COPPER POISONING: A CASE REPORT IN A SHEEP FLOCK IN SARDINIA XXII International Congress of Mediterranean Federation for Health and Production of Ruminant s Sassari, June 17th-20 th, 2015 COPPER POISONING: A CASE REPORT IN A SHEEP FLOCK IN SARDINIA Mulas M .(1), Boeddu L.(1), Cocco M. (1), Solinas C.(1), Tanda B.(1), Rosu V.(1), Manconi G. (2) , Bandino E. (1) Istituto Zooprofilattico Sperimentale della Sardegna “G. Pegreffi”, SC Diagnostico Territotoriale di Nuoro Associazione Regionale Allevatori della Sardegna INTRODUCTION Copper plays a number of important biological roles in animals through several Cu-dependent enzymes (Xin et al., 1991), in fact is an essential metal for cellular homeostasis and for the normal function of different animal body systems. High level copper concentrations in diet could lead to severe intoxication. Sheep have a strong ability to accumulate copper in their livers reduced rate of bile clearence more readly than do other animal species (Smith, 1996; Maxie, 2007). These factors make sheep one of the most susceptible species for copper toxicity (Sameh Youssef, 2009). The most common form of copper toxicosis in sheep is the chronic form (Fuentalba and Aburto, 2003; Maxie, 2007 and Mendel, 2007). Copper poisoning in sheep is characterized to gradual copper storage in the liver and its release into the blood stream due to stressors as transport or diet changing. This lead to severe intoxications, often fatal (Kimberling 1988 - NRC 1980). The aim of the present study is to describe a case of chronic copper poisoning occurred in a sardinian sheep flock, subsequent epidemiological investigation and the use of specific laboratory tests for a correct diagnostic approach. MATERIAL AND METHODS A case of high mortality occurred in the fall of 2014 in a flock of about 400 sheep bred extensively in Sardinia is reported. Within a few days died 35 adult animals after a short depressive symptoms. N. 3 head were promptly delivered to the Istituto Zooprofilattico della Sardegna (IZS) in Nuoro for further investigations. Autopsies were carried out in the 3 carcasses and various samples were collected to perform bacteriological, histological and toxicological assays. A first inspection was carried out on the farm and blood samples were collected from 5 head which showed clinical signs to carry out toxicologic test to detect heavy metals and to biochemical analysis. Ten days later during a second inspection further blood samples were collected from 6 sheep showing symptoms and from 4 head without any signs. Besides feed and water samples were collected to perform toxicologic tests. FIG. 1 FIG. 2 RESULTS AND DISCUSSION   Autopsy of the three sheep showed mucosae sub-icterus, serum-blood fluid nasal discharge, perineum and hind limbs smeared with very dark urine. Severe icterus in all districts of the organism. Hepatomegaly and yellow-orange liver pigmentation (Fig. 3); swollen, dark brown edematous kidneys (Fig. 4); greatly thickened bladder wall and presence of coffee grounds urine. Splenomegaly with softening of splenic pulp. The inspection carried out on the farm the day after, allowed to detect clinical symptoms on five head characterized by apathy, loss of appetite, head down, apparent mucosal icterus, perineal region and hind limbs stained by very dark urine (Fig. 1). A tank containing high concentrations of copper sulfate used as a foot bath for Foot rot’s prevention and treatment was situated at the exit of the milking parlor (Fig. 2). The tank, the only source of copper in the farm, was placed so that the animals would mandatory pass through it after milking to