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Comparative Aspects of Plasma Antioxidant Status in Sheep and Goats, and the Influence of Experimental Abomasal Nematode Infection
J. Comp. Path. 2001, Vol. 124, 192–199 doi:10.1053/jcpa.2000.0453, available online at http://www.idealibrary.com on
Comparative Aspects of Plasma Antioxidant Status in Sheep and Goats, and the Influence of Experimental Abomasal Nematode Infection J. H. Lightbody, L. M. Stevenson, F. Jackson∗, K. Donaldson and D. G. Jones∗ Napier University, 10 Colinton Road, Edinburgh EH10 5DT and ∗Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 OPZ, Scotland, UK
Summary This paper provides, for the first time, comparative data on the plasma antioxidant status of two ruminant species, namely sheep and goats. In addition, the influence of experimental infection with Teladorsagia circumcincta on antioxidant status in the same two species is compared and contrasted. In general terms, antioxidant status was significantly higher in uninfected kids than in lambs. Differences in protein sulphydryl groups and vitamin E concentrations were particularly noteworthy; trends were similar, however, for albumin, vitamin A and total antioxidant capacity (TAC). Parasitological results, based on worm burden, faecal egg counts and peripheral blood eosinophil numbers, confirmed that goat kids were more susceptible than lambs to experimental T. circumcincta infection. “Trickle infection” had a variable impact on both total and individual antioxidant status; particularly during the early weeks, the trend was for reduced values in lambs and increased values in kids, as compared with uninfected controls. Subsequent challenge infection was associated with a transient decrease in TAC and albumin in trickle-infected animals of both species, and in appropriate control animals. The observed differences in plasma antioxidant capacity between sheep and goats may have important implications in terms of the comparative resilience of sheep 2001 Harcourt Publishers Ltd and goats to parasite infection.
Introduction Recent evidence suggests that tissue damage mediated by oxidative stress and reactive oxygen species (ROS) plays a role in the pathogenesis of many inflammatory (Barnes, 1990) and other diseases (Young et al., 1993; Loughrey et al., 1994). Many potentially toxic ROS are generated through normal oxidative metabolism (Halliwell and Gutteridge, 1989) and the body has adapted to these by developing a complex system of protective antioxidants, including redox enzymes and small, readily oxidizable molecules (Halliwell and Gutteridge, Correspondence to: D. G. Jones. 0021–9975/01/020192+08 $35.00
1990). Oxidative stress is thought to result from an imbalance between ROS and antioxidants. In human allergic inflammatory diseases, such as asthma and helminth infections, the associated influx of eosinophils has been implicated as a primary source of tissue damage (Corrigan and Kay, 1992), possibly via their potent ROS production (Yamashita et al., 1985; Petreccia et al., 1987). Ruminant gastrointestinal nematode infections are common and widespread, and their immunopathology closely resembles that of asthma and human helminthiasis. In man, smoking, chronic obstructive pulmonary disease (COPD) and allergic asthma (Rahman et al., 1996, 1997) have all been associated with depleted systemic antioxidant capacity and, therefore, indirectly with oxidative stress. However, the potential role of antioxidants and 2001 Harcourt Publishers Ltd
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oxidative stress in the aetiology of ruminant nematodiasis has not been explored. Interestingly, responsiveness to nematode infection varies considerably between ruminant species, with goats being markedly more susceptible and less capable of developing immune resistance than sheep (Le Jambre, 1984; Huntley et al., 1995). In this study the aim was to establish and compare plasma concentrations of several individual antioxidant components and total antioxidant capacity (TAC) in lambs and goat kids, and then to monitor the impact of a controlled experimental nematode infection on systemic antioxidant status in the two species. Materials and Methods Animals and Experimental Design Groups of 10 Suffolk cross lambs and 10 Cashmere goat kids, approximately 6 months old and reared worm-free, were each divided into two subgroups of five animals, matched for weight and sex. Animals were housed throughout the experiment and fed a maintenance diet of pelleted concentrate together with hay and water ad libitum. One subgroup of each species was dosed orally with 2000 third stage larvae (L3) of Teladorsagia circumcincta five times weekly for 7 weeks (“trickle infection”). The other two subgroups served as uninfected controls. On day 49 all animals were given a single challenge dose of 50 000 L3 T. circumcincta orally. The animals were killed 10 days post-challenge. Faecal Egg Counts (FECs) FECs were carried out with a modified flotation method. (Christie and Jackson, 1982). Worm Burden The animals were stunned by captive bolt and exsanguinated. The abomasum was ligated, removed and washed, and its contents and washings were collected. The abomasum was then incubated in 2 litres of physiological saline (0·85%) for 4 h at 37°C. The resultant saline digest was pooled with the contents and previous washings, fixed by adding formalin 2% ( Jackson et al., 1984), and then made up to a total volume of 5 litres with saline. To determine the number and stage of development of nematodes present, 100 ml (2%) of this pool were stained with Gram’s iodine and examined by light microscopy at ×40 magnification.
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Eosinophil Counts Eosinophil numbers were estimated in peripheral blood samples fixed with Carpentier’s stain. Briefly, heparinized whole blood (50 l) was mixed with 450 l of stain (prepared by mixing 2 ml of eosin [2% eosin Y in distilled water], 3 ml of 37% formaldehyde [saturated with CaCO3 by adding 0·5 g/ 100 ml] and 95 ml of water). Samples of this mixture were counted in an improved Neubauer chamber. Total Antioxidant Capacity (TAC) Plasma TAC was analysed in a microcentrifugal analyser (Monarch 2000; Allied Instrumentation Laboratory, Warrington, UK) by a modified (Lightbody, 1999) version of the method of Miller et al. (1993). Plasma samples were diluted 1:1 with phosphate-buffered saline (PBS; pH 7·4) before addition to a reaction mixture containing 141 l of chromogen (3·05 M metmyoglobin, 3·05 mM 2,2′-azino-bis[3-ethylbenzthiazoline-6-sulphonic acid] in PBS, pH 7·4) and 3 l of calibrator, or diluted plasma, in a total volume (including analyser diluents) of 288 l. The reaction was initiated by adding of 28 l of hydrogen peroxide (0·6 mM). Absorbance was monitored at 650 nm over a total run time of 6 min. TAC was standardized against bovine serum albumin (BSA) (Lightbody et al., 1998), over a concentration range of 0–80 g/litre, and expressed as BSA equivalents (g/litre BSA Equiv). Albumin Plasma albumin was analysed spectrophotometrically in a microcentrifugal analyser (Monarch 2000) by an automated procedure (Instrumentation Laboratory, Cat. No. 181620–80) based on selective binding of bromocresol green. Protein Sulphydryl (-SH) Groups The presence of reduced -SH groups, on cysteine amino-acid residues of proteins, was determined by the method of Ellman (1959). Briefly, the concentration of -SH groups was calculated from the change in absorbance at 412 nm, over a 30-min period, in a reaction mixture containing 1·4 ml of PBS (pH 7·4), 100 l of plasma and 20 l of 5,5′dithio-bis(2-nitrobenzoic acid) (DTNB; 10 mM in PBS, pH 7·0). Vitamins E (D--tocopherol) and A (Retinol) The lipid-soluble antioxidant vitamins A and E were determined by high performance liquid
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Fig. 1. Faecal egg counts (mean±SEM, n=5) in kids (Μ) and lambs (•) trickle-infected with Teladorsagia circumcincta.
