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Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med.121
KEY WORDS: aflatoxicosis, aflatoxin B1,
diagnostic model, evaluation, sheep
ABSTRACT
The aim of this research is to evaluate a
diagnostic model for uncovering an afla-
toxicosis outbreak in sheep. The diagnostic
model is based on differentiation between
an aflatoxicosis suspected herd (ASH) and
a control herd (CH) including, clinical find-
ings, feed analysis for aflatoxin and other
nutrients, hemato-chemical and biochemical
profiles, histopathologic changes, and re-
sidual aflatoxin levels in their organs. There
was a significant difference between the
sheep of the ASH and CH in relation to mor-
tality percent, symptoms, aflatoxin B1 level
in their feed and tissues, hematological and
biochemical parameters, liver and kidney
enzymes and metabolites, serum electro-
lytes, and vitamins A and E. The relationship
of the histopathological lesions of affected
Evaluation of a Diagnostic Model for
Aflatoxicosis in Sheep: A Prerequisite for
Future Adoption of National Surveillances
E.K. Barbour1
,*
M.E. Abou-Alsaud2
N.A. Gheith3
M.A. Abdel-Sadek2,4
H.M.A. Heba2
S. Harakeh5
G.I.A. Karrouf 6
1
Department of Animal and Veterinary Sciences,
Faculty of Agricultural and Food Sciences, American University of Beirut,
P.O. Box 11-0236, Beirut, Lebanon; Adjunct Professor at the Biochemistry Department,
King Abdulaziz University (KAU), Jeddah, Kingdom of Saudi Arabia (KSA)
2
Biological Science Department, Faculty of Science,
King Abdulaziz University, Jeddah 21589, Saudi Arabia.
3
Public Administration Departments, Faculty of Economic and Administration,
King Abdulaziz University, Kingdom of Saudi Arabia.
4
Vacsera, Agouza, Giza, Egypt.
5
Special Infectious Agents Unit – Biosafety Level 3,
King Fahd Medical Research Center, King Abdulaziz University,
Jeddah 21589, Saudi Arabia
6
King Fahd Medical Research Center King, Abdulaziz University,
P.O. Box 80216, Jeddah 21589, Saudi Arabia; Surgery,
Anaesthesiology and Radiology Department, Faculty of Veterinary Medicine,
Mansoura University, 35516 Mansoura, Egypt
*Corresponding author: Elie K. Barbour, Department of Animal and Veterinary Sciences, Faculty of Ag-
ricultural and Food Sciences, American University of Beirut, P.O. Box 11-0236, Beirut, Lebanon, Fax:
961-1-744470, Tel: 961-1-350000 Ext. 4460, Email:eb01@aub.edu.lb
Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 122
tissues to the aflatoxin B1 is discussed. This
diagnostic model resulted in significant dif-
ferences among many assigned parameters
in ASH compared to the CH, allowing for its
future adoption in national surveillances of
aflatoxicosis in sheep.
INTRODUCTION
Aflatoxins are secondary metabolites
produced by the fungus Aspergillu.s1-3
The
produced aflatoxins are acutely toxic, im-
munosuppressive, mutagenic, teratogenic,
and carcinogenic compounds, targeting
mainly the liver organ, inducing toxicity and
carcinogenicity.4
In livestock, the consumption of
aflatoxin-contaminated feed causes many
health problems. Aflatoxin B1 (AFB1) acts
as a hepatotoxicant, hepatocarcinogen and
mutagen.5
The acute toxic effects of AFB1
include hemorrhage and death. Chronic
exposure to the aflatoxin affects growth rate,
feed efficiency and susceptibility towards
bacterial and