitic infection in sheep is that of Hucker and Yong (1986). They used repeated injections of ammonium tetrathiomolybdate to deplete wether lambs of copper and subsequently challenged them with a mixed infec-

tion of Trichostrongylus axei and T colubriformis. Copper-depleted lambs showed transiently increased susceptibility to infection in the form of higher faecal egg counts and blood lymphocyte counts (Yong et al 1985). The infection also lowered plasma copper concentrations (Hucker and Yong 1986). To test the hypothesis that molybdenum can inhibit the establishment or maintenance of nematode infections by depriving the parasite of available copper from the digesta, the authors examined the effects of a dietary molybdenum supplement on the outcome of a trickle infection of the intestinal nematode, T vitrinus in lambs. Materials and methods

Animals and experimental design Four groups of six (three male and three female) Greyface × Suffolk lambs, reared wormfree from birth and weighing 23 kg on average, were given a basal diet with (groups Mo) or without (groups O) a dietary supplement of 0.05 mmol molybdenum kg-1 dry matter (DM) (as Sodium molybdate) each with (groups Mo/I and O/I) or without (groups Mo/O and O/O) trickle infections of 2500 T vitrinus third stage larvae, five days a week for four weeks, in a 2 × 2 × 2 factorial design. The basal diet was a pelleted mixture (kg kg -1) of whole barley (0.60), whole oats (0.17), oat feed (0-10), dried skimmed milk (0.12), urea (0.01), calcium sulphate (0-0136), sodium chloride (0.010) with added cobalt, zinc, iron, iodine and vitamins A, D and E. The diet contained 79 ~rnol copper and 1 ~rnol molybdenum kg -1 DM and was fed at 80 g kg-1 W °75. Each lamb was penned individually and given free access to water. After 10 days' feeding the diets with or without added molybdenum, a liver 224

Molybdenum effects in T vitrinus infection sample was taken for copper analysis by aspiration biopsy and dosing with Tvitrinus larvae was commenced (day 0). Progress of the infection was monitored at approximately weekly intervals from the third week of infection by estimating phosphorus, albumin and globulin concentrations in plasma; faecal egg counts were made 39, 46 and 56 days after the start of infection. After nine weeks (that is, five weeks after the last larval dose), the lambs were killed by intravenous inoculations of sodium pentobarbitone and worms retrieved from the small intestine. At the same time the liver weight and dressed carcase weights were recorded. Samples of small intestine were taken for histology.

Infective larvae Larvae for infection were obtained from faeces collected from donor lambs infected with T vitrinus; faeces were cultured at 20°C for 10 days and the third stage larvae recovered by a standard Baermann technique. Larvae were stored at 4 to 7°C and used within three weeks.

Worm recovery The small intestine was removed at slaughter and processed according to the procedures described by Coop et al (1986). Intestinal contents and intestinal digests were washed separately on a 300 mesh sieve (53 ~am), the retentates made up to 2 litres and worm burdens were estimated from 2.5 to 5 per cent aliquots. Mean worm length was determined using an x-y plotter to measure the images projected with a camera lucida from a random sample of 25 adult worms per lamb.

225

[Jencons Scientific])from freshly retrieved parasites to study the effects of dietary molybdenum on parasite biochemistry. Parasite copper status was estimated by measuring the reactivity of homogenates with bathocuproin (Boehringer). Superoxide dismutase (SOD), cytochrome oxidase (co) and acetylcholinesterase (ACHE)activities were determined by previously described methods (SOD, Jones and Suttle [1981]; co, Horton 1968; ACHE, Ellman et al [1961]). Proteinase activity was determined essentially as described by Knox and Kennedy (1988) and protein estimations were performed using BCA protein assay reagent (Pierce). Enzyme assays were also performed on the supernatants of homogenates centrifuged at 100,000 g for one hour at 4°C to distinguish the contribution of soluble (presumably secretable) enzymes. Some of the adult T vitrinus obtained at slaughter were cultured for eight hours at 37°C in sterile PBS (150 worms m1-1) containing 10 ~tg glucose, 500 iu penicillin and 5 mg streptomycin m1-1, to examine the effects of previous exposure to added dietary molybdenum on their secretory activities. Following culture, parasites were pelleted by brief centrifugation at low speed (100 rpm) and the supernatant stored at -50°C before protein and enzyme analyses as for the worm homogenates.

