SCVMJ, X (1), 2006 17

Clinical and Haemato-Biochemical Studies On Cases Of Alope- cia In Sheep Due To Deficiency Of Some Trace Elements

Abd El-Raof, Y.M. and Ghanem, M.M. Department of Animal Medicine, Fac. Vet. Med., Benha University

ABSTRACT This study was carried out on twenty Balady sheep collected from two flocks of sheep at Kalubia Governorate. Ten sheep of them (from one flock) suffered from different degrees of alopecia, unthriftiness, lameness and diarrhea. The other ten sheep (from other flock) were clinically healthy and considered as control. Skin scrapings were taken from alopecic sheep for microscopic exami- nation after addition of KOH 10%, but there were no mites. Two blood samples were obtained from each animal. The first sample was collected with anticoagu- lant for estimation of blood picture, while the other was collected without anti- coagulant for separation of serum on which some biochemical analysis was per- formed. Alopecic sheep showed a significant decrease (P ≤ 0.01) of haemoglobin and erythrocytic count, while total leucocytic count did not change significantly. Differential leucocytic count indicated a non significant decrease of lymphocytes and a non-significant increase of neutrophils. Bio- chemical analysis of serum showed a highly significant decrease (P ≤ 0.01) of copper and zinc and a significant decrease (P ≤ 0.05) of manganese, iron, total proteins, albumin and A/G ratio. On the other hand, there was a significant in- crease (P ≤ 0.05) in serum ALT, AST and urea. It was concluded that alopecia syndrome commonly occurs due to multiple trace element deficiencies, particu- larly copper and zinc and deficiency of a single element seldom occurs under field condition. INTRODUCTION tant factors in the aetiology of alope- Deficiency of hair or wool in com- cia in sheep. parison to normal pilosity of the skin Among the important nutritional area is referred to as alopecia. There factors causing alopecia are deficien- are two kinds of alopecia; one is cies of micro and macro-elements caused by follicle dysfunction and the (Akgul et al., 2000). Alopecia and de- other by injury to the fiber as ring- ficient fiber growth are consistent out- worm and trauma. The capacity of the comes of deficiencies of biotin, ri- follicular epithelium to produce a boflavin, pyridoxine, folate and pan- fiber may be congenitally defective or tothenic acid. Copper has direct ef- may temporarily be reduced because fects on the activity of an unidentified of nutritional deficiency (Radostits et enzyme on oxidation of thiol groups al., 2000). Bacteria, virus, parasites, to form disulphide linkage, so wool in toxic agents, metabolic disorders and copper deficient sheep becomes weak, nutritional insufficiencies are impor- lustrous and lack crimps. Moreover, zinc like copper is required for the 18 Abd El-Raof and Ghanem normal keratinization of fibers (Hynd, several metalloenzymes and other 2000). Deficiency of a single trace el- metalloproteins (such as ceruloplas- ement rarely occurs in domestic ani- min, superoxide dismutase, cy- mals in the field, however a combina- tochr-ome c oxidase, lysyl oxidase, tion of mineral deficiencies are more and metallothionein) (Minatel and common (Hidiroglou, 1980). Carfagnini, 2002). Primary copper Zinc and Copper are very important deficiency developed when the copper for sheep health and production. Zinc content of the ration is less than ani- is a constituent of numerous met- mal requirements. Secondary defi- aloenzymes and required for normal ciency produced when copper of the protein synthesis and metabolism ration is marginal but absorption and (Church and Pond, 1988). Zinc defi- utilization of ingested copper is im- ciency may be primary due to inade- paired by other minerals (Wikse et al., quate levels in the ration or secondary 1992). These antagonistic minerals in- as a result of the presence of a sub- clude molybdenum, sulfur and iron stance interfering with its absorption (phillipo et al., 1987) or metabolism, in spite of the normal The symptoms of copper deficiency diet concentration (Wikse et al., in sheep include wool abnormalities 1992). as the fine wool becomes limp and Clinical signs of zinc deficiency in- glossy and losses its crimps. More- clude alopecia and thickening or kera- over, the black wool showed depig- tinization of epithelial cells. Addition- mentation. Additional signs included al signs included growth retardation, anaemia, scouring, swayback and swelling of the coronet, hock and knee bone deformities (Radostits et al., joints, rough coat and congestion of 2000). Copper deficiency affects vari- the eye mucous membrane (El-Attar, ous physiological characteristics that 1979, Church and Pond, 1988, and may be important in immunological Radostits et al., 2000). In zinc defi- defense against pathogenic challenge ciency, there was an elevation in (Stable et al., 1993). Copper deficien- haematocrit value. However, leukope- cy induces hypochromic macrocytic nia and lymphocytopenia were record- anemia (Church and Pond, 1988). ed in albino rats (Macapinlac et al., Haemoglobin level and erythrocytic 1966 and Miller et al., 1968). More- count were depressed in advanced over, in zinc deficiency, there was a cases of primary copper deficiency reduction in serum glucose, while, (Radostits et al., 2000). there was an increase in serum lipids, This study was planned to reach a AST and bilirubin values (Garcia- correct diagnosis of alopecia syn- Partida et al., 1985). drome among sheep depending on the Copper is required for the activity of clinical and laboratory examinations, enzymes associated with ferrous me- which included skin scraping, bio- tabolism, elastin and collagen forma- chemical analysis of serum to estimate tion, melanin production and integrity the levels of some blood parameters of central nervous system (McDonald and some liver and kidney function et al., 1984). Copper is a cofactor of parameters. SCVMJ, X (1), 2006 19

