The effect of gastrointestinal parasitism on blood copper and hemoglobin levels in sheep A. Adogwa, A. Mutani, A. Ramnanan, C. Ezeokoli Abstract — Endoparasitism is a problem in Trinidad, as it is in most tropical countries. Parasite infection has been suspected to contribute to the pathogenesis of swayback disease (which is also prevalent in Trinidad), but the mode of action has not been clearly defined, although it has been suggested that parasites interfere with the absorption of copper from the gastrointestinal tract. The objectives of the study were to assess the effect of endoparasitism on blood copper levels and hemoglobin (Hb) concentration in sheep in Trinidad. Copper was administered intramuscularly to parasite infected and noninfected animals. The results showed that parasitism has a depressing effect on blood copper and Hb levels, even when administered parenterally. It is concluded that parasitism can aggravate existing hypocupremia and possibly influence the expression of swayback disease. Résumé — Effet du parasitisme gastro-intestinal sur les taux de cuivre sanguin et d’hémoglobine chez le mouton. L’endoparasitisme constitue un problème à Trinidad comme dans la majorité des pays tropicaux. On a soupçonné que l’infection parasitaire pouvait contribuer à la pathogenèse de l’ataxie enzootique de l’agneau (également prévalente à Trinidad) mais le mécanisme n’a pas été clairement identifié même si on a évoqué la possibilité que les parasites puissent interférer avec l’absorption du cuivre au niveau du tractus gastro-intestinal. Les objectifs de cette étude étaient d’évaluer les effets de l’endoparasitisme sur les taux sanguins de cuivre et d’hémoglobine (Hb) chez le mouton à Trinidad. Du cuivre a été administré par voie intramusculaire à des animaux parasités et non parasités. Les résultats démontrent que le parasitisme diminue les niveaux de cuivre sanguin et d’Hb même lorsque le cuivre est administré par voie parentérale. Il a été conclu que le parasitisme peut aggraver une hypocuprémie existante et possiblement influencer l’expression de la maladie de l’ataxie enzootique de l’agneau. (Traduit par Docteur André Blouin) Can Vet J 2005;46:1017–1021
Introduction
wayback disease is caused by copper deficiency and S it affects several species of domestic and wild animals (1). Clinical signs in animals include decreased growth rate, anemia, ataxia, bone disorders, diarrhea, abnormal pigmentation, and poor reproductive performance. The clinical disease is affected by several factors, including the species, age, and sex of the affected animals, and the duration and severity of the copper deficiency; ruminants are the species most highly susceptible to copper deficiency. Swayback occurs in 2 forms in lambs and kids. In the neonatal form, it causes stillbirth, whereas in the delayed form, the lamb or kid is normal at birth but shows clinical signs within the first 6 mo (2). In primary copper deficiency, the intake of copper is inadequate because of low levels of copper in the feed, whereas in secondary copper deficiency, the feed has adequate levels of copper, but due to the presence of excess molybdenum, sulphur, iron, or zinc, which form complexes with copper, adequate levels of copper are not available to the tissues (3). Swayback disease is widespread and of considerable importance in Trinidad. Most of the cases in Trinidad School of Veterinary Medicine, Faculty of Medical Sciences, The University of the West Indies, St. Augustine, West Indies. Address all correspondence and reprint requests to Dr. Andrew Adogwa; e-mail:
[email protected] Can Vet J Volume 46, November 2005
occur during the dry season, between February and May. Swayback disease and the factors causing hypocupremia have attracted significant research interest in Trinidad. Youssef (4,5) reported low levels of copper in the forage and in the blood of small ruminants. Mohammed et al (6) described the lesions of swayback disease in lambs and kids in Trinidad. Alleyne et al (7) showed that the mitochondria were defective in lambs and kids affected by swayback disease. The association between swayback disease and parasitism has been suspected, but not investigated seriously, even though both conditions coexist in Trinidad. It has been reported that blood copper levels are depressed in ruminants infected with nematodes (8,9,11). Several studies on the interaction of copper and parasitism have been reported in which copper was administered orally. There is a paucity of information on the relationship of copper and parasitism when the copper is administered parenterally. In this study, copper was administered, IM, as copper heptonate; blood copper levels and common hematological parameters were compared between nematode infected and noninfected animals.
