Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
356
CHRONIC COPPER POISONING OF SHEEP AT BASRAH
GOVERNORATE, IRAQ
Hussein Ali Naji*, Jihad Abdulameer Ahmed** and Kamal M. Alsaad*
*Department of internal and preventive medicine, College of Veterinary Medicine, University of
Basrah, Basrah, Iraq
**Department of pathology and Poultry diseases,College of Veterinary Medicine, University of
Basrah, Basrah, Iraq
Keywords:Sheep, gall bladders, copper poisoning.
Corresponding Author: kamalsad58@yahoo.com
ABSTRACT
Chronic copper poisoning had been detected in sheep of Basrah governorate . The study was
conducted on (52) local sheep breeds. Forty two (42) local sheep breeds of different ages and of
both sexes with the indoor system of feeding at Basrah governorate, Iraq, show different clinical
manifestations belong to chronic copper poisoning. Ten clinical healthy local sheep breeds of
same ages were considered as controls. Complete clinical examinations have been applied to all
animals in the study. Diseased sheep show different clinical manifestations such as complete loss
of appetite (90.4%), depression, Weakness and Lethargy (78.5%), Increase abdominal respiration
(78.5%), Moreover, Pale and / or icteric mucous membranes were registered in (71.4%) of
animals when examining the vaginal mucous membranes, conjunctiva and nictitating membrane.
Furthermore, discolored urine of coffee like appearance was detected in (66.6%), salivation
(52.3%) and grinding of teeth (28.5%). Furthermore, clinical examinations of diseased animals
show a significant increase (P<0.05) of respiratory and heart rate. However ruminal contractions
and capillary refill time were decreased significantly (P<0.05). Evaluation of hemogram of
diseased sheep and controls indicated a clear anemia of macrocytic hypochromic type, which
occur due to a significant decrease (P<0.05) of the total erythrocyte count, hemoglobin
concentration and packed cell volume. However, total and differential leukocyte count values
were all within normal limit when compared with controls. A significant difference of copper
concentration in plasma, liver and kidneys was also indicated.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
357
Cross examinations of dead sheep reveal that the blood of dead animals was more darker than the
normal. Moreover, autopsied sheep show clear enlargement of the liver, which appear yellow,
fragile and brittle. However, gallbladders was distended and contain a dense, dark bile.
Furthermore, the little enlargement has been detected on both kidneys with dark appearance
which become a gun like. In addition, The histopathological results of autopsied animals
revealed a mild atrophied glomerulus with thickening of glomerular basement membrane.
Moreover, there is an accumulation of inflammatory cells near the glomerulus and between renal
cortical tubules. Additionally, a cystic like dilation in the renal interstitium was also detected.
Furthermore, a coagulative necrosis appeared in some renal tubules, as well to present of hyaline
like cast in the kidney. It has been concluded that chronic copper poisoning of sheep was a
dangerous type of disease with both high morbidity and mortalities as well as it's considered as
an economic loss.
INTRODUCTION
Copper is an important nutrient metallic mineral that needed in a very small amount for all
species of farm animals. Since, Is essential for life for different types of animal species, As, most
domestic animals have a dietary requirement for copper (1). Moreover, Copper is required for
normal physical and chemical processes that occur normally in the body, where, it's necessary
for erythrocyte formation and normal iron metabolism, as well as it aid in the production of both
hair pigments and skin or the melanin’s. Furthermore, copper is essential for the formation of
the myelin sheath that supports and protects the nerve structure of the central nervous system (2),
On the other hand, copper is also necessary for cross-linking collagen and elastin that is required
for normal bone formation. Additionally, copper is assisted in wool production and is involved in
the animal’s immune response to disease challenges (3).
Copper can also be poisonous if ingested in excessive amount that exceed the animal’s
requirement. There is a large variation in the amount of copper needed by different species of
farm animals. Similarly, there is a massive variation in the susceptibility to copper poisoning
among the farm animal species (4).
It had been documented that the sheep is the most susceptible farm animal species to copper
poisoning, However, the goats being less susceptible than sheep and cattle being less susceptible
than either sheep or goats (5). Poisoning, mostly occur when sheep or even the goats ingest small
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
358
amounts of copper over a prolonged time of period. Since, Copper that is ingested is always
stored in the animal’s liver, and repeated ingestion of small amounts of copper above the
animal’s requirement may cause accumulation of what finally becomes a toxic amount of copper
for the animal (6).