Analyses of host diet, tissues and body fluids Total protein and albumin concentrations in plasma were measured by the methods described by Coggins and Field (1968). Copper in diets was determined by atomic absorption spectrophotometry and molybdenum by a catalytic method as described by Suttle (1983).

Histological methods Faecal egg counts Faeces were taken from the rectum and the number of eggs per gram of fresh weight (epg) estimated by the flotation method of Christie and Jackson (1982) using collapsible cellulose acetate tubes to separate the eggs following flotation in saturated sodium chloride.

The surface topography of 16 cm 2 pieces of duodenum and small intestine, taken at 0.5 m and 1.5 m from the pylorus and fixed in 10 per cent formol saline, was examined by using a dissecting microscope as described by Coop et al (1979). Selected areas from each site were later processed to paraffin-wax, and 6 gm sections cut and stained by Mayer's haemalum and eosin.

Analysis of worm homogenates and secretions Whole worm homogenates (150 worms m1-1 in 0.1 M phosphate buffered saline [PBSl,pH 7.4, were prepared in a 1 ml glass/glass homogeniser

Statistical analysis Normally distributed data were analysed by two or three way analysis of variance with F

226

N. F. Suttle, D. P. Knox, F. Jackson, R. L. Coop, K. W. Angus

ratio tests of significance or by Student's t test and means are given with standard errors unless stated otherwise. Abnormal distributions were compared by the Mann-Whitney U test and their median values are given with ranges.

TABLE 2: Soluble constituents of homogenates of 150 adult T vitrinus retrieved from the small intestine after prolonged trickle larval infection of lambs given diets with (group Mo/1)or without (group O/1) a molybdenum supplement Group O/I

Results Adult worm character&tics

Adult worm pop-lations were well established in both infected groups when the first faecal egg counts were conducted after 39 days of larval infection (809 + 147 and 1187 + 144 epg in groups O/I and Mo/I, respectively). Although differences in epg were not significant between dietary treatments or sexes at any time during the experiment, values decreased in group Mo/I (-388 + 186) but not in group O/I (+165 _+280 epg) between days 39 and 56. At slaughter (Table 1), 23 per cent fewer adult T vitrinus were retrieved from group Mo/I and their length was significantly (P< 0.05) reduced compared with group O/I and both effects were most marked in female worms from female lambs (P < 0-01). The effects of dietary molybdenum on markers of copper status in the adult worms are shown in Fig 1 and Table 2. There was significantly less 'reactive' copper in the whole homogenate of worms from group Mo/I (17-4 compared with 28.0 + 2.27 kanol copper ml-l: P < 0.01) and significant correlations with co and SOD activities across groups (Fig 1). The activity of 'soluble' SODwas decreased by molybdenum only if results were expressed per mg soluble protein but such comparisons are biased since molybdenum supTABLE 1: Characteristics of male (m) and female (f) adult worms retrieved from male (M) and female (F) lambs given diets with (group Mo/1)or without (group O/1)supplementary molybdenum (0,05 mmol kg-1 DM),five weeks after ceasing a four-week trickle larval infection Lamb sex Group O/I Mo/I

M F M F SE

Mean worm length (mm)

Mean worm Mean* weight (~tg) worm count

m

f

150m+f

m

f

5.02 a 4.80 a 4-76 a 4.40 c 0.107

6.22 a 6.01 a 5.99 a 5.31 b 0.181

45 a 68 a 43 a 37 a 10.3

5533 a 5600 a 4093 a 4947 a 733

6640a 7213a 5267 b 4933b 472

*Almost exclusively adult worms Differences in superscripts within columns indicate significant differences between subgroups (P < 0.05)