chromatography (HPLC) after extraction, with 2 ml hexane, from a mixture of 400 l of plasma and 400 l of internal standard (tocol, 1 mol/l). Samples were separated by isocratic HPLC, in a 1% isopropanol-in-hexane (both HPLC grade; Rathburn Chemicals Ltd, Walkerburn, Scotland) mobile phase (flow rate 2·8 ml/minute), on a cyanobonded silica cartridge column ( Jones Chromatography, Mid-Glamorgan, UK). Results were obtained by dual wavelength fluorescence at excitation and emission wavelengths of 296 and 330 nm for vitamin E and tocol, and 326 and 480 nm for vitamin A. Total run time was 5·9 min. Statistics Results were subjected to repeated measures analysis (Kenward, 1987) to assess trends over the time course of the experiment. Where differences were apparent, means of groups were examined by analysis of variance (ANOVA) or two-sample Student’s t-test within individual time points. Results Faecal Egg Counts The FECs of trickle-infected lambs were higher than those of kids (Fig. 1) from the time that eggs first appeared at day 21. Egg output by lambs peaked at day 35 and sharply declined thereafter. In kids, the FECs peaked earlier than in lambs and, except in the late stages of the trickle infection period, were markedly lower than in lambs. The differences reached statistical significance on days 21 (P=0·042) and 35 (P=0·018)
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Fig. 2. Worm burdens (mean, n=5) in control and trickleinfected kids and lambs post mortem, 10 days after challenge with T. circumcincta. Abbreviations: EL4 (Ε), ML4 (C), LL4 (Γ), early, mid and late 4th stage larvae respectively; EL5 (∨), early 5th stage larvae; adults (Φ).
Worm Burden Figure 2 shows the distribution of larval stages comprising the worm burden harboured by kids and lambs at post-mortem examination. After challenge, trickle-infected kids (P<0·05), but not lambs, had total worm burdens greater than those of equivalent challenge controls. Moreover, the composition of the worm burdens differed markedly in terms of the distribution of developmental stages. Trickle-infected kids harboured predominantly adult worms and early 4th stage larvae (EL4), whereas their lamb counterparts had mostly late 4th stage larvae (LL4) and virtually no adults. Total worm burdens in the trickle-infected lambs were comparable with those in challenge control groups from both species, but again composition was quite distinct. Challenge control kids contained predominantly mid-4th stage larvae (ML4) and few adults, but worms recovered from challenge control lambs were predominantly adults. Eosinophils Figure 3 illustrates the influence of trickle and challenge infections on circulating eosinophil numbers. Challenge control lambs had a low baseline eosinophil count throughout the trickle-infection period and there was no increase in numbers after challenge. Trickle-infected lambs showed a typical response, with initially low baseline eosinophil counts rising from around 3 weeks after the start of infection. After challenge, the numbers transiently increased even further. In comparison with the
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Fig. 3. Whole blood eosinophil counts (mean±SEM, n=5) in control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
lambs, both kid groups had high baseline eosinophil counts; within each species, however, the challenge controls initially had counts higher than those of the trickle-infected animals. The latter showed a substantial eosinophilic response from day 21 onwards, which peaked at day 35 and then gradually subsided, with counts returning to pre-infection levels by day 49. After challenge, there was a comparable but transient increase in eosinophil numbers in both challenge control and trickleinfected kids. Statistical analysis revealed no overall significant influence of infection on eosinophil numbers, but the analysis may have been masked by significant (P<0·05) group differences, particularly the high baseline values in the challenge control kids, which were apparent throughout. Since it was possible that the kid results might have masked a real comparison of parasitic treatment effects in the lambs, data from the latter were analysed separately by ANOVA. This showed that infected lambs had a significantly (P=0·005) higher blood eosinophilia than controls during the trickle-infection period, and a similar difference was also evident after challenge (P=0·018 on day 51). Total Antioxidant Capacity There were no consistent differences in plasma TAC between species or treatment groups up to day 14 (Fig. 4). After day 14, however, a striking divergence in TAC emerged between species (P<0·001), the TAC values of control kids being consistently higher than those of control lambs. Trickle infection had an inconsistent impact on TAC. After day 35, values in the infected groups
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Fig. 4. Plasma TAC (mean±SEM, n=5) in control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
41 39 Albumin (g/litre)
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Fig. 5. Plasma albumin concentrations (mean±SEM, n=5) in control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
tended to be lower than in the challenge control groups. On day 51, immediately after challenge, TAC values in the trickle-infected groups were significantly (P<0·05) lower than in the corresponding challenge control groups (123·5± 1·7 vs 118·5±1·8 and 123·3±1·3 vs 118·0±2·4 g/ litre BSA Equiv for kids and lambs, respectively); these differences were no longer apparent at day 56. Albumin As illustrated in Fig. 5, there were clear differences in plasma albumin concentrations between the challenge control and trickle-infected groups from the two species, and this was statistically significant (P<0·001) from day 21 onwards. Trickle infection
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Fig. 6. Plasma protein sulphydryl (−SH) group levels (mean±SEM, n=5) in control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
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Fig. 7. Plasma vitamin E concentrations in pooled (n=5) samples from control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
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Sulphydryl (-SH) Groups As with albumin, the most striking feature of the data for -SH groups (Fig. 6) was the difference in plasma values between species. The mean concentrations in lambs were significantly lower than in kids (P<0·001), especially from day 21, the contrast between sheep and goats being even greater than with albumin concentrations. In kids there was a significant (P<0·05) reduction in -SH groups in trickle-infected animals from day 35. A similar trend was observed in lambs, but the differences were not statistically significant.
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appeared to exacerbate species differences, the albumin concentrations being lower than in the corresponding challenge controls for lambs, and vice versa for kids. Challenge infection was followed by a marked decrease in albumin concentrations in all groups.
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Fig. 8. Plasma vitamin A concentrations in pooled (n=5) samples from control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
for vitamin A, however, there were marked differences between group baseline values for both kids and lambs.