The most important sources of copper that might cause poisoning of animals are, trace mineral
supplemented salt that is formulated for cattle or horses, vitamin and mineral supplements
intended for horses, cattle, swine, or poultry, complete feeds for swine, horses, poultry, or cattle,
Pasture that has been fertilized with swine manure and poultry litter, copper-containing
disinfectant foot baths for cattle (1).
The most common type of copper toxicity in sheep is called chronic copper poisoning (when the
animal consume a doses of copper below the acutely toxic level) and is indeed the result of a two
stage process. The static accumulation of copper in the liver over a time and then, the release of
copper from the liver into the bloodstream (7). In fact, toxicity results when stress, such as a
sudden change of the weather, disease or transportation of animals causes the liver to release the
copper directly into the bloodstream with rapid and severe consequences, where, the breakpoint
upon which the liver can no longer keep or hold its copper load is hard to expect (8). Moreover,
Christodoulopoulos and Roubies (9) added that normally, copper will absorb from the diet and
transported in the blood circulation to the liver for storage. However, excess copper from the diet
is stored in the liver and is released into the blood as needed for regular body functions. The
circulating copper level tends to remain constant regardless of the amount of excess copper
accumulating in the liver. This makes it difficult to ascertain a sheep’s copper status.
When animals, especially sheep, affected with copper poisoning they are simply found dead.
However, the most obvious post mortem lesions includes jaundice and what call, the gun metal
blue kidneys. Furthermore, In the live animals, pale and also icteric mucous membranes, with
deep red or brown urine (coffee like), loss of appetite, weakness and recumbency are the
common clinical manifestations exhibited by diseased animals (10).
It had been mentioned that, treatment of copper poisoning is very difficult and may be
challenged in most toxic cases, Thereby, severely affected animals often die despite treatment.
Nevertheless, medications are administered to diseased animals in order to increase the rate of
excretion of the toxic copper from the liver. However, administration Oxygen therapy and other
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
359
supportive care may also require. On the other hand, feeding of sodium sulfate and ammonium
molybdate might help to reduce the rate of absorption of copper from the digestive system (9).
Sheep with signs of chronic copper toxicosis was suspected in Basrah governorate, therefore,
clinical and laboratory study was applied for diagnosis.
MATERIALS AND METHODS
Study design and animals :-
The study was conducted on (52) local sheep breeds. Forty two (42) local sheep breeds of
different ages and of both sexes with the indoor system of feeding at Basrah governorate, Iraq
show different clinical manifestations belong to chronic copper poisoning. Ten clinical healthy
local sheep breeds of same ages were considered as controls. Complete clinical examinations
have been applied to all animals in the study.
Diseased sheep show signs of complete loss of appetite, depression, increase abdominal
respiration, weakness, lethargy, polydipsia. Moreover, discolored urine of coffee like appearance
was detected. Pale and / or icteric mucous membranes were registered when examining the
vaginal mucous membranes, conjunctiva and nictitating membrane.
Collection of blood samples :-
Ten (10 ml) of blood was collected via jugular vein puncture from each sheep. Complete blood
analysis of EDTA blood, was used on an automatic full digital cell counter (Beckman, USA), to
evaluate the total erythrocyte count (TRBc), hemoglobin concentration (HB), packed cell volume
PCV, and total leukocyte count (TLC). However, differential leukocyte count was done using
Giemsa stain blood smears according to Weiss and Wardrop, (11).
Estimation of copper :-
Plasma copper concentration was estimated using Flame atomic absorption spectrophotometer
(FAAS) according to the procedure mention by, Xueping and Reny, 2002, (12). As, the sample
was digested by adding 2 ml of concentrated nitric acid (HNO3) and 1 ml of concentrated
Perchloric acid (HClO4) to 0.5 ml serum in Pyrex tube. Heated for 1 hour at 160 ºC using oil
bath, then samples cooled and the volumes were completed to 10 ml of 0.3 N HCl. The
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
360
absorbance of copper in standards and sample solutions were measured by flame atomic
absorption spectrophotometer at wavelength 324 nm .