reach a contiguous paddock where they standed and had free access to the tank. Toxicological analysis revealed high concentrations of copper in liver, kidneys and blood (Table 1 and 2). Histological examinations in liver showed a lobular architecture disorganization, severe necrotic lesions mainly centrilobular accumulation of yellow-brown pigments in various hepatocytes and macrophages, lympho-macrophage inflammatory infiltrates (Fig. 5) and in kidneys revealed a tubulonefrosis haemoglobinuria (Fig. 6), confirming what has already been described by other authors (Sameh Youssef, 2009-Ortolani et al 2004 and Mendel et al 2007) Biochemical parameters showed an alteration in liver enzyme activities, with a significant increase in the values of GPT, GOT and GGT. For these latter parameters alteration of enzyme activity was found in three of five sheep which showed symptoms during the first inspection on the farm and in seven out of ten sheep, both sick and apparently healthy, taken in the second inspection (Table 3). Pathological lesions, biochemical profiles, histological and toxicological tests confirmed the hypothesized diagnosis of chronic copper poisoning. FIG.3 FIG.4 FIG.5 FIG.6 Biological sample Normal range Concentrations head 1 concentrations head 2 concentrations head 3 Liver 25-150 mg/kg 468mg/kg 455 mg/Kg 622 mg/Kg Kidneys 3-6 mg/kg 32 mg/kg 10,6 mg/Kg 48 mg/kg Copper in blood samples REFERENCE RANGE 0,6-1,5 µg/l HEAD 1 1475 µg/l HEAD 2 1623 µg/l HEAD 3 1359 µg/l HEAD 4 782 µg/l HEAD 5 602 µg/l HEAD 6 1014 µg/l HEAD 7 942 µg/l HEAD 8 580µg/l HEAD 9 917µg/l HEAD 10 1358µg/l HEAD 11 Not det. HEAD 12 HEAD 13 2121µg/l HEAD 14 1682µg/l HEAD 15 Normal Range ALB 2-3,5 g/dl ALP 45-250 U/L BT 0,15-0,65 mg/dl CRE 0,3-0,9 mg/dl GGT 60-120 U/L GOT 70-200 U/L GPT 15-45 U/L PRO 6-8,5 g/dl UR 25-60 mg/dl HEAD 1 1.7 g/dl 95 U/L 0.2 mg/dl 0.4 mg/dl 122 U/L 265 U/L 25 U/L 6 g/dl 15 mg/dl HEAD 2 175 U/L 0.8 mg/dl 145 U/L 852 U/L 74 U/L 5.8 g/dl 24 mg/dl HEAD 3 2 g/dl 117 U/L 0.5 mg/dl 3.1 mg/dl 223 U/L 343 U/L 17 U/L 7.2 g/dl 117 mg/dl HEAD 4 2.3 g/dl 56 U/L 0.1 mg/dl 0.6 mg/dl 103 U/L 170 U/L 24 U/L 8.2 g/dl 31 mg/dl HEAD 5 2.7 g/dl 105 U/L 30 U/L 93 U/L 20 U/L 6.7 g/dl 34 mg/dl HEAD 6 2.2 g/dl 193 U/L 0.7 mg/dl 190 U/L 404 U/L 54 U/L 7.1 g/dl 23 mg/dl HEAD 7 2.4 g/dl 246 U/L 311 U/L 305 U/L 34 U/L 7 g/dl HEAD 8 118 U/L 102 U/L 182 U/L 27 U/L 22 mg/dl HEAD 9 2.5 g/dl 129 U/L 180 U/L 261 U/L 38 U/L 7.6 g/dl 19 mg/dl HEAD 10 442 U/L 353 U/L 484 U/L 48 U/L 7.3 g/dl 13 mg/dl HEAD 11 Not Det. HEAD 12 2.6 g/dl 62 U/L 9.5 mg/dl 181 U/L 605 U/L 114 U/L 7.8 g/dl 81 mg/dl HEAD 13 140 U/L 6 mg/dl 480 U/L 805 U/L 58 U/L 8.4 g/dl 290 mg/dl HEAD 14 2.1 g/dl 1.2 mg/dl 224 U/L 796 U/L 7.5 g/dl 29 mg/dl HEAD 15 TABLE 1 CONCLUSION   The solution of copper sulfate used as a foot bath for Foot rot’s prevention can determine, accidentally, the onset of a chronic copper poisoning in sheep sardinian breed. We can confirm that this species is particularly susceptible to copper poisoning and that prolonged exposure to this metal can cause severe hemolytic crisis often lethal (Pouliquen H. et a. Douart 1999). The correct application of diagnostic protocol used has led to the diagnosis confirmation in a short time; the prompt elimination of the tank, source of the intoxication, and the treatment with hepatoprotectors led to a gradual symptoms resolution in involved animals and to an improvement of the general health status. TABLE 2 TABLE 3 ennio.bandino@izs-sardegna.it Istituto Zooprofilattico Sperimentale della Sardegna – SC Diagnostico Territoriale di Nuoro - Via F.lli Kennedy 2 Tel.: 0784-233899 – Fax: 0784-399006