Vitamins E and A Figures 7 and 8 show vitamin E and vitamin A concentrations, respectively, in pooled plasma samples for each treatment group at each sampling time. The need to pool samples for analysis precluded informative statistical evaluation. However, as with the other antioxidant parameters described, there were obvious and distinct species differences in the plasma concentrations of both vitamins, kids having consistently higher concentrations than lambs throughout the experimental period. Trickle infection had little impact on either vitamin E or vitamin A concentrations within treatment groups;
Discussion This study describes and compares, for the first time, relative aspects of antioxidant status in healthy lambs and goat kids, and the impact of helminth infection thereon. Perhaps the most striking finding was the marked species differences in antioxidant status between sheep and goats, as judged by the parameters measured in this study. In several instances, these differences were further influenced by experimental nematode infection. Moreover, an intriguing relationship emerged between antioxidant status and differences in the
Plasma Antioxidant Status in Sheep and Goats
relative susceptibility of sheep and goats to nematode infection. The comparative nature of the experiment was based on the previous observation that goats are more susceptible than sheep to nematode infection (Le Jambre and Royal, 1976; Le Jambre, 1984), due to inability to regulate worm burden, a fact confirmed in the current study. At post-mortem examination, the worm burdens of trickle-infected and subsequently challenged kids were greater than those of comparable lambs (Fig. 2). A large proportion of this extra burden was due to adult worms and it is possible that these had persisted from the trickle infection. Comparison of the results from the trickle-infected groups suggests that lambs were more efficient in controlling the challenge infection than kids, since the latter harboured both greater total burdens and higher numbers of adult worms. These findings may reflect a relatively more efficient development of immunity in the lambs. The “nonimmune” controls had relatively similar total worm burdens following challenge; again, however, the distribution of developmental stages differed. The reasons for this are unclear but may also reflect species differences in defence mechanisms against invading nematodes. The lower FECs in the kids (Fig. 1) is not readily explained. FECs in the lambs declined sharply after day 35 and this probably reflected the development of an effective immunity. The results of antioxidant analysis produced an interesting comparison which, even in the absence of parasitic infection, highlighted striking innate differences between sheep and goats. Compared with lambs, kids had consistently higher plasma concentrations of all the individual antioxidants measured, i.e., albumin, protein -SH groups and the antioxidant vitamins E and A (Figs 5–8), as well as a higher TAC (Fig. 4); a group of yearling goats from the same experiment (data not shown) gave similar results. The differences observed were clearly species-related but their basis remains uncertain. One possibility is that underlying nutritional effects contributed. For example, even though the same diets were offered to all animals, intake and utilization may have varied between species. Experimental infection with T. circumcincta was associated with a number of alterations in antioxidant status. TAC levels were variable, but towards the end of the trickle-infection period and post-challenge they were generally reduced, particularly in the lambs. Reduced TAC was paralleled by decreased plasma albumin concentrations and, after day 35, by a reduction in protein -SH groups;
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these findings are consistent with previous observations on tissue damage (Coop et al., 1979) and protein loss (Abbott et al., 1986; KatungukaRwakishaya, 1997) during parasitic infection. It is tempting to speculate that the effects on systemic antioxidant status reflected increases in oxidative stress related to the gut nematode infection. Since exposed -SH groups on the surface of albumin provide a bulk source of antioxidant protection, loss of this protein due to nematode-mediated mucosal damage might result in lowered antioxidant capacity. A consistent feature for all antioxidants measured was the detection of increased values in the trickleinfected kids at about days 21 to 28, the values falling thereafter below those of the corresponding challenge controls. The reasons for this are unclear; possibly, however, an initial mobilization of antioxidant resources was followed by a depletion, due to the continuing presence of parasites, resulting in increased mucosal damage and local oxidative stress. The reasons for the striking and consistent differences in antioxidant status between kids and lambs are uncertain, but a genetic basis is possible. As Le Jambre and Royal (1976) pointed out, goats evolved as browsers and, therefore, were less likely than sheep (typically grazers) to be exposed to parasitic larvae. Consequently, kids may have been under less evolutionary pressure to develop mechanisms to resist infection. However, modern farming techniques have forced goats to graze far more, thus increasing the potential for exposure to infective larvae. In evolutionary terms, therefore, it is possible that goats developed a rather inefficient immune response to parasitism, but a higher antioxidant capacity as a means of withstanding low level infections. An alternative explanation for the species differences might be found in the characteristic eosinophilic response to parasitic infection (Stevenson et al., 1994; Pfeffer et al., 1996; Winter et al., 1997). Activation of eosinophils by a number of mechanisms, including the release of histamine and interleukin (IL)-4 from mast cells (Butterworth and Thorne, 1993), leads to the release of cytotoxic enzymes (Butterworth et al., 1979; Gleich et al., 1979) and, via respiratory burst activity (Babior, 1978; Baehner et al., 1982), to the production of ROS such as superoxide anion and hydrogen peroxide. Clearly, therefore, in conditions associated with eosinophilia, the risk of oxidative stress is potentially heightened. There is little direct evidence of increased oxidative stress during nematode infections. Increased lipid peroxidation has