Moll

Protease substrate Azocaseinl 5.21 (2-17-8.47) Azocoll 8.4 (2.1-12.4) Elastin-orcein 0.87 (2.43-4-0) AChE (iu ml-1) * 0-12 soo (U ml-1)* 2.35 Protein (p.g m1-1) 172

Mann Whitney U test

Pooled SE

1-34 (0.11-5-85) 3.3 (0.11-5.85) 0.12 (0-0-41) 0.10 1.59 327

P < 0.06 P < 0.03 P < 0.005 0.026 0-373 P < 0.05

*ACHEAcetylcholinesterase measured at 25°C, SOD Superoxide dismutase measured at 37°C with one unit = 50 per cent inhibition of colour formation [Change in absorbance (determined at 380 nm (Azocasein), 520 nm (Azocoll) or 550 nm (Elastin-orcein)) per ml homogenate following 16 hours assay incubation at 25°C Median values given with ranges in brackets if distributions are abnormal 200

8000 •

150

~-

~

OO

:~e• .."a'"

100

,."

a, 0

6000 4000 O

O

O9

50

2000

0 0

' 20

0 40

60

80

100

120

Reactive copper FIG 1: Correlations between cuproenzyme activity and reactive copper concentrations (RCu, ~mol mg-1 protein) in homogenates of T vitrinus retrieved from lambs either given ( A , O) or not given (/',, O) a dietary molybdenum supplement. Cytochrome oxidase (co; O, Q) and superoxide dismutase (SOD, /k, A ) are expressed as iu of activity mg -1 protein. The regression equations are co = 31.7 + 1.15 +_ 0.242 RCu (R = 0.81); - - : SOD = 2981 + 29-5 -+ 9.67 RCu (r = 0.69); - - -

plementation increased soluble protein concentrations in the worm tissue (Table 2). The effects of treatments on protease activities in worm homogenates were restricted to the major, 'soluble' protease component and data for total and insoluble protease activity are not presented. Soluble protease activities were highly variable but significantly lower in group Mo/I than in group O/I (Table 2): differences were proportionately and statistically greatest with elastinorcein and least with 'azocasein' as substrate.

227

Molybdenum effects in T vitrinus infection When worms were cultured for eight hours, less azocaseinolytic and azocollolytic activity was recovered in culture fluids from group Mo/I while the amount of protein recovered was not affected (Table 3). There were no significant effects of, or interactions with, sex of host in the data for protease activity or secretion.

Host characteristics During the final weeks of the experiment clinical signs of nematodiasis appeared in all female lambs in group Me/I: a gradual reduction in plasma albumin concentrations (Fig 2; P < 0.001 at day 56) was accompanied by diarrhoea, loss of appetite and low final dressed carcase weights (Table 4) (P < 0.01). There was also a reduction in dressed carcase weight in lambs given molybdenum whether infected or not (molybdenum effect: P < 0.05). Plasma P was not affected by the treatments but plasma globulin had been increased from 36.2 in uninfected to 42.9 + 1.92 g litre -1 in infected groups when the experiment ended (P < 0.0l). There was no detectable effect o f dietary molybdenum on topographical or histological findings in infected lambs. Both the duodenum and jejunum showed extensive villous atrophy, crypt elongation, goblet cell proliferation and profuse mononuclear cell infiltration. Numerous worms, typically sited in epithelial channels at the mucosal surface, were seen in all lambs. Intraepithelial mast cells were not seen at slaughter in any lamb in either group. TABLE 3: Mean secretion of protein and of protease and acetylcholinesterase (ACHE) activity by 150 adult T vitrinus during eight hours culture. Worms were retrieved from groups of lambs given diets supplemented (group Mo/I) or not supplemented (group O/I) with molybdenum Group Protein secretion (mg g-1 worm) Protease substrate Azocaseint Azocollagent Elastin-orceinl-

AChE (iu ml-1)

Residual SD Significance

O/I

Mo/I

23.5

21.5

5-99

NS

20.5

4.0

-

P < 0.02*

(5-52)

(o-9)