Moreover, evaluation of copper in tissues was done according to method of Akinloye et al
,2011, (13), Since, 0.5 gm from the liver and kidneys was placed in a 250 ml digestion tube and
3.5 ml of concentrated sulfuric acid was added. The mixture was allowed to stand for 30 min at
room temperature, then 3.5 ml of 30% Hydrogen peroxide was added to the digestive tube and
then samples were heated by electro-thermal at 200 ºC for 45 minutes. After that they were
cooled and when the solution became clear, they were filtered twice through Whatman No. 42
filter paper and transferred to a 25 ml volumetric flask and adding distilled water to the volume.
Histopathological examination:-
Four sheep were dead after sampling, Thereby, tissue samples for histopathological examination
are taken from most of body tissues from autopsied sheep, Fixed at 10% buffered formalin
solution for 72 hrs, then trimmed to apposite and suitable sizes and washed, dehydrated and
cleared in xylol. In addition, the tissue samples were embedded in paraffin wax, after that it
sectioned at 4-5 μ thickness, stained with hematoxyline and eosin, and examined under a light
microscope (14).
Statistical analysis :-
Evaluation of statistics was done according to Leech, et al (15), using SPSS programs, the
student t- test.
RESULTS
Diseased sheep show different clinical manifestations (Table 1) such as, complete loss of
appetite (90.4%), depression, weakness and lethargy (78.5%), increase abdominal respiration
(78.5%). Moreover, pale and / or icteric mucous membranes were registered in (71.4%) of
animals when examining the vaginal mucous membranes, conjunctiva and nictitating membrane.
Furthermore, discolored urine of coffee like appearance was detected in (66.6%), salivation
(52.3%) and grinding of teeth (28.5%).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
361
Table 1: Clinical manifestations of diseased sheep with chronic copper poisoning
Clinical manifestation No.of diseased sheep %
Complete loss of appetite 38 90.4
Depression, Weakness and Lethargy 33 78.5
Increase abdominal respiration 33 78.5
Pale and /or icteric mucus membranes 30 71.4
Discolored urine of coffee like appearance was
detected
28 66.6
Salivation 22 52.3
Grinding of teeth 12 28.5
Moreover, clinical examinations of diseased animals show a significant increase (P<0.05) of
respiratory and heart rate compared to control. However, ruminal contractions and capillary refill
time were decreased significantly (P<0.05) in the diseased sheep when compared to control.
Table 2.
Table 2 : Clinical parameters of diseased sheep with chronic copper poisoning and controls
Parameters Control n= 10 Diseased sheep n= 42
Body temperature C 39.45 ± 1.26 39.23± 1.22
Respiratory rate / mint 24.38 ±7.53 88.32± 9.56*
Heart rate / mint 87.3 ±8.4 132.54±6.42*
Ruminal contractions/ 5 mint 3.2± 1.44 1.26± 1.33*
Capillary refill time / Sec 1.1±0.41 5.33±2.56*
Values are mean ± standard error of mean. * (P<0.05).
Evaluation of hemogram of diseased sheep and controls indicated a clear machrocytic
hypochromic anemia, which occur due to a significant decrease (P<0.05) of the total erythrocyte
count, hemoglobin concentration and packed cell volume. However, total and differential
leukocyte count values were all within normal limit in comparison with controls.Table 3
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table 3 : Hemogram of diseased sheep with chronic copper poisoning and controls
Parameters Control n=10 Diseased sheep n=42
TRBc x106 8.11± 1.76 5.56 ± 1.89 *
Hb g/dl 10.3 ± 1.73 6.51 ± 1.69 *
PCV % 30.12± 5.22 23.33 ± 3.71 *
MCV fl 37.13 ± 2.58 41.96±3.22 *
MCHC 34.19 ± 4.24 27.9 ± 4.38*
TLC x103 9.858 ± 2.45 9.504 ± 4.23
Nutrophils % 4122±777.38 4174.35 ± 423.21
Lymphocytes % 5320 ± 521.11 5421.3± 215.12
Eosinophiles % 170 ± 56.23 173 ± 62.92
Monocytes % 211± 84.21 201.75± 122.47
Basophiles % 35 ± 7.33 36.67± 17.32
Values are mean ± standard error of mean. * (P<0.05).