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been demonstrated in gut tissue of parasitized rats by Smith and Bryant (1989), and the same authors showed that peroxidation was due to free radicals derived from host leucocytes. Results from the present study provided partial support for a relationship between peripheral blood eosinophilia and lowered antioxidant status. Certainly, in control animals the generally higher basal antioxidant status in kids than in lambs corresponded with their higher baseline eosinophil counts. The lack of statistical significance limits the interpretative value of the data but, nevertheless, in trickle-infected lambs decreases in TAC from day 14 to 28 coincided with the onset of increased eosinophil numbers. Moreover, in the same animals there was a further decrease in TAC immediately after challenge, which corresponded with a transient rise in eosinophil counts (Figs 3 and 4). In contrast, in trickle-infected kids TAC rose during the development of eosinophilia; levels had decreased markedly, however, on day 35, when eosinophil numbers were highest. As in the lambs, challenge was accompanied by a rapid rise in eosinophil counts, paralleled by a decrease in TAC. A similar pattern was seen with albumin, but relationships between -SH groups, vitamins E and A and peripheral eosinophil numbers were less obvious. Relating the current findings to the earlier hypothesis of evolutionary pressure, one conclusion might be that high antioxidant status in goats evolved to counteract the potentially greater oxidative challenge provided by the greater numbers of circulating eosinophils in kids than in lambs, and to combat the inevitably greater eosinophilic response to nematode infection in the more susceptible goat. In conclusion, this study has highlighted potentially important differences in antioxidant status, as reflected by assessment of TAC and selected individual antioxidants, between both healthy and parasitized lambs and goat kids. Further studies are planned to assess other important antioxidant components such as uric acid and ascorbic acid in sheep and goats. Attempts, including a comparative evaluation of lymph antioxidant status, are also in progress to provide further information on the role of antioxidants and oxidative stress in parasitic infections of ruminants. Acknowledgments The authors thank members of the Clinical Division and the Division of Parasitology at the Moredun Research Institute for animal husbandry, parasitological sampling and analysis. Funding of the principal author ( J. H. L.) by The Wellcome Trust,
and others by the Scottish Executive Rural Affairs Department, is gratefully acknowledged. References Abbott, E. M., Parkins, J. J. and Holmes, P. H. (1986). The effect of dietary protein on the pathophysiology of acute ovine haemonchosis. Veterinary Parasitology, 20, 291–306. Babior, B. M. (1978). Oxygen-dependent microbial killing by phagocytes I. New England Journal of Medicine, 298, 659–668. Baehner, R. L., Boxer, L. A. and Ingraham, L. M. (1982). Reduced oxygen by-products and white blood cells. In: Free Radicals in Biology, Vol. V, W. A. Pryor, Ed., Academic Press, London, pp. 91–113. Barnes, P. J. (1990). Reactive oxygen species and airway inflammation. Free Radicals in Biology and Medicine, 9, 234–243. Butterworth, A. E. and Thorne, K. J. I. (1993). Eosinophils and parasitic diseases. In: Immunopharmacology of Eosinophils, H. Smith and R. M. Cook, Eds, Academic Press, London, pp.119–151. Butterworth, A. E., Wassom, D. L., Gleich, G. J., Loegering, D. A. and David, J. R. (1979). Damage to schistosomula of Schistosoma mansoni induced directly by eosinophil major basic protein. Journal of Immunology, 122, 221–229. Christie, M. and Jackson, F. (1982). Specific identification of strongyle eggs in small samples of sheep faeces. Research in Veterinary Science, 32, 113–117. Coop, R. L., Angus, K. W. and Sykes, A. R. (1979). Chronic infection with Trichostrongylus vitrinus in sheep. Pathological changes in the small intestine. Research in Veterinary Science, 26, 363–371. Corrigan, C. J. and Kay, A. B. (1992). T-cells and eosinophils in the pathogenesis of asthma. Immunology Today, 13, 501–507. Ellman, G. L. (1959). Tissue sulphydryl groups. Archives of Biochemistry and Biophysics, 82, 70–77. Gleich, G. J., Frigas, E., Loegering, D. A., Wassom, D. L. and Steinmuller, D. (1979). Cytotoxic properties of the eosinophil major basic protein. Journal of Immunology, 123, 2925–2927. Halliwell, B. and Gutteridge, J. C. M. (1989). Free Radicals in Biology and Medicine, 2nd Edit., Clarendon Press, Oxford. Halliwell, B. and Gutteridge, J. M. C. (1990). The antioxidants of human extracellular fluids. Archives of Biochemistry and Biophysics, 280, 1–8. Huntley, J. F., Patterson, M., Mackellar, A., Jackson, F., Stevenson, L. M. and Coop, R. L. (1995). A comparison of the mast cell and eosinophil responses of sheep and goats to gastrointestinal nematode infections. Research in Veterinary Science, 58, 5–10. Jackson, E., Jackson, F. and Smith, W. D. (1984). Comparison of saline incubation and pepsin digestion as methods for recovering Ostertagia circumcincta larvae from the abomasum of sheep. Research in Veterinary Science, 36, 380–381. Katunguka-Rwakishaya, E. (1997). The influence of
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dietary protein on some blood biochemical parameters in Scottish Blackface sheep experimentally infected with Trypanosoma congolense. Veterinary Parasitology, 68, 227–240. Kenward, M. G. (1987). A method for comparing profiles of repeated measurements. Applied Statistics – Journal of the Royal Statistical Society, Series C, 36, 296–308. Le Jambre, L. F. (1984). Stocking rate effects on the worm burdens of angora goats and merino sheep. Australian Veterinary Journal, 61, 280–282. Le Jambre, L. F. and Royal, W. M. (1976). A comparison of worm burdens in grazing merino sheep and angora goats. Australian Veterinary Journal, 52, 181–183. Lightbody, J. H. (1999). Relationships between antioxidant status, eosinophilia and the aetiology of ruminant gastrointestinal nematode infection. Ph.D. Thesis, Napier University, Edinburgh, pp.1–238. Lightbody, J. H., Stevenson, L. M., Jones, D. G. and Donaldson, K. (1998). Standardisation of a spectrophotometric assay for plasma antioxidant capacity. Analytical Biochemistry, 258, 369–372. Loughrey, C. M., Young, I. S., Lightbody, J. H., McMaster, D., McNamee, P. T. and Trimble, E. R. (1994). Oxidative stress in haemodialysis. Quarterly Journal of Medicine, 87, 679–683. Miller, N. J., Rice-Evans, C., Davies, M. J., Gopinathan, V. and Milner, A. (1993). A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clinical Science, 84, 407–412. Petreccia, D. C., Nauseef, W. M. and Clark, R. A. (1987). Respiratory burst of normal human eosinophils. Journal of Leukocyte Biology, 41, 283–288. Pfeffer, A., Douch, P. G. C., Shaw, R. J., Gatehouse, T. K., Rabel, B., Green, R. S., Shirer, C. L., Jonas, W. E. and Bisset, S. (1996). Sequential cellular and humoral responses in the abomasal mucosa and blood
of Romney sheep dosed with Trichostrongylus axei. International Journal for Parasitology, 26, 765–773. Rahman, I., Morrison, D., Donaldson, K. and MacNee, W. (1996). Systemic oxidative stress in asthma, COPD, and smokers. American Journal of Respiratory and Critical Care Medicine, 154, 1055–1060. Rahman, I., Skwarska, E. and MacNee, W. (1997). Attenuation of oxidant/antioxidant imbalance during treatment of exacerbations of chronic obstructive pulmonary disease. Thorax, 52, 565–568. Smith, N. C. and Bryant, C. (1989). Free-radical generation during primary infections with Nippostrongylus brasiliensis. Parasite Immunology, 11, 147–160. Stevenson, L. M., Huntley, J. F., Smith, W. D. and Jones, D. G. (1994). Local eosinophil-related and mast cell-related responses in abomasal nematode infections of lambs. FEMS Immunology and Medical Microbiology, 8, 167–173. Winter, M. D., Wright, C. and Lee, D. L. (1997). The mast cell and eosinophil response of young lambs to a primary infection with Nematodirus battus. Parasitology, 114, 189–193. Yamashita, T., Someya, A. and Hara, E. (1985). Response of superoxide anion production by guinea-pig eosinophils to various soluble stimuli – comparison to neutrophils. Archives of Biochemistry and Biophysics, 241, 447–452. Young, I. S., Purvis, J. A., Lightbody, J. H., Adgey, A. A. J. and Trimble, E. R. (1993). Lipid-peroxidation and antioxidant status following thrombolytic therapy for acute myocardial infarction. European Heart Journal, 14, 1027–1033.