Materials and methods Twenty ewes were used for the study. These were selected from ewes purchased from different farms in Trinidad, and then they were housed in 6 concrete pens, each pen approximately 3.4 m 2 in dimension and enclosed by 1017
Infected_no Cu Infected_C Not_Infected_no Cu NotInfectedCu
Figure 1. Serum copper levels in ewes under various treatments.
metal bars. On the basis of fecal analysis, 10 ewes that were infected with nematodes and another 10 that were free of infection were selected for the study. The animals were separated into 4 groups of 5. Two groups contained infected animals, the other 2 contained animals that were free of infection. The noninfected pens were always cleaned out, and the animals in those pens were fed before the animals in the infected pens. The animals were acclimatized for 2 wk before the start of the experiment; fecal samples were taken weekly during the period of acclimatization and throughout the study to check for changes in the infection status of the animals. The noninfected groups remained free of helminths throughout the period of the study, and the infected animals similarly retained their infection status throughout the study period. Blood samples ethylenediaminetetraacetic acid (EDTA)-treated and serum were collected by venipuncture and transported on ice to the laboratory and stored there at 4ºC until processed. The level of infection was quantified by the fecal egg count, using the number of eggs per low power (10x) microscopic field. The animals were grouped as follows: Group 1: Infected animals that did not receive copper; group 2: Infected animals that received copper; group 3: Noninfected 1018
animals that did not receive copper; and group 4: Noninfected animals that received copper. Copper heptonate (Cuvine; Pfizer, Sandwich, UK), 31.25 mg (2 mL), was administered, IM, in the lateral aspect of the caudal neck region. The EDTA-treated blood and serum samples were obtained weekly for 12 wk for the evaluation of serum copper levels and hemoglobin (Hb) concentrations. Three blood samples were taken at 2-week intervals before the administration of copper, and 3 samples also at 2-week intervals after the administration of copper. The clotted blood was centrifuged for 10 min at 1047 g, and the serum was removed with a Pasteur pipette into labeled plastic vials and stored at -70°C till analyzed. The EDTA-treated blood was used to determine the Hb levels by standard procedures at the School of Veterinary Medicine hematology laboratory. The blood copper levels were measured in the EDTA blood, with an atomic absorption spectrophotometer (PYE UNICAM sp 2900, PYE UNICAM; Philips Scientific, Cambridge, UK) equipped with a data graphics system. The fecal egg count was done by the flotation method, using a 33% zinc sulphate solution. The number of eggs per field under the 100 magnification was used as an indication of the severity of infection, which was scored Can Vet J Volume 46, November 2005
Infected No Cu Infected Cu Not Infected No Cu Not Infected Cu
Figure 2. Hemoglobin levels in ewes under various treatments.
1 to 4, with the 4 indicating the most severe infection. The severity of the infection was scored as follows: 1 = 1 to 2 eggs/field; 2 = 3 to 4 eggs/field; 3 = 5 to 6 eggs/field; and 4 = 6 eggs/field. All the animals had mild infection (1). The data were subjected to a one-way analysis of variance (ANOVA) to determine if there were any significant differences between the various treatment options.
Results The results are presented in Figures 1 and 2, and Tables 1 and 2. The noninfected animal groups showed significantly (P 0.01) higher levels of Hb and serum copper than the infected animals groups. The noninfected animals that did not receive copper still had higher levels of Hb and serum copper than the infected animals that did receive copper supplement. Administration of copper did not significantly change the serum copper levels in the infected animal groups; both groups of infected animals had low serum copper levels throughout the experiment. There was a gradual increase in the serum copper levels of the noninfected animal group that did not receive copper supplementation, which may have been due to the copper contained in the feed. The increase in the serum copper levels was more pronounced in the noninfected animal group that received copper suppleCan Vet J Volume 46, November 2005
ment. There was no significant difference in the increase in values of the serum copper levels between the 2 noninfected animal groups. The results of the Hb determinations were similar to the results of the serum copper levels. The mean Hb values of the infected animals to which copper was not administered remained relatively low (Figure 2) and, in fact, were decreasing towards the end of the experiment. The mean Hb values of the animals that were not infected continued to increase throughout the experiment, with the values of the noninfected animals that received copper being higher than those of the animals that did not. The results showed that the infected animal groups had significantly lower Hb values than the noninfected animal groups (P 0.05). The difference in the values of Hb between the non infected groups was not significant. The difference in values of Hb between the groups of infected animals was also not significant.