3.16 1.00 10-7

1.42 0.82 13.3

1.218 0.69 5.52

P < 0.05 NS NS

Enzyme activity expressed as change in absorbance per mg secreted protein during a 60 hours incubation *Median (range) values given for data subject to Mann Whitney U test tUnits described in Table 2

TABLE 4: Effects of molybdenum, infection and sex of the lamb on dressed carcase weight, plasma albumin and change (A) in liver copper content at the end of the experiment

Dressed carcase (kg) Plasma albumin (g d-1) A Liver copper (mmol)

No infection

Infection

Host Me

M

F

M

F

O + 0 + 0 +

15.6 14.4 29.4 29.9 124 32

16-2 15.3 33.3 30-8 428 -91

15-4 15-1 30-0 29.5 229 -88

15.1 12.0 29-4 21-2 138 150

SED

0-92 2.2 115.3

M Male F Female

4O

Y

20 28

, 35

~ , 42 49 Days after first infection

I. 56

FIG 2: Divergence in mean (_+ SE) plasma albumin concentrations between female ( A ) and male ( & ) lambs infected with T vitrinus larvae for 28 days and given before and after a continuous dietary molybdenum supplement (group Me/I). No divergence was seen in a comparable group not given molybdenum (group O/I; O, • for male and female). Pooled means given for 28 days and 35 days

Mean plasma copper concentrations remained normal (over 9 grnol litre -1) throughout the study in all groups and no individual became hypocupraemic. Liver copper concentrations remained within the normal range but showed some marked and unexpected changes (Table 4). Molybdenum reduced liver copper accumulation in all but one of the subgroups, the infected females (molybdenum x sex x infection effect: P < 0-001). The mean liver copper concentrations when infection began had been 793 __ 200 gmol kg -1 DM with no difference between the two dietary groups. Discussion The experiment confirmed an impression gained from field studies that worm burdens can be lower than expected when the ovine host's

228

N. F. Suttle, D. P. Knox, F. Jackson, R. L. Coop, K. W. Angus

diet is enriched with molybdenum, and provides additional evidence that the growth of surviving adult worms is reduced. The authors' initial hypothesis that dietary molybdenum might inhibit gut parasites by depriving them of available copper from the digesta is supported by the reduced concentration of 'reactive copper' and activity of c o seen in worms from lambs given molybdenum. However, the results for protease activities in or secreted by the parasite raise another possibility: that molybdenum in the digesta was toxic to the parasite. Previous exposure to dietary supplements of molybdenum reduced 'azocaseinolytic' activity in secretions of adult worms in culture: this could be due to enzyme inactivation by thiomolybdates, the rumen-derived metabolites of molybdates which have a strong affinity for various proteins (Woods and Mason 1987, Allen and Gawthorne 1988). However, conversion to thiomolybdate may not be essential for proteinase inhibition because molybdate per se inhibits the activity of proteinases obtained from cultured T vitrinus adults and fourth stage larvae retrieved from sheep not given molybdenum (Knox and Jones 1988). The reduced proteinase activities did not appear to be secondary consequences of reduced parasite viability, because AChE activities, either in homogenates or recovered in culture fluids, were not reduced in worms from lambs given molybdenum. The consistency with which the molybdenum supplement reduced proteolytic activity in the tissues of recovered worms varied with substrate, elastin-orcein degradation being particularly sensitive. T vitrinus contains and secretes multiple proteolytic enzymes with a variety of substrate specificities (Knox and Jones 1990). Recent experiments (D. P. Knox, unpublished) suggest that molybdate preferentially inhibits the activity of cysteinyl proteinases, one of the four major proteinase classes, probably by reacting with a 'thiol' group essential for catalytic activity. It is possible that the decrease in proteolytic digestion of elastin-orcein, observed here, is primarily due to the inhibition ofa cysteinyl proteinase by molybdenum. The greater reduction in number and size of female than male worms in lambs given molybdenum might be related to the inhibition of co or proteases and the greater requirements of the female worm for copper or protein for egg pro-