Received, July 31st, 2000 Accepted, October 24th, 2000
Comparative Aspects of Plasma Antioxidant Status in Sheep and Goats, and the Influence of Experimental Abomasal Nematode Infection J. H. Lightbody, L. M. Stevenson, F. Jackson∗, K. Donaldson and D. G. Jones∗ Napier University, 10 Colinton Road, Edinburgh EH10 5DT and ∗Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 OPZ, Scotland, UK
Summary This paper provides, for the first time, comparative data on the plasma antioxidant status of two ruminant species, namely sheep and goats. In addition, the influence of experimental infection with Teladorsagia circumcincta on antioxidant status in the same two species is compared and contrasted. In general terms, antioxidant status was significantly higher in uninfected kids than in lambs. Differences in protein sulphydryl groups and vitamin E concentrations were particularly noteworthy; trends were similar, however, for albumin, vitamin A and total antioxidant capacity (TAC). Parasitological results, based on worm burden, faecal egg counts and peripheral blood eosinophil numbers, confirmed that goat kids were more susceptible than lambs to experimental T. circumcincta infection. “Trickle infection” had a variable impact on both total and individual antioxidant status; particularly during the early weeks, the trend was for reduced values in lambs and increased values in kids, as compared with uninfected controls. Subsequent challenge infection was associated with a transient decrease in TAC and albumin in trickle-infected animals of both species, and in appropriate control animals. The observed differences in plasma antioxidant capacity between sheep and goats may have important implications in terms of the comparative resilience of sheep 2001 Harcourt Publishers Ltd and goats to parasite infection.
Introduction Recent evidence suggests that tissue damage mediated by oxidative stress and reactive oxygen species (ROS) plays a role in the pathogenesis of many inflammatory (Barnes, 1990) and other diseases (Young et al., 1993; Loughrey et al., 1994). Many potentially toxic ROS are generated through normal oxidative metabolism (Halliwell and Gutteridge, 1989) and the body has adapted to these by developing a complex system of protective antioxidants, including redox enzymes and small, readily oxidizable molecules (Halliwell and Gutteridge, Correspondence to: D. G. Jones. 0021–9975/01/020192+08 $35.00
1990). Oxidative stress is thought to result from an imbalance between ROS and antioxidants. In human allergic inflammatory diseases, such as asthma and helminth infections, the associated influx of eosinophils has been implicated as a primary source of tissue damage (Corrigan and Kay, 1992), possibly via their potent ROS production (Yamashita et al., 1985; Petreccia et al., 1987). Ruminant gastrointestinal nematode infections are common and widespread, and their immunopathology closely resembles that of asthma and human helminthiasis. In man, smoking, chronic obstructive pulmonary disease (COPD) and allergic asthma (Rahman et al., 1996, 1997) have all been associated with depleted systemic antioxidant capacity and, therefore, indirectly with oxidative stress. However, the potential role of antioxidants and 2001 Harcourt Publishers Ltd
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oxidative stress in the aetiology of ruminant nematodiasis has not been explored. Interestingly, responsiveness to nematode infection varies considerably between ruminant species, with goats being markedly more susceptible and less capable of developing immune resistance than sheep (Le Jambre, 1984; Huntley et al., 1995). In this study the aim was to establish and compare plasma concentrations of several individual antioxidant components and total antioxidant capacity (TAC) in lambs and goat kids, and then to monitor the impact of a controlled experimental nematode infection on systemic antioxidant status in the two species. Materials and Methods Animals and Experimental Design Groups of 10 Suffolk cross lambs and 10 Cashmere goat kids, approximately 6 months old and reared worm-free, were each divided into two subgroups of five animals, matched for weight and sex. Animals were housed throughout the experiment and fed a maintenance diet of pelleted concentrate together with hay and water ad libitum. One subgroup of each species was dosed orally with 2000 third stage larvae (L3) of Teladorsagia circumcincta five times weekly for 7 weeks (“trickle infection”). The other two subgroups served as uninfected controls. On day 49 all animals were given a single challenge dose of 50 000 L3 T. circumcincta orally. The animals were killed 10 days post-challenge. Faecal Egg Counts (FECs) FECs were carried out with a modified flotation method. (Christie and Jackson, 1982). Worm Burden The animals were stunned by captive bolt and exsanguinated. The abomasum was ligated, removed and washed, and its contents and washings were collected. The abomasum was then incubated in 2 litres of physiological saline (0·85%) for 4 h at 37°C. The resultant saline digest was pooled with the contents and previous washings, fixed by adding formalin 2% ( Jackson et al., 1984), and then made up to a total volume of 5 litres with saline. To determine the number and stage of development of nematodes present, 100 ml (2%) of this pool were stained with Gram’s iodine and examined by light microscopy at ×40 magnification.
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Eosinophil Counts Eosinophil numbers were estimated in peripheral blood samples fixed with Carpentier’s stain. Briefly, heparinized whole blood (50 l) was mixed with 450 l of stain (prepared by mixing 2 ml of eosin [2% eosin Y in distilled water], 3 ml of 37% formaldehyde [saturated with CaCO3 by adding 0·5 g/ 100 ml] and 95 ml of water). Samples of this mixture were counted in an improved Neubauer chamber. Total Antioxidant Capacity (TAC) Plasma TAC was analysed in a microcentrifugal analyser (Monarch 2000; Allied Instrumentation Laboratory, Warrington, UK) by a modified (Lightbody, 1999) version of the method of Miller et al. (1993). Plasma samples were diluted 1:1 with phosphate-buffered saline (PBS; pH 7·4) before addition to a reaction mixture containing 141 l of chromogen (3·05 M metmyoglobin, 3·05 mM 2,2′-azino-bis[3-ethylbenzthiazoline-6-sulphonic acid] in PBS, pH 7·4) and 3 l of calibrator, or diluted plasma, in a total volume (including analyser diluents) of 288 l. The reaction was initiated by adding of 28 l of hydrogen peroxide (0·6 mM). Absorbance was monitored at 650 nm over a total run time of 6 min. TAC was standardized against bovine serum albumin (BSA) (Lightbody et al., 1998), over a concentration range of 0–80 g/litre, and expressed as BSA equivalents (g/litre BSA Equiv). Albumin Plasma albumin was analysed spectrophotometrically in a microcentrifugal analyser (Monarch 2000) by an automated procedure (Instrumentation Laboratory, Cat. No. 181620–80) based on selective binding of bromocresol green. Protein Sulphydryl (-SH) Groups The presence of reduced -SH groups, on cysteine amino-acid residues of proteins, was determined by the method of Ellman (1959). Briefly, the concentration of -SH groups was calculated from the change in absorbance at 412 nm, over a 30-min period, in a reaction mixture containing 1·4 ml of PBS (pH 7·4), 100 l of plasma and 20 l of 5,5′dithio-bis(2-nitrobenzoic acid) (DTNB; 10 mM in PBS, pH 7·0). Vitamins E (D--tocopherol) and A (Retinol) The lipid-soluble antioxidant vitamins A and E were determined by high performance liquid
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Fig. 1. Faecal egg counts (mean±SEM, n=5) in kids (Μ) and lambs (•) trickle-infected with Teladorsagia circumcincta.
