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Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 51 Catharanthus roseus Toxicity in Sheep
Clinical and Pathological Investigations of Accidental
Catharanthus roseus Toxicity in Sheep
Aydogan, A.,
1
* Sezer, K.,
3
Ozmen, O.,
1
Haligur, M.
2
and Albay, M.K.
3
1
University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine, Department of Pathology, Istiklal Yerleskesi, 15030,
Burdur-Turkey.
2
University of Cukurova, Faculty of Ceyhan Veterinary Medicine, Department of Pathology, 01000, Adana-Turkey.
3
University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine, Department of Internal Medicine, Istiklal Yerleskesi,
15030, Burdur-Turkey.
*
Corresponding Author: Assoc. Prof. Ahmet Aydogan, DVM, PhD, University of Mehmet AkifErsoy,Faculty of Veterinary Medicine, Department of
Pathology, 15030, Istiklal Yerleskesi, Burdur-Turkey. Telephone: +90248 2132173, Fax: +902482132005. E-mail: aaydogan@mehmetakif.edu.tr
ABSTRACT
Clinical signs, blood parameters, postmortem and histopathological fndings in sheep that were accidentally
poisoned with Catharanthus roseus are described. Afected animals (40 sheep) showed acute clinical signs
such as salivation, dyspnea, anorexia, bloody diarrhea and dehydration. Blood samples were collected from
5 sheep prior to death. Marked increase in Activated Partial Tromboplastin Time (APTT), D-DIMER,
hemoglobin, urea and creatinine levels with the decrease in cholinesterase activity and calcium levels were
observed in the blood parameters of afected sheep. Necropsy examination was performed in17 sheep and all
organs were examined pathologically. According to blood parameters and pathologic fndings, Disseminated
Intravascular Coagulation (DIC) was observed in diferent organs supported by the increases in APTT,
Prothrombin time (PT) and D-dimer levels in blood. Grossly, linear mucosal hemorrhages in the jejunum and
ileum, auricular petechial hemorrhages in the heart were common fndings. Te livers were pale and friable.
Microscopically, the fbrinous exudate consisting of fbrin networks were seen in the lumen of jejunum and
ileum. In addition, clumps of fbrin in many small vessels were noted in the brain, lungs, liver, kidneys and
intestines. Focal periacinar and mid-zonal necrosis with hemorrhages were observed in the liver. Marked
tubular necrosis was seen in the kidneys.
Keywords: Catharanthus roseus; Pathology; Blood Parameters; Accidental Toxicity; Sheep
INTRODUCTION
Catharanthus roseus (Madagascar periwinkle) or previously
known as Vinca rosea is a herb of the Apocynaceae family
with green leaves and pale pink to white fowers (1-2). Te
plant contains alkaloids such ascartharathine, lochnenine,
vindoline, vindolinenine, vincristine, vinblastine, tetrahydroal-
stronine, reserpine and serpentine (3). Especially, vincristine
and vinblastine are used as chemotherapeutic drugs for some
forms of cancer such as lymphomas and testicular carcinoma
(4). Tese drugs can induce axonal degeneration in cancer
patients (5). In addition, the organic extract of C. roseus is
used in some countries for treatment of diabetes, malaria,
wasp stings, sore throat, eye irritation, low blood pressure,
insomnia and infections (2). Some cases of neurotoxicity
have been previously reported with the use of vincristine in
patients with acute lymphoblastic leukemia (6). According
to another report the use of vincristine at normal dosage
resulted in the development of peripheral neurotoxicity in ten
out of 20 children. In these patients, weakness of lower limbs,
arefexia, neuropathic pain, sensory loss, constipation, urinary
retention, seizures, confusion, aphasia, transient blindness
and malnutrition were observed (7). In vincristine toxicity,
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Aydogan, A. 52
neuropathy of peripheral nervous system was reported in cats
and dogs, however, bone marrow, kidney and gastrointestinal
tract are also afected (5). Recently, toxicity studies with the
use of C. roseus have been reported in experimental animals.
In a study, remarkable liver damage was noted in rabbits after
taking 0.1 g/kg of aqueous leaf extract of C. roseus for 9 days
(8). In another study, the oral LD
50
value of methanol leaf
extract of C. roseus was reported as 2.1 g/kg in mice (9).
Prolonged treatment of repeated doses of 0.5 g/kg and 1.0
g/kg extract of C. roseus cause diarrhea and mortality in rats
(10).