and AST (Reitman and Frankel, 1957) and urea (Patton and Crouch, MATERIALS AND METHODS 1977). Serum iron, zinc, copper and Animals and experimental design magnesium were estimated by atomic absorption spectrophotometer AAS, N The present investigation was car- as described by Meret and Henkin ried out on ten diseases Balady sheep (1971). (3-6 years old) of both sexes from a flock of sheep at Kalubia Gover- Electrophoresis of sera norate. Diseased individuals showed The sera were freshly used for elec- different degrees of alopecia, un- trophoresis of serum proteins by thriftiness, lameness and diarrhea. An- Agarose Gel electrophoresis accord- other ten clinically healthy Balady ing to Alper (1974). sheep related to another flock at the same locality fed on balanced concen- Statistical Analysis trate mixture and roughage were Statistical analysis was performed served as a control group. according to Petric and Waston (1999). A student t test was performed Skin scrapings to compare values in the diseased Skin scrapings were taken from group to the control group. Data were alopecic areas. The scraped material represented as means ± standard error was mixed with 10% potassium hy- of the mean. The difference between droxide and then examined micro- the means was considered significant scopically for detection of mites (Kel- if P ≤ 0.05. ly, 1984). RESULTS AND DISCUSSION Haematological analysis Careful clinical examination of dis- One ml of blood was withdrawn eased sheep revealed the presence of from jugular vein into heparinized test alopecia at the neck, shoulder and ab- tubes. Haemoglobin concentration domen, easily detached wool, rough (Hb), erythrocytic (RBCs) count, wool (Fig. 1 and Fig. 2), steely wool white blood cell (WBCs) count and Fig. (3) and achromatrichia (wool dis- the differential leucocytic count were coloration). Also, there was enlarge- estimated according to Coles (1986). ment of knee joint (Fig. 4). Additional Biochemical analysis signs included inappetance, poor Five ml of blood were withdrawn by growth, illthrift, pale mucous mem- puncturing the jugular vein, into test brane, lameness, staggering gait and tube without anticoagulant, left to clot diarrhea. These signs were nearly sim- and centrifuged for serum separation. ilar to those recorded by Fahmy et al. Commercially available diagnostic (1980), Taha et al. (1993), Radostits kits were used for colorimetric deter- et al. (2000); Metwalli et al. (1997) mination of serum calcium (Glinder and Mobarak (1998). Wool abnor- and King, 1972), inorganic phospho- malities were usually related to defi- rus (El-Merzabani et al., 1977), total ciency of copper and zinc (Church proteins (Doumas et al., 1981), ALT and Pond, 1988). The alopecia and 20 Abd El-Raof and Ghanem the loss of crimp (steely wool) are at- values of total leucocytic count tributed to defective keratinization showed a non-significant change. This (Davis and Mertiz, 1987 and Linder, result was coincided with those of 1991). The polypeptide chain of ker- Mobarak (1998). Differential leuco- atin fibers are cross-linked by disul- cytic count indicated a non-significant phide bonds which are formed by oxi- decrease of lymphocytes and a non- dation of the –SH group of the cys- significant increase of neutrophils. teinyl residue present in the polypep- This result was nearly similar to those tide chain. This process is copper-de- mentioned by Arthington et al. pendent and affects the chemical and (1996). physical properties of wool and hair Biochemical analysis of serum sam- (Linder, 1991 and Frank et al., ples of alopecic animals (Table 2) re- 2000). Locomotor disturbance and vealed a highly significant decrease in lameness with enlar-gement of hock copper and zinc levels, while a signifi- and knee joints might be regarded to cant decrease of manganese and iron deficiency of manganese, zinc and was recorded. Similar results were ob- copper (Mobarak, 1998). It has been tained by Fahmy et al. (1980) who found that copper deficiency inter- recorded a significant