chromatography (HPLC) after extraction, with 2 ml hexane, from a mixture of 400 l of plasma and 400 l of internal standard (tocol, 1 mol/l). Samples were separated by isocratic HPLC, in a 1% isopropanol-in-hexane (both HPLC grade; Rathburn Chemicals Ltd, Walkerburn, Scotland) mobile phase (flow rate 2·8 ml/minute), on a cyanobonded silica cartridge column ( Jones Chromatography, Mid-Glamorgan, UK). Results were obtained by dual wavelength fluorescence at excitation and emission wavelengths of 296 and 330 nm for vitamin E and tocol, and 326 and 480 nm for vitamin A. Total run time was 5·9 min. Statistics Results were subjected to repeated measures analysis (Kenward, 1987) to assess trends over the time course of the experiment. Where differences were apparent, means of groups were examined by analysis of variance (ANOVA) or two-sample Student’s t-test within individual time points. Results Faecal Egg Counts The FECs of trickle-infected lambs were higher than those of kids (Fig. 1) from the time that eggs first appeared at day 21. Egg output by lambs peaked at day 35 and sharply declined thereafter. In kids, the FECs peaked earlier than in lambs and, except in the late stages of the trickle infection period, were markedly lower than in lambs. The differences reached statistical significance on days 21 (P=0·042) and 35 (P=0·018)
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Fig. 2. Worm burdens (mean, n=5) in control and trickleinfected kids and lambs post mortem, 10 days after challenge with T. circumcincta. Abbreviations: EL4 (Ε), ML4 (C), LL4 (Γ), early, mid and late 4th stage larvae respectively; EL5 (∨), early 5th stage larvae; adults (Φ).
Worm Burden Figure 2 shows the distribution of larval stages comprising the worm burden harboured by kids and lambs at post-mortem examination. After challenge, trickle-infected kids (P<0·05), but not lambs, had total worm burdens greater than those of equivalent challenge controls. Moreover, the composition of the worm burdens differed markedly in terms of the distribution of developmental stages. Trickle-infected kids harboured predominantly adult worms and early 4th stage larvae (EL4), whereas their lamb counterparts had mostly late 4th stage larvae (LL4) and virtually no adults. Total worm burdens in the trickle-infected lambs were comparable with those in challenge control groups from both species, but again composition was quite distinct. Challenge control kids contained predominantly mid-4th stage larvae (ML4) and few adults, but worms recovered from challenge control lambs were predominantly adults. Eosinophils Figure 3 illustrates the influence of trickle and challenge infections on circulating eosinophil numbers. Challenge control lambs had a low baseline eosinophil count throughout the trickle-infection period and there was no increase in numbers after challenge. Trickle-infected lambs showed a typical response, with initially low baseline eosinophil counts rising from around 3 weeks after the start of infection. After challenge, the numbers transiently increased even further. In comparison with the
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Fig. 3. Whole blood eosinophil counts (mean±SEM, n=5) in control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
lambs, both kid groups had high baseline eosinophil counts; within each species, however, the challenge controls initially had counts higher than those of the trickle-infected animals. The latter showed a substantial eosinophilic response from day 21 onwards, which peaked at day 35 and then gradually subsided, with counts returning to pre-infection levels by day 49. After challenge, there was a comparable but transient increase in eosinophil numbers in both challenge control and trickleinfected kids. Statistical analysis revealed no overall significant influence of infection on eosinophil numbers, but the analysis may have been masked by significant (P<0·05) group differences, particularly the high baseline values in the challenge control kids, which were apparent throughout. Since it was possible that the kid results might have masked a real comparison of parasitic treatment effects in the lambs, data from the latter were analysed separately by ANOVA. This showed that infected lambs had a significantly (P=0·005) higher blood eosinophilia than controls during the trickle-infection period, and a similar difference was also evident after challenge (P=0·018 on day 51). Total Antioxidant Capacity There were no consistent differences in plasma TAC between species or treatment groups up to day 14 (Fig. 4). After day 14, however, a striking divergence in TAC emerged between species (P<0·001), the TAC values of control kids being consistently higher than those of control lambs. Trickle infection had an inconsistent impact on TAC. After day 35, values in the infected groups
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Fig. 4. Plasma TAC (mean±SEM, n=5) in control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
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Fig. 5. Plasma albumin concentrations (mean±SEM, n=5) in control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
tended to be lower than in the challenge control groups. On day 51, immediately after challenge, TAC values in the trickle-infected groups were significantly (P<0·05) lower than in the corresponding challenge control groups (123·5± 1·7 vs 118·5±1·8 and 123·3±1·3 vs 118·0±2·4 g/ litre BSA Equiv for kids and lambs, respectively); these differences were no longer apparent at day 56. Albumin As illustrated in Fig. 5, there were clear differences in plasma albumin concentrations between the challenge control and trickle-infected groups from the two species, and this was statistically significant (P<0·001) from day 21 onwards. Trickle infection
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Fig. 6. Plasma protein sulphydryl (−SH) group levels (mean±SEM, n=5) in control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
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Fig. 7. Plasma vitamin E concentrations in pooled (n=5) samples from control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
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Sulphydryl (-SH) Groups As with albumin, the most striking feature of the data for -SH groups (Fig. 6) was the difference in plasma values between species. The mean concentrations in lambs were significantly lower than in kids (P<0·001), especially from day 21, the contrast between sheep and goats being even greater than with albumin concentrations. In kids there was a significant (P<0·05) reduction in -SH groups in trickle-infected animals from day 35. A similar trend was observed in lambs, but the differences were not statistically significant.
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appeared to exacerbate species differences, the albumin concentrations being lower than in the corresponding challenge controls for lambs, and vice versa for kids. Challenge infection was followed by a marked decrease in albumin concentrations in all groups.
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Fig. 8. Plasma vitamin A concentrations in pooled (n=5) samples from control kids (Α) and lambs (Β), and trickle-infected kids (Μ) and lambs (Χ), before and after challenge (at day 49) with T. circumcincta.
for vitamin A, however, there were marked differences between group baseline values for both kids and lambs.