duction. Fecundity was not affected by molybdenum supplementation, however, suggesting that any additional requirement was met at the expense of other needs. Sex also influenced the outcome of the molybdenum x infection interaction in the host, female lambs being most affected. Other reports of sex x infection interactions involving parasites have been reported. Windon and Dineen (1981) found that female lambs developed a stronger immunity than males when vaccinated against trichostrongylid worms. Murray et al (1971) found that female rats expelled Nippostrongylus brasiliensis earlier than males and associated the effect with earlier rises in intraepithelial mast cell counts. However, intraepithelial mast cells were not seen in male or female lambs with T vitrinus and normally appear at a much later stage of infection (nine to 13 weeks, Jackson et al 1983, Coop et al 1979). At slaughter, mononuclear cell infiltration was pronounced and the hypoalbuminaemia (Fig 2) indicated that the inflammatory reaction had probably caused protein leakage to occur in females for some weeks. Passage of protective antibodies into the gut lumen might also have been enhanced, contributing to the reduction of worm growth and reduction of worm number in the female lamb host. It is unclear how the molybdenum-supplemented female might develop the stronger inflammatory reaction to Tvitrinus infection but the suggestion that she does so is strengthened by her unexpected increase in liver copper (Table 4) since liver copper can increase during inflammation (Kishore et al 1984). An increased protein loss associated with improved immunity may explain why molybdenum impaired growth of the infected female host despite reducing her worm burden. An interaction between dietary molybdenum and parasitic infection may have important implications for animal production from high molybdenum pastures where stunted growth has been observed in normocupraemic lambs (Suttle et al 1992). The molybdenum supplement significantly retarded lamb growth in this study without inducing copper deficiency although the females in group Mo/I contributed most to the effect. It is possible that when infected pasture contains plentiful molybdenum, enhanced inflammatory reactions result in protein loss and growth retardation so that poor lamb production is not predicted

Molybdenum effects in T vitrinus infection by measures of induced copper deficiency. In uninfected lambs localised copper depletion of the intestinal mucosa (Suttle el al 1989) may also have impaired mucosal function in some way. In summary, the biochemical observations here clearly show that molybdenum can have direct inhibitory effects on nematode metabolism which may reduce worm growth and viability. In addition there is indirect evidence that molybdenum could enhance immunity against parasite infection by modulating the inflammatory response. Possible mechanisms for such an effect are considered in a related paper containing histological evidence of enhanced inflammatory reactions in abomasally infected lambs given molybdenum supplements (Suttle et al 1992). References ALLEN, J. D. & GAWTHORNE, J. M. (1988) In Proceedings of the Sixth International Symposium on Trace Element Metabolism in Man and Animals. Ed. L. S. Hurley, C. Keen, C. Lonnerdal and R. B. Rucker. Plenum Press. pp 315-316 CHRISTIE, M. & JACKSON, F. (1982) Specific identification of strongyle eggs in small samples of sheep faeces. Research in Veterinary Science 32, 113-117 COGGINS, C. R. E. & FIELD, A. C. (1976) Diurnal variation in the chemicat composition of plasma from lactating beef cows on three dietary energy intakes. Journal of Agricultural Science 86, 595-602 COOP, R. L., ANGUS, K. W. & SYKES, A. R. (1979) Chronic infection with Trichostrongylus vitrinus in sheep. Research in Veterinary Science 26, 363-371 COOP, R. L., FIELD, A. C., GRAHAM, R. B., ANGUS, K. W. & JACKSON, F. (1986) Effect of concurrent infection with Ostertagia circumcincta and Trichostrongylus vitrinus on the performance of lambs. Research in Veterinary Science 40, 241-245. ELLMAN, G. L., COURTNEY, K. D., ANDRES, V. & FEATHERSTONE, R. M. (196I) A new rapid determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88-95 HORTON, R. (1968) A spectrophotometric method for the determination of cytochrome oxidase activity in biological samples. Analytical Biochemistry 24, 334-335 HUCKER, D. A. & YONG, W. K. (1986) Effects of concurrent copper deficiency and gastro-intestinal nematodiasis on circulating copper and protein levels, liver copper and bodyweight in sheep. Veterinary Parasitology 19, 67-76 JACKSON, F., ANGUS, K. W. & COOP, R. L. (i983) Development of morphological changes in the small intestine of lambs continually infected with Trichostrongylus vitrinus. Research in Veterinary Science 34, 301-304 JONES, D. G., SUTTLE, N. F., JONES, G. E., WOOLLIAMS, C. & WIENER, G. (1985) In Proceedings of the Fifth International