Furthermore, the results were also indicated that diseased sheep show a significant difference
(P<0.05), of the values of copper concentration in both plasma liver and kidneys, as mentioned
in table 4.
Table 4: Copper concentration in serum, Liver and kidneys of diseased sheep with chronic
copper poisoning and controls
Parameters Controls n= 10 Diseased sheep n= 42
Plasma Cu μg/dl 40.8±11.34 167.48±6.89
Liver Cu μmol/kg 2461±20.56 6475±16/78
Kidney Cu μmol/kg 253.11.54±8.12 744.72±8.12
Values are mean ± standard error of mean. ** (P<0.05).
Cross examination of dead sheep reveals that the blood of dead animals was more darker than the
normal. Moreover, autopsied sheep show a clear enlargement of the liver, which appear yellow,
fragile and brittle. Moreover, the gallbladders was distended and contain a dense, dark bile,
furthermore, little enlargement has been detected on both kidneys with dark appearance which
become a gun like. Fig 1.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
363
Fig. (1): A gun like appearance kidney of chronic copper poisoning sheep
However, histopathological results of autopsied animals revealed a mild atrophied glomerulus
with thickening of glomerular basement membrane. Moreover, there is an accumulation of
inflammatory cells near the glomerulus and between renal cortical tubules. In addition to a cystic
like dilation in the renal interstitium. Furthermore, a coagulative necrosis appeared in some renal
tubules, as well to present as to hyaline like cast in the kidney. Fig. (2) and Fig.(3).
Figure (2): Histopathological section of the kidney in the chronic cooper poisoning group showed
mild atrophied glomerulus with thickening of glomerular basement membrane (blue arrows), also
there is an accumulation of inflammatory cells near the glomerulus and between renal cortical
tubules (black arrow), in addition to cystic like dilation in the renal interstitium (green arrow).
H&E stain. 10X
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
364
Figure (3): Histopathological section of the kidney in chronic cooper poisoning group showed
coagulative necrosis in some renal tubules, as well to present of hyaline like cast (black arrow).
H&E stain. 40X.
On the other hand, results of histopathological sections of the liver revealed a multiple petechial
hemorrhages in the liver parenchyma with dilation of sinusoids with the vacuolation of some
mid-zonal hepatocytes. Fig (4) and Fig. (5).
Figure (4): Histopathological section of liver in chronic cooper poisoning group showed active
macrophage in the central vein (black arrow), as well to dilation of sinusoids with the vacuolation of
some mid-zonal hepatocytes (blue arrows), also there are pyknotic nuclei of hepatocytes (green
arrows) . H&E stain. 10X.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
365
Figure (5): Histopathological section of liver in chronic cooper poisoning group showed multiple
petechial hemorrhages in the liver parenchyma (black arrows), also there is a cytoplasmic
vacuolation (blue arrows). H&E stain. 40X.
DISCUSSION
To our knowledge, this is the first record for chronic copper toxicity at Basrah Governorate, Iraq.
Sheep are the most susceptible species to chronic copper toxicity, because their liver cells have a
high affinity for copper and they excrete copper into the bile at a very low rate, leading to a
build-up of liver copper concentration over time (1,2). Furthermore, Sheep are more susceptible
to the effects of copper toxicity than other species of farm animals because their copper
excretory mechanism is less efficient (16).
Chronic copper poisoning is essentially a problem of sheep under practical farm conditions,
Since, sheep accumulate copper in their livers when fed copper slightly in excess of
requirements. Sheep of all ages are affected but a higher incidence is reported in housed than in
pastured sheep.( 5,17). One of the most common causes of toxicity in sheep is the accidental
feeding of foodstuffs intended for other livestock (18). It had been documented that
Molybdenum will reduce the accumulation of copper in the liver, Thereby, the ratio of copper to
molybdenum in the feed is, therefore, an important factor determining the risk of copper
poisoning (4, 19). On the other hand, It had been shown that sheep could be protected from
chronic copper poisoning by increasing the crude protein intake, As, high protein might not be
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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the only factor in decreasing copper storage, Since, the supplemental protein, contained high
molybdenum levels which could affect the metabolism and retention of copper (20).