Vitamins E and A Figures 7 and 8 show vitamin E and vitamin A concentrations, respectively, in pooled plasma samples for each treatment group at each sampling time. The need to pool samples for analysis precluded informative statistical evaluation. However, as with the other antioxidant parameters described, there were obvious and distinct species differences in the plasma concentrations of both vitamins, kids having consistently higher concentrations than lambs throughout the experimental period. Trickle infection had little impact on either vitamin E or vitamin A concentrations within treatment groups;
Discussion This study describes and compares, for the first time, relative aspects of antioxidant status in healthy lambs and goat kids, and the impact of helminth infection thereon. Perhaps the most striking finding was the marked species differences in antioxidant status between sheep and goats, as judged by the parameters measured in this study. In several instances, these differences were further influenced by experimental nematode infection. Moreover, an intriguing relationship emerged between antioxidant status and differences in the
Plasma Antioxidant Status in Sheep and Goats
relative susceptibility of sheep and goats to nematode infection. The comparative nature of the experiment was based on the previous observation that goats are more susceptible than sheep to nematode infection (Le Jambre and Royal, 1976; Le Jambre, 1984), due to inability to regulate worm burden, a fact confirmed in the current study. At post-mortem examination, the worm burdens of trickle-infected and subsequently challenged kids were greater than those of comparable lambs (Fig. 2). A large proportion of this extra burden was due to adult worms and it is possible that these had persisted from the trickle infection. Comparison of the results from the trickle-infected groups suggests that lambs were more efficient in controlling the challenge infection than kids, since the latter harboured both greater total burdens and higher numbers of adult worms. These findings may reflect a relatively more efficient development of immunity in the lambs. The “nonimmune” controls had relatively similar total worm burdens following challenge; again, however, the distribution of developmental stages differed. The reasons for this are unclear but may also reflect species differences in defence mechanisms against invading nematodes. The lower FECs in the kids (Fig. 1) is not readily explained. FECs in the lambs declined sharply after day 35 and this probably reflected the development of an effective immunity. The results of antioxidant analysis produced an interesting comparison which, even in the absence of parasitic infection, highlighted striking innate differences between sheep and goats. Compared with lambs, kids had consistently higher plasma concentrations of all the individual antioxidants measured, i.e., albumin, protein -SH groups and the antioxidant vitamins E and A (Figs 5–8), as well as a higher TAC (Fig. 4); a group of yearling goats from the same experiment (data not shown) gave similar results. The differences observed were clearly species-related but their basis remains uncertain. One possibility is that underlying nutritional effects contributed. For example, even though the same diets were offered to all animals, intake and utilization may have varied between species. Experimental infection with T. circumcincta was associated with a number of alterations in antioxidant status. TAC levels were variable, but towards the end of the trickle-infection period and post-challenge they were generally reduced, particularly in the lambs. Reduced TAC was paralleled by decreased plasma albumin concentrations and, after day 35, by a reduction in protein -SH groups;
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these findings are consistent with previous observations on tissue damage (Coop et al., 1979) and protein loss (Abbott et al., 1986; KatungukaRwakishaya, 1997) during parasitic infection. It is tempting to speculate that the effects on systemic antioxidant status reflected increases in oxidative stress related to the gut nematode infection. Since exposed -SH groups on the surface of albumin provide a bulk source of antioxidant protection, loss of this protein due to nematode-mediated mucosal damage might result in lowered antioxidant capacity. A consistent feature for all antioxidants measured was the detection of increased values in the trickleinfected kids at about days 21 to 28, the values falling thereafter below those of the corresponding challenge controls. The reasons for this are unclear; possibly, however, an initial mobilization of antioxidant resources was followed by a depletion, due to the continuing presence of parasites, resulting in increased mucosal damage and local oxidative stress. The reasons for the striking and consistent differences in antioxidant status between kids and lambs are uncertain, but a genetic basis is possible. As Le Jambre and Royal (1976) pointed out, goats evolved as browsers and, therefore, were less likely than sheep (typically grazers) to be exposed to parasitic larvae. Consequently, kids may have been under less evolutionary pressure to develop mechanisms to resist infection. However, modern farming techniques have forced goats to graze far more, thus increasing the potential for exposure to infective larvae. In evolutionary terms, therefore, it is possible that goats developed a rather inefficient immune response to parasitism, but a higher antioxidant capacity as a means of withstanding low level infections. An alternative explanation for the species differences might be found in the characteristic eosinophilic response to parasitic infection (Stevenson et al., 1994; Pfeffer et al., 1996; Winter et al., 1997). Activation of eosinophils by a number of mechanisms, including the release of histamine and interleukin (IL)-4 from mast cells (Butterworth and Thorne, 1993), leads to the release of cytotoxic enzymes (Butterworth et al., 1979; Gleich et al., 1979) and, via respiratory burst activity (Babior, 1978; Baehner et al., 1982), to the production of ROS such as superoxide anion and hydrogen peroxide. Clearly, therefore, in conditions associated with eosinophilia, the risk of oxidative stress is potentially heightened. There is little direct evidence of increased oxidative stress during nematode infections. Increased lipid peroxidation has