Discussion The results of our study indicate significant differences in the mean serum copper and the mean Hb levels between the infected and the noninfected animals. Within the infected groups, copper supplementation did not improve the Hb or blood copper levels. It is therefore clear that parasitic infection significantly lowers these parameters, even when the copper is administered 1019
Table 1. Mean serum copper (g/L) levels in ewes following various infection and treatment conditions Week 2 4 6 8 10 12
Infected, No Cu Mean and standard deviation
Infected, Cu Mean and standard deviation
Not infected, No Cu Mean and standard deviation
Not infected, Cu Mean and standard deviation
4.1, s = 0.52 4.32, s = 0.33 4.16, s = 0.53 4.72, s = 0.47 4.56, s = 0.35 4.72, s = 0.52
4.56, s = 0.72 4.24, s = 0.45 3.84, s = 0.2 5.04, s = 0.21 5.12, s = 0.52 4.72, s = 0.59
4.56, s = 0.22 5.28, s = 0.52 6.32, s = 0.52 6.48, s = 0.71 6.32, s = 0.43 6.56, s = 0.35
4.88, s = 0.18 5.68, s = 0.59 6.24, s = 0.53 6.9, s = 0.38 7.2, s = 1.00 7.8, s = 0.69
Table 2. Mean and standard deviation(s) of hemoglobin levels (g/L) in ewes treated with copper (cu) Week 2 4 6 8 10 12
Infected, No Cu Mean and standard deviation
Infected, Cu Mean and standard deviation
Not infected, No Cu Mean and standard deviation
Not infected, Cu Mean and standard deviation
10.2, s = 11 112, s = 14 108, s = 9 106, s = 9 100, s = 8 97, s = 8
103, s = 10 10.4, s = 8 107, s = 5 104, s = 11.4 99, s = 17 86, s = 15
113, s = 9 116, s = 12 120, s = 10 121, s = 10 121, s = 8 123, s = 8
11.2, s = 8 117, s =10 118, s = 7 125, s = 7 125, s = 8 131, s = 6.2
parenterally. Our findings are consistent with the results of previous studies in which copper was supplemented by the oral route (9–11). Depressions of whole blood copper levels have been demonstrated in sheep infected with Trichostrongylus spp. (11) and with Haemonchus spp. (12). A study of the effect of ostertagiasis on the copper status of sheep led to the conclusion that gastrointestinal nematodes interfered with copper metabolism by causing an increase in pH in abomasal and duodenal digesta (13). Poppi et al (15) also reported that the effectiveness of oral copper supplementation depended on the increase in soluble copper concentrations in the abomasal digesta, which was reduced by nematode-induced increase in pH. Ortolani et al and Frandsen (10,11) showed that plasma copper was decreased by 50% in lambs challenged with infective larvae of Haemonchus contortus and that parasitism influenced the molybdenum and sulphur antagonism. According to Ivan (14), availability of copper in sheep was decreased by helminthiasis and the magnitude of the effect was independent of copper concentrations in the diet. Hucker and Young (12) and Frandsen (11) showed that when hypocupremic sheep were challenged with the 3rd stage larvae of Trichostrongylus axei and Trichostrongylus colubriformis, the liver copper, plasma, and ceruloplasmin activities were decreased. They concluded that gastrointestinal nematodiasis could significantly exacerbate an existing copper deficiency in sheep. Most of the work reported to date on the relationship between endoparasitism and copper deficiency have been based on the oral copper supplementation (9,13,15). Underwood (17) suggested that copper is absorbed in the stomach and small and large intestines in the sheep. Gastrointestinal parasites were therefore thought to affect copper metabolism by interference with copper absorption from the gastrointestinal tract by increasing the pH of the gastric environment (15). According to this view, the effect of parasitism would be seen only when the copper supplementation is via the oral route. 1020
In the study reported here, the copper was administered, IM, but we were still able to show that parasitism depressed Hb and serum copper. Our results suggest that interference with absorption in the gastrointestinal tract is not the only mechanism by which the gastrointestinal parasites impede copper metabolism in the sheep. Whitelaw et al (18) reported that the periodic injections of parental copper at a dose-rate related to body weight did not maintain adequate normocupremia in lambs. Our study may provide a possible explanation for the observation, if the lambs were infected with helminth parasites. Our study also confirms the views of McPhee and Cawley (19), who showed that ewes treated with copper heptonate had significantly higher blood copper levels than did untreated control ewes. Copper deficiency is widely known to cause anemia. The decline in the Hb in parasite-infected animals was also reported in copper deficient sheep (20). Friedsen (21) has suggested that such anemia is indicative of concurrent interference with iron metabolism. Our studies conclude that parasitism negatively impacted on blood copper and Hb levels. The mechanism of parasite interference with copper may not be entirely by blocking the absorption of copper from the gastrointestinal tract, since the copper in this study was adminCVJ istered parenterally.