Up until now, there have been no studies documenting
poisoning due to accidental ingestion of C. roseus in animals.
To the best knowledge of the authors, this is the frst study to
investigate the clinical signs, blood parameters and pathologic
fndings resulting from accidental acute poisoning by C. roseus
in sheep.
MATERIALS AND METHODS
In November 2012, the Burdur Municipality located in the
southwest of Turkey cultivated fowers of C. roseus in the city
gardens. After the fowers had faded, they were collected
from gardens by municipal gardeners. Due to the lack of
fresh grass in the winter season in Burdur, animal owners
usually feed their animals with these collected green plants.
Tese collected plants were fed ad libitum to sheep without
knowing that the plant was poisonous. Feeding of the leaves
and fowers of C. roseus to a fock of 40 sheep (31 female and
9 male) caused an acute toxicosis within 24 h of ingestion
of the plant with all animals manifesting salivation, incoor-
dination, staggering, recumbency, dyspnea, anorexia, bloody
diarrhea and dehydration. All the sheep died within two days
after the start of the signs.
For diagnostic purposes, 17 of the 40 sheep carcasses were
presented to Department of Pathology of the University of
Mehmet Akif Ersoy, Burdur-Turkey. In addition, speci-
mens of the plants were collected for identifcation by the
director of Municipal gardens. Necropsy was performed on
all presented sheep and all organs were examined grossly.
Tissue samples were taken from the organs and fxed in 10%
bufered formalin. Using standard methods, tissues were
blocked in parafn and cut to 5µm thickness. Sections were
stained with hematoxylin and eosin (H&E) and examined
microscopically.
After the death of 10 animals, the farm was visited and
blood samples were collected from 10 live afected ani-
mals. MS9 blood counting equipment (Melet Schloesing
Laboratories, France) was used for hematological analysis
of the blood drawn in EDTA tubes. Serum biochemical
analyses were made using by Dade Behring (Dade Behring/
Dimension RXL MAX, Germany) equipment and reagents.
In order to measure PT (Prothrombin time) and APTT
(Activated Partial Tromboplastin Time) levels, 9 ml blood
from each animal was collected in 1ml 3.8% sodium citrate
containing tubes and centrifuged at 1500 x g for 15 minutes.
Te plasma samples were separated from blood within 30
minutes, followed by blood collection. APTT and PT were
measured by Diagnostica Stago STA analyzer (USA).
Gesan Chem 200 autoanalyser (Campobello-Italy) was
used for detecting serum cholinesterase levels by Bireagent-
cholorimetric method.
RESULTS
C. roseus was identifed by its characteristic fowers and leaves
(Figure 1). Te blood parameters such as APTT, D-DIMER,
hemoglobin, urea and creatinine levels showed a marked in-
crease. In addition we found a severe decline in cholinesterase
activity and calcium levels in the blood of afected sheep
(Table 1.)
At the clinical examination, salivation, incoordination,
staggering, recumbency, dyspnea, anorexia, bloody diarrhea
and dehydration were the common clinical symptoms.
At necropsy the afected sheep were found to be in good
body condition. Poorly masticated leftovers of leaves of
Figure 1: Te plant C. roseus.
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Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 53 Catharanthus roseus Toxicity in Sheep
C. roseus were found in their mouths. Some carcasses showed
early and rapid autolytic and putrefactive changes especially
of the liver, brain, spleen and kidneys. Lesions were most
severe in the gastrointestinal tract. Te main macroscopic
fnding of gastrointestinal tract was linear mucosal hemor-
rhages in the jejunum and ileum together with watery to
mucoid diarrhea with or without blood. Masses of fbrin were
present in the form of cylindrical casts in the intestinal lumen
which tended to cause complete obstruction (Figure 2). Te
gut mucosa was hyperemic and edematous. Externally, the
serosa of the afected portion of ileum and jejunum was red in
color and blood vessels were congested. Severe hyperemia was
noted in the brain. Marked auricular petechial hemorrhages
were seen in the hearts of fve of the sheep examined. Te
lungs were dark purple-red in color, swollen and oozed a large
amount of sero-sanguineous fuid from the cut surface in six
of the sheep. Te livers were enlarged, pale and friable in all
afected sheep. Pale and swollen kidneys were commonly
observed.