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been demonstrated in gut tissue of parasitized rats by Smith and Bryant (1989), and the same authors showed that peroxidation was due to free radicals derived from host leucocytes. Results from the present study provided partial support for a relationship between peripheral blood eosinophilia and lowered antioxidant status. Certainly, in control animals the generally higher basal antioxidant status in kids than in lambs corresponded with their higher baseline eosinophil counts. The lack of statistical significance limits the interpretative value of the data but, nevertheless, in trickle-infected lambs decreases in TAC from day 14 to 28 coincided with the onset of increased eosinophil numbers. Moreover, in the same animals there was a further decrease in TAC immediately after challenge, which corresponded with a transient rise in eosinophil counts (Figs 3 and 4). In contrast, in trickle-infected kids TAC rose during the development of eosinophilia; levels had decreased markedly, however, on day 35, when eosinophil numbers were highest. As in the lambs, challenge was accompanied by a rapid rise in eosinophil counts, paralleled by a decrease in TAC. A similar pattern was seen with albumin, but relationships between -SH groups, vitamins E and A and peripheral eosinophil numbers were less obvious. Relating the current findings to the earlier hypothesis of evolutionary pressure, one conclusion might be that high antioxidant status in goats evolved to counteract the potentially greater oxidative challenge provided by the greater numbers of circulating eosinophils in kids than in lambs, and to combat the inevitably greater eosinophilic response to nematode infection in the more susceptible goat. In conclusion, this study has highlighted potentially important differences in antioxidant status, as reflected by assessment of TAC and selected individual antioxidants, between both healthy and parasitized lambs and goat kids. Further studies are planned to assess other important antioxidant components such as uric acid and ascorbic acid in sheep and goats. Attempts, including a comparative evaluation of lymph antioxidant status, are also in progress to provide further information on the role of antioxidants and oxidative stress in parasitic infections of ruminants. Acknowledgments The authors thank members of the Clinical Division and the Division of Parasitology at the Moredun Research Institute for animal husbandry, parasitological sampling and analysis. Funding of the principal author ( J. H. L.) by The Wellcome Trust,
and others by the Scottish Executive Rural Affairs Department, is gratefully acknowledged. References Abbott, E. M., Parkins, J. J. and Holmes, P. H. (1986). The effect of dietary protein on the pathophysiology of acute ovine haemonchosis. Veterinary Parasitology, 20, 291–306. Babior, B. M. (1978). Oxygen-dependent microbial killing by phagocytes I. New England Journal of Medicine, 298, 659–668. Baehner, R. L., Boxer, L. A. and Ingraham, L. M. (1982). Reduced oxygen by-products and white blood cells. In: Free Radicals in Biology, Vol. V, W. A. Pryor, Ed., Academic Press, London, pp. 91–113. Barnes, P. J. (1990). Reactive oxygen species and airway inflammation. Free Radicals in Biology and Medicine, 9, 234–243. Butterworth, A. E. and Thorne, K. J. I. (1993). Eosinophils and parasitic diseases. In: Immunopharmacology of Eosinophils, H. Smith and R. M. Cook, Eds, Academic Press, London, pp.119–151. Butterworth, A. E., Wassom, D. L., Gleich, G. J., Loegering, D. A. and David, J. R. (1979). Damage to schistosomula of Schistosoma mansoni induced directly by eosinophil major basic protein. Journal of Immunology, 122, 221–229. Christie, M. and Jackson, F. (1982). Specific identification of strongyle eggs in small samples of sheep faeces. Research in Veterinary Science, 32, 113–117. Coop, R. L., Angus, K. W. and Sykes, A. R. (1979). Chronic infection with Trichostrongylus vitrinus in sheep. Pathological changes in the small intestine. Research in Veterinary Science, 26, 363–371. Corrigan, C. J. and Kay, A. B. (1992). T-cells and eosinophils in the pathogenesis of asthma. Immunology Today, 13, 501–507. Ellman, G. L. (1959). Tissue sulphydryl groups. Archives of Biochemistry and Biophysics, 82, 70–77. Gleich, G. J., Frigas, E., Loegering, D. A., Wassom, D. L. and Steinmuller, D. (1979). Cytotoxic properties of the eosinophil major basic protein. Journal of Immunology, 123, 2925–2927. Halliwell, B. and Gutteridge, J. C. M. (1989). Free Radicals in Biology and Medicine, 2nd Edit., Clarendon Press, Oxford. Halliwell, B. and Gutteridge, J. M. C. (1990). The antioxidants of human extracellular fluids. Archives of Biochemistry and Biophysics, 280, 1–8. Huntley, J. F., Patterson, M., Mackellar, A., Jackson, F., Stevenson, L. M. and Coop, R. L. (1995). A comparison of the mast cell and eosinophil responses of sheep and goats to gastrointestinal nematode infections. Research in Veterinary Science, 58, 5–10. Jackson, E., Jackson, F. and Smith, W. D. (1984). Comparison of saline incubation and pepsin digestion as methods for recovering Ostertagia circumcincta larvae from the abomasum of sheep. Research in Veterinary Science, 36, 380–381. Katunguka-Rwakishaya, E. (1997). The influence of
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dietary protein on some blood biochemical parameters in Scottish Blackface sheep experimentally infected with Trypanosoma congolense. Veterinary Parasitology, 68, 227–240. Kenward, M. G. (1987). A method for comparing profiles of repeated measurements. Applied Statistics – Journal of the Royal Statistical Society, Series C, 36, 296–308. Le Jambre, L. F. (1984). Stocking rate effects on the worm burdens of angora goats and merino sheep. Australian Veterinary Journal, 61, 280–282. Le Jambre, L. F. and Royal, W. M. (1976). A comparison of worm burdens in grazing merino sheep and angora goats. Australian Veterinary Journal, 52, 181–183. Lightbody, J. H. (1999). Relationships between antioxidant status, eosinophilia and the aetiology of ruminant gastrointestinal nematode infection. Ph.D. Thesis, Napier University, Edinburgh, pp.1–238. Lightbody, J. H., Stevenson, L. M., Jones, D. G. and Donaldson, K. (1998). Standardisation of a spectrophotometric assay for plasma antioxidant capacity. Analytical Biochemistry, 258, 369–372. Loughrey, C. M., Young, I. S., Lightbody, J. H., McMaster, D., McNamee, P. T. and Trimble, E. R. (1994). Oxidative stress in haemodialysis. Quarterly Journal of Medicine, 87, 679–683. Miller, N. J., Rice-Evans, C., Davies, M. J., Gopinathan, V. and Milner, A. (1993). A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clinical Science, 84, 407–412. Petreccia, D. C., Nauseef, W. M. and Clark, R. A. (1987). Respiratory burst of normal human eosinophils. Journal of Leukocyte Biology, 41, 283–288. Pfeffer, A., Douch, P. G. C., Shaw, R. J., Gatehouse, T. K., Rabel, B., Green, R. S., Shirer, C. L., Jonas, W. E. and Bisset, S. (1996). Sequential cellular and humoral responses in the abomasal mucosa and blood
of Romney sheep dosed with Trichostrongylus axei. International Journal for Parasitology, 26, 765–773. Rahman, I., Morrison, D., Donaldson, K. and MacNee, W. (1996). Systemic oxidative stress in asthma, COPD, and smokers. American Journal of Respiratory and Critical Care Medicine, 154, 1055–1060. Rahman, I., Skwarska, E. and MacNee, W. (1997). Attenuation of oxidant/antioxidant imbalance during treatment of exacerbations of chronic obstructive pulmonary disease. Thorax, 52, 565–568. Smith, N. C. and Bryant, C. (1989). Free-radical generation during primary infections with Nippostrongylus brasiliensis. Parasite Immunology, 11, 147–160. Stevenson, L. M., Huntley, J. F., Smith, W. D. and Jones, D. G. (1994). Local eosinophil-related and mast cell-related responses in abomasal nematode infections of lambs. FEMS Immunology and Medical Microbiology, 8, 167–173. Winter, M. D., Wright, C. and Lee, D. L. (1997). The mast cell and eosinophil response of young lambs to a primary infection with Nematodirus battus. Parasitology, 114, 189–193. Yamashita, T., Someya, A. and Hara, E. (1985). Response of superoxide anion production by guinea-pig eosinophils to various soluble stimuli – comparison to neutrophils. Archives of Biochemistry and Biophysics, 241, 447–452. Young, I. S., Purvis, J. A., Lightbody, J. H., Adgey, A. A. J. and Trimble, E. R. (1993). Lipid-peroxidation and antioxidant status following thrombolytic therapy for acute myocardial infarction. European Heart Journal, 14, 1027–1033.
Received, July 31st, 2000 Accepted, October 24th, 2000