decrease in the feres with osteoblast activity in bone levels of copper, zinc, manganese and because of the inactivation of lysyl iron in sheep with alopecia and wool oxidase activity, which is a copper-de- eating habit. Moreover, Ali (2000) re- pendent enzyme (Goonratne et al., ported a significant decrease of the 1989). Unthriftiness and anemia values of serum copper, iron, zinc, (manifested by paleness of ocular and cobalt and manganese in sheep show- oral mucous membranes) could be at- ing alopecia. On the other hand, tributed to deficiency of iron, zinc and serum calcium and inorganic phos- copper (Radostits et al., (2000). phorus showed a non-significant Result of skin scraping demonstrat- change. These results coincided with ed no mite infestation and therefore those of Farahat (1994). hematological and biochemical analy- The values of serum total proteins sis were subsequently conducted for and albumin (Table 2) recorded a sig- further investigation to reach accurate nificant decrease, which was followed diagnosis. by a compensatory increase in the Results of blood picture (Table 1) globulin levels. This result was nearly indicated a significant decrease of the similar with those of Akgul et al. values of haemoglobin (Hb) and ery- (2000); Radostits et al. (2000) and throcytic count (RBCs). This decrease Mobarak (1998). This decrease of to- might be due to disturbance in the reg- tal proteins and albumin might be at- ular metabolism of iron as copper de- tributed to inappetence and albumin ficiency decreases the absorption of loss as a result of increased capillary iron, releasing of iron from body permeability in copper deficient and stores and utilization in haemoglobin alopecic animals, O’Dell (1976) and synthesis (McDonald et al., 1984 and Rucker and Tinker (1977). The activ- Church and Pond, 1988). The mean ities of serum alanine aminotrans- SCVMJ, X (1), 2006 21 ferase and aspartate aminotransferase duction in the activity of superoxide (Table 2) increased significantly in dismutase that enhances the oxidative alopecic sheep. The increased activity stress within hepatic cells. of liver enzymes indicates liver dam- Serum urea concentration (Table 2) age. This could be explained by the shows a significant increase in results of Randhawa and Brar (1998) alopecic sheep. This increase in urea who demonstrated fatty changes concentration suggests that trace ele- around the central vein in the liver of ments deficiency, particularly copper hypocupraemic sheep. It has been is associated with kidney dysfunction found that absorbed copper appears in sheep. The exact mechanism by first in plasma as cupric ion loosely which copper alters kidney function is bound to albumin. During hepatic not fully understood. However, Rand- synthesis of ceruloplasmin, copper is hawa and Brar (1998) observed vac- bound to this metaloprotein, which is uolar degeneration and coagulative then released to general circulation necrosis in the kidneys of sheep with (Scheinberg and Sternlieb, 1960). Ul- hypocupraemia. Consequently, cha- timately cuproprotein is present in nges in kidney function could be at- brain, erythrocyte and liver as cere- tributed to these histopathological brocuprein, erythrocuprein and hepa- changes. tocuprein, respectively (Scheinberg In summary, alopecia and wool ab- and Sternlieb, 1960). Therefore, it is normalities observed in sheep were at- possible that copper deficiency re- tributed to copper and zinc deficiency. duces the hepatocuprein in liver and In addition, deficiency of iron and adversely affects liver function. In ad- manganese occurs concurrently sug- dition, zinc is essential for maintain- gesting that nutritional deficiency ing the activity of superoxide dismu- problem is mostly caused by deficien- tase, a copper-dependant enzyme, in cy of more than one element and defi- the liver that function as antioxidant ciency of a single element seldom oc- (Sidhu et al., 2005). Ther-efore, cop- curs under field condition. per and zinc deficiency results in re-