At the microscopic examination, the fbrinous exudate
in the lumen of jejunum and ileum was found to consist
of a fbrin network with varying quantities of mucus, neu-
trophils and desquamated epithelial cells. Clumps of fbrin
Table 1: Blood parameters of sheep poisoned by C.roseus
Parameters Sheep Number References
Values (11)
1 2 3 4 5
APTT (s) 58.7* 64.5* 70.6* 44.4* 61.1* 25.92
PT (s) 51.7* 41.2* 51.9* 42.8* 45.9* 23.29
Fibrinogen (mg/dL) 378 220 454 248 350 200-500
D-DIMER(ng/mL) 953* 381* 781* 681* 316* 162-222
Cholinesterase (nmol/min/ml plasma) 350* 361* 377* 287* 475* 514-1465
ALT (IU/L) 39* 12 22 14 62* 22-38
ALP (IU/L) 350 96 99 191 231 70-390
AST (IU/L) 220 161 203 143 139 60-280
GGT (IU/L) 55* 54* 39 42 56* 20-52
Hemoglobin (g/dL) 17.2* 16.8* 15.5* 17.8* 15.8* 9.0-15.0
PCV (%) 29.6 38.36 37.63 35.12 38.17 27-45
RBC (x10
12
/L) 9.31 13.15 12.43 10.22 12.96 9.0-15.0
WBC (x10
9
/µL) 6.9 6.8 5.5 4.0 4.2 4.0-12.0
PLT (x10
9
/µL) 7.5* 66* 77* 112* 99* 250-750
Urea (mg/dL) 666* 828* 720* 576* 540* 72-122.4
Creatinine (mg/dL) 3.51* 6.76* 4.69* 2.04* 2.25* 1.2-1.9
T Bil (mg/dL) 0.10 0.11 0.13 0.12 0.15 0.1-0.5
D. Bil (mg/dL) 0.25 0.12 0.14 0.12 0.21 0-0.27
Ca (mg/dL) 4.15* 8.72* 6.34* 7.47* 3.25* 11.5-13.0
Mg (mg/dL) 2.25 2.98 3.53 2.63 2.12 2.2-2.8
P (mg/dL) 2.20 4.41* 5.86 2.58* 3.11 5.0-7.3
Sodium (mEq/L) 110* 142* 126* 111* 141* 145-152
* Abnormal results
Figure 2: Fibrin masses in the intestinal lumen which blocked
the lumen.
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Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Aydogan, A. 54
were present in the brain, lungs, liver, kidneys and intestines
in numerous small vessels. Many arterioles and capillaries
contained clumps of fbrin without evidence of organization
or invasion of fbroblasts.
Focal periacinar and midzonal necrosis and fatty degen-
eration with hemorrhages were observed in the liver (Figure
3). Te capillaries in the alveolar walls were congested with
the alveoli flled with eosinophilic material characteristic for
pulmonary edema (Figure 4). Hyperemia, edema and slight
mononuclear cell infltrations consisting of lymphocytes,
plasma cells and macrophages with extensive hemorrhages
of the villi, propria mucosa and submucosa were observed in
the other parts of intestines.
Acute tubular necrosis was commonly seen in the kid-
neys. Te presences of pyknosis and karyolysis with intense
eosinophilic homogenous cytoplasm in the tubular epithe-
lium are regarded as a characteristic feature for acute tubular
necrosis. Lumens of tubules contained necrotic cellular debris
and proteinous materials (Figure 5).
DISCUSSION
C. roseus is a medicinal plant from which secondary metabo-
lites are used for treatment of certain diseases. One hundred
and thirty alkaloids can be found in this plant and these may
cause toxicities in humans and animals in the course of treat-
ment (2, 12).Te alkaloid of C. roseus, vincristine cause vin-
cristine neuropathy and also afect other organs and systems
among them bone marrow, kidneys and the gastrointestinal
tract (5). A report documents a 67‐year‐old woman with
hepatitis C-related liver cirrhosis and hepatoma treated with
C. roseus as an alternative anticancer treatment which caused
severe bone marrow suppression. Vomiting, diarrhea, oral
ulceration, severe gastrointestinal disturbances, bacteremia,
urinary tract infection and fever were noted about 1 week
after a 5‐days' course of C. roseus. In addition,bone marrow
biopsy material showed rapid autolysis (13). In the present
study, early and rapid autolytic and putrefactive changes were
observed in some carcasses. Tis condition was thought to
be mainly due to bacteremia. In addition, some gastrointes-
tinal tract disturbances such as linear mucosal hemorrhages,
bloody diarrhea, dehydration, masses of fbrin in the lumen
of intestines were observed.