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Symposium on Trace Elements in Man and Animals. Ed. C. F. Mills, I. Bremner and J. K. Chesters. Farnham Royal Commonwealth Agricultural Bureaux. pp 46-48 JONES, D. G. & SUTTLE, N. F. (1981) Some effects of copper deficiency on leucocyte function in sheep and cattle. Research in Veterinary Science 31, 151-156 KISHORE, V., LATMAN, N., ROBERTS, D. W., BARNETT, J. B. & SORENSEN, J. R. J. (1984) Effects of nutritional copper deficiency on adjuvant arthritis and immuno-competence in the rat. Agents and Actions 14, 274-282 KNOX, D. P. & JONES, D. G. (1988) Modulatory effects of molybdenum on proteolytic enzyme activity of two ovine gastro-intestinal nematodes, Trichostrongylus vitrinus and Ostertagia circumcincta during in vitro culture. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 937 KNOX, D. P. & JONES, D. G. (1990) Studies on the presence and release of proteolytic enzymes (proteinases) in gastro-intestinal nematodes of ruminants. International Journal for Parasitology 20, 243-249 KNOX, D. P. & KENNEDY, M. W. (1988) Proteinoses released by the parasitic larval stages of Ascaris suum and their inhibition by antibedies. Molecular and Biochemical Parasitology 28, 207-216 MURRAY, M., JARRETT, W. F. H. & JENNINGS, F. W. (1971) Mast cells and macromolecular leaks in intestinal immunological reactions. The influence of sex of rats infected with N brasiliensis. Immunology 21, 17-31 SUTTLE, N. F. (1983) Effects of molybdenum concentration in fresh herbage, hay and semi-purified diets on the copper metabolism of sheep. Journal of Agricultural Science, Cambridge 100, 651-656 SUTTLE, N. F., KNOX, D. P., ANGUS, K. W., JACKSON, F. & COOP, R. L. (1989) Dietary molybdenum may enhance the inflammatory reaction to and hence rejection of gut nematodes in lambs. Proceedings of the Nutrition Society 48, 71A SUTTLE, N. F., KNOX, D. P., ANGUS, F., JACKSON, F. & COOP, R. L. (1992). Effects of dietary molybdenum on nematode and host during Haemonchus contortus infection in lambs. Research in Veterinary Science 52, 230-235 SUTTLE, N. F., MacPHERSON, A., PHILLIPS, P. & WRIGHT, C. L. (1992) The influence of trace element status on the preweaning growth of lambs on improved hill pastures in Scotland. Journal of Agricultural Science, Cambridge (in press) WINDON, R. G. & DINEEN, J. K. (1981) The effect of selection of both sire and dam on the response of F~ generation lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology 11, 11-18 WOOLLIAMS, C., SUTTLE, N. F., WOOLLIAMS, J. A., JONES, D. G. & WIENER, G. (1986) Studies on lambs from lines genetically selected for low and high copper status. 1. Differences in mortality. Animal Production 43, 293-301 WOODS, M. & MASON, J. (1987) Spectral and kinetic studies on the binding of trithiomolybdate to bovine and canine serum albumin in vitro: the interaction with copper. Journal of Inorganic Biochemistry 30, 261-272 YONG, W. K., EDWARDS, L. D. & HUCKER, D. A. (1985) Peripheral blood white cell responses during concurrent copper deficiency and gastrointestinal nematodiasis in sheep. Australian Journal of Experimental Biology and Medical Science 63, 273-8I

Received April 8, 1991 Accepted October 14, 1991