Fig. (1): A sheep showing scattered areas of alopecia at neck, shoulder and abdomen (black arrows). 22 Abd El-Raof and Ghanem

Fig. (2): A sheep showing alopecia (black arrow) and rough wool.

Fig. (3): (A) showing steely wool, and (B) demonstrating wool with normal crimps from apparently healthy ram.

(A) (B)

Fig. (4): A lamb showing loss of hair at the knee joints (arrows).

Table (1): Haemoglobin, erythrocytic count, leucocytic count and differen- tial leucocytic count in alopecic and control sheep. Parameter Control sheep Alopecic sheep Hemoglobin (Hb) gm/dl 12.60 ± 0.24 8.40 ± 0.47** Erythrocytic count (RBCs) 106/C mm 11.97 ± 0.25 8.16 ± 0.19** White blood cells count (WBCs) 103/C mm 9.30 ± 0.25 8.87 ± 0.17 Lymphocytes % 55.60 ± 2.30 52.30 ± 1.90 Monocytes % 2.40 ± 0.28 2.70 ± 0.33 Neutrophils % 40.20 ± 2.10 43.10 ± 2.10 Eosinophils % 1.80 ± 0.35 1.90 ± 0.29 Basophils % 0 0 SCVMJ, X (1), 2006 23

The statistical test is the student t test. Data are presented as means ± SE ** denotes highly significant different from control at P ≤ 0.01 * denotes significant different from control at P ≤ 0.05 Table (2): Some biochemical parameters in the sera of alopecic and control sheep. Parameters Control sheep Alopecic sheep Copper (ug/dl) 95.66 ± 3.1 38.30** ± 6.32 Zinc (ug/dl) 95.7 ± 2.85 60.63** ± 5.27 Manganese (ug/dl) 3.80 ± 0.37 2.96* ± 0.15 Iron (ug/dl) 105.75 ± 5.60 81.08* ± 4.55 Calcium (mg/dl) 9.62 ± 0.58 9.80 ± 0.51 Inorganic phosphorus (mg/dl) 5.91 ± 0.21 6.14 ± 0.31 Total proteins (gm/dl) 6.38 ± 0.33 5.04* ± 0.28 Alanine aminotransferase (u/L) 52.71 ± 1.13 88.10* ± 2.59 Aspartate aminotransferase (u/L) 38.96 ± 2.22 59.89* ± 3.00 Urea (mg/dl) 32.45 ± 2.15 48.58* ± 3.16 The statistical test is the student t test. Data are presented as means ± SE ** denotes highly significant different from control at P ≤ 0.01 * denotes significant different from control at P ≤ 0.05 Table (3): Serum electrophoretic pattern in the sera of alopecic and control sheep. Parameters Control sheep Alopecic sheep Albumin (%) 37.28 ± 1.23 33.26* ± 0.92 Alpha globulins (%) 18.62 ± 0.90 20.12 ± 1.16

1-globulin (%) 8.10 ± 0.65 9.41 ± 0.42

2-globulin (%) 10.52 ± 0.67 10.71 ± 0.83 Beta globulins (%) 11.64 ± 0.78 12.15 ± 0.83

1-globulin (%) 5.76 ± 0.54 6.83 ± 0.66

2-globulin (%) 5.88 ± 0.36 5.32 ± 0.41 Gamma globulins (%) 32.45 ± 1.38 34.35 ± 1.18

1-globulin (%) 24.28 ± 1.04 26.35 ± 0.85

2-globulin (%) 8.17 ± 0.56 8.00 ± 0.44 Total globulins (%) 62.72 ± 1.23 66.62* ± 0.92 A/G ratio 0.59 ± 0.03 0.50* ± 0.02 The statistical test is the student t test. Data are presented as means ± SE ** denotes highly significant different from control at P ≤ 0.01 * denotes significant different from control at P ≤ 0.05 24 Abd El-Raof and Ghanem

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