Vincristine neuropathy of the peripheral nervous system
Figure 5: Histopathologic appearance of acute tubular necrosis in
kidney, proteinous materials in tubules (arrows). H&E, bar = 50 µm.
Figure 4: Eosinophilic material in lungs that characteristic for
pulmonary edema. H&E, bar = 50 µm.
Figure 3: Periacinar and midzonal necrosis with hemorrhages in the
liver. HE, bar = 30 µm.
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Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 55 Catharanthus roseus Toxicity in Sheep
has been reported in vincristine toxicity (C. roseus) especially
in cats and dogs (5). In this study, poorly masticated remains
and leaves of C. roseus were found in the mouth of most
sheep the digestion of which may indicate the possibility of
neuropathic efects of this plants alkaloids in the sheep (14).
A histological study to ascertain the toxic efect of C.
roseus on liver and kidneys in New Zealand rabbits revealed
centrilobular hemorrhagic necrosis in the liver and glomeru-
lonephritis, without cardiac pathology (8). Generally, the
toxic efects of alkaloids are due to their metabolites produced
by biotransformation in the liver, and not by the alkaloids
themselves (15). In this study, periacinar or centrilobular
hemorrhagic necrosis were seen in the liver which may have
been due to the toxic efect of C. roseus alkaloids. In addition,
acute tubular necrosis in the kidney, auricular petechial hem-
orrhages in the heart and pulmonary edema were also noted.
Te APTT measures the integrity of the intrinsic and
common pathways of coagulation. D-dimer is a fbrin deg-
radation product, a small protein fragment present in the
blood after a blood clot is degraded by fbrinolysis. It is so
named because it contains two cross-linked D fragments of
the fbrinogen protein. D-dimers are not normally present in
blood plasma, except when the coagulation system has been
activated, for instance due to the presence of thrombosis or
disseminated intravascular coagulation (DIC) (16). APTT
and D-dimer levels were elevated in all fve examined animals
compared to reference levels. Lower cholinesterase activity
levels were attributed to central nervous system damage (17).
Marked increase of the urea and creatinine levels supported
kidney failure.
According to pathological fndings a process in which
DIC may have been present in the diferent organs was ap-
parent. Tese included sinusoidal platelet or fbrin thrombi,
hemorrhages and focal necrosis in the liver; acute focal
tubular necrosis in the kidneys; capillary platelet and fbrin
thrombi with focal infarcts in the brain; hemorrhages, focal
ulcers and gastroenteritis in the gastrointestinal tract; capil-
lary fbrin thrombi and alveolar hemorrhages in the lungs
(18). We also observed capillary fbrin thrombi in the liver,
brain and lungs and focal periacinar and midzonal necrosis
and hemorrhages in the liver. At necropsy, hemorrhages and
enteritis were observed in the gastrointestinal tract along with
fbrin aggregates in jejunal and ileal lumen. Pulmonary edema
was observed in the afected lung while changes observed in
the kidney included acute tubular necrosis. It is considered
that these fndings may have occurred due to DIC efects
of the alkaloids of C. roseus. Clinical and pathological fnd-
ings showed that coagulopathy was a marked fnding in this
poisoning.
DIC diagnosis can be made based on the various tests
results (19). Traditionally diagnosis of overt DIC in animals
include a combination of two or more test abnormalities, spe-
cifcally thrombocytopenia, prolonged coagulation times (PT
and APTT), hypofbrinogenemia, low antithrombin activity
and high fbrin degradation product or D-dimer (20). DIC
can be diagnosed when at least three of fve tests included in
a coagulation profle are abnormal (21). Te most common
cause of prolonged APTT and PT is increased consumption
of clotting factors during DIC (22). In this study, higher
APTT, PT and D-dimer levels supported DIC formation.
To the best knowledge of the authors no study of C. roseus
poisoning in sheep in the veterinary medicine literature has
been documented. In the present study, accidental C. roseus
poisoning of blood parameters and pathological efects were
investigated in sheep.Tis study demonstrates that C. roseus
can cause toxicosis in sheep resulting in mortality probably
due to DIC and circulatory disturbances related to diarrhea
and dehydration. In conclusion, results of this study suggested
that the diagnosis of this poisoning depends upon a combina-
tion of anamnesis, clinical signs, laboratory parameters and
pathological examination.
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