Chronic copper toxicity were described in sheep grazing on pasture and the conditions classified
according to their primary effect on sheep, as, phytogenic chronic copper poisoning occurred
when sheep consumed forages with an imbalance of copper and molybdenum, This type of
poisoning was related to lush growth of specific plants growing at early autumn, These plants
contained little or no molybdenum and sheep grazing on them stored high levels of copper in the
liver which predisposed them to the hemolytic crisis of chronic copper poisoning(2,7,19).
Diseased sheep show different clinical manifestations which will mentioned by others
(6,9,10).There are two recognizable phases in chronic copper poisoning, an asymptomatic phase
when copper is accumulating in the liver and a toxic phase of an acute hemolytic crisis.
Kumaratilake, 2014(1) and and Villar et al , 2002 (2), recorded a loss of appetite, fatigue and
difficulty breathing at the onset of hemolytic crisis. Extensive hemolysis resulted in the presence
of free hemoglobin in plasma and urine.
Results of hematological parameters indicated a clear anemia of diseased sheep which
manifested with macrocytic hypochromic cells of diseased sheep compared with controls, the
same results were also indicated by (21,22), Since anemia and weakness are the most common
clinical signs, As it were documented that, In copper toxicosis, the concentration of copper
increased suddenly, causing lipid peroxidation and intravascular hemolysis. A hemolytic crisis
may be promoted by many factors including transportation, pregnancy, lactation, strenuous
exercise, or a deteriorating plane of nutrition. (6,7).
Methemoglobinemia occurs and may be the cause of the chocolate brown color of the blood and
mucous membranes at the start of hemolysis. The mucous membranes become jaundiced, and
this is more prominent if the animals survive for over two days. The temperature may or may not
be elevated. Death from consumption of a large dose of copper is rapid and preceded by diarrhea,
dyspnea and marked weakness(9,21).
Copper is a strong oxidizing agent. It binds to proteins and stored within the hepatocytes (1). As
the copper remains stored in the hepatocytes it does not cause any tissue damages, However,
copper could be released at the times of stress, including a sudden change of the weather, at the
time of shearing and animal transportation. Therefore, chronic copper poisoning could be
described as a stress-related disease (2,6). Moreover, when copper enters the blood, it will
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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fractionated with the red cells, Thereby, elevating erythrocyte copper levels more than 20 times,
whereas plasma copper levels only will increased more than 2-3 times, Therefore, it results in
oxidative injury to hemoglobin, inducing erythrocytes inclusions such as, Heinz-body formation
and converting the hemoglobin to methemoglobin, which cannot bind the oxygen,( that why
diseased animals will suffer from increase respiratory rate with increased abdominal respiration
as mentioned in the results of the current study). Moreover, The sulfhydryl groups of the
erythrocyte membrane also might have an oxidative changes, resulting in significant hemolysis
and anemia. Finally, this massive release of hemoglobin can result in hemoglobinuric nephrosis
and renal failure.(21,22).
Chronic copper toxicity was related mostly to the ingestion of feeds that have a high copper -
molybdenum ratio, Since, any feed which have copper levels more than 25-30 ppm is will
strongly consider as a toxic food for sheep. (19). In addition, when copper is ingested in large
amounts in the diet, it may accumulate within the liver over a period of a few weeks to more than
a year without clinical signs followed by a sudden release of liver copper stores with final result
in the state of toxicity, As different factors could alter the copper metabolism and can influence
chronic copper toxicosis by enhancing the absorption or retention of the copper (9)
Studies of hepatic histochemistry of copper loaded animals showed sequestration of excess
copper in the lysosomes, However, It was observed a diffuse copper distribution in the
hepatocyte in early stages whereas in later stages deposits observed near the bile canaliculi and
in the Kupffer cells were in lysosomes(2,3).
Degenerative changes of hepatocytes were observed early in the course of copper poisoning , As
centrilobular necrosis of the parenchymal cells was an early observation and corresponded to
areas of increased acid phosphatase activity and copper deposition (4,8).Moreover, swollen
Kupffer cells were also seen early, and they exhibited increased acid phosphatase activity and
copper deposition. Massive necrosis in the centrilobular and periportal areas was evident in
hemolytic crisis and changes in Kupffer cells were more pronounced. Such massive necrosis may
be associated with copper release into the bloodstream. Centrilobular necrosis observed in
chronic copper poisoning were also observed in experimental acute copper poisoning without
elevated hepatic copper levels (14,18).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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