References 1. Suttle NF, Field AC. Production of swayback by experimental copper deficiency. In: Mills CF, ed. Trace Elements Metabolism in Animals. London: E & S Livingston, 1970:110–113. 2. Patterson DSP, Foulkes JA, Sweasey D, Glancyand EM, Terkecki S. A neurochemical study of field cases of the delayed spinal form of swayback (enzootic ataxia) in lambs. J Neurochem 1974;123: 1245–1253. 3. Dick AT, Dewey DW, Gawthorne JM. Thiomolybdates and copper — molybdenum — sulphur interaction in ruminant nutrition J Agric Sci 1975;85:567–568. 4. Youssef FG. A preliminary mineral blood profile of tropical goats and sheep in Trinidad. In: Mills CF, Bremner I, Chesters JK, eds. Trace Elements in Man and Animals (TEMA 5): TEMA: Farnham Royal, 1985:857–859. Can Vet J Volume 46, November 2005
5. Youssef FG, Brathwaite RA. The mineral profile of some tropical grasses in Trinidad. Trop Agric 1987;64:122–128. 6. Mohammed A, Adogwa A, Youssef FG. Pathological and mineral status investigation in quadriplegic lambs. Mol Chem Neuropathol 1995;24:257–261. 7. Alleyne T, Adogwa A, Lalla A, Joseph J, John R, Mohammed A. Novel mitochondrial proteins and decreased intrinsic activity of cytochrome-c-oxidase. Characteristics of swayback diseases in sheep. Mol Chem Neuropathol 1996;28:285–293. 8. Bremner KC. Parasitic gastro-enteritis and its effect on the blood and liver copper levels of dairy calves. Aust J Agric Res 1959;10: 471–486. 9. McCosker P J. Observations on blood copper in the sheep. Res Vet Sci 1968;9:91–97. 10. Ortolanie E, Knox D, Jackson J, et al. Abomasal parasitism lowers liver copper status and influences the copper, molybdenum and sulphur antagonism in lambs. In: Anke M, Meissner D, eds. Trace Elements in Man and Animals (TEMP 8). Verlag Media Touristik: Gersdorf, Germany, 1993:331–332. 11. Frandsen JC. Effects of concurrent subclinical infections by coccidian (Eimeria Chistenseni) and intestinal nematodes (Trichostrongylus colubriformis) on apparent nutrient digestibilities, serum copper and zinc, and bone mineralization in the pigmy goat. Am J Vet Res 1982;43:1951–1953. 12. Hucker DA, Young WK. Effects of concurrent copper deficiency and gastrointestinal nematodiasis on circulating copper and protein levels, liver copper and body weight in sheep. Vet Parasitol 1985;19:67–76.
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13. Bank KS, Familton AS, Sykes AR. Effect of ostertagiasis on copper status in sheep: A study involving use of copper oxide wire particles. Res Vet Sci 1990;49:306–314. 14. Ivan M. Effects of faunation on ruminal solubility and liver content of copper in sheep fed low or high copper diets. J Anim Sci 1988; 66:1496–1501. 15. Poppi DP, Sykes AR, Dynes RA. The effect of endoparasitism on host nutrition — the implications for nutrient manipulation. Proc NZ Soc Anim Prod 1990;50:237–243. 16. Copeman DB, Sumburg FP, Poothongtong P, Hag S, Armstrong JR. Effect of molasses supplementation and copper, cobalt and antihelmintic therapy on weight gain of beef yearling steers grazing improved pastures in a wet tropical environment. Aust J Exp Agric and Anim Husb 1977;17:538–544. 17. Underwood EJ. Trace elements in human and animal nutrition. New York: Acad Pr. 1977:56–108. 18. Whitelaw A, Russel AJ, Armstrong RH, Evans CC, Fawcett AR, Macdonald AJ. Use of cupric oxide needles in the prophylaxis of induced copper deficiency in lambs grazing improved hill pastures. Vet Rec 1989;112:382–384. 19. McPhee M, Cawley GD. Copper heptonate for the treatment of hypocupraemia in sheep. Vet Rec 1988;122:483–485. 20. Howell JM. The effect of experimental copper deficiency on growth, reproduction and haemopoiesis in sheep. Vet Rec 1968; 83:226–227. 21. Frieden E. Caeruloplasmin, a link between copper and iron metabolism. Adv Chem Ser 1971;100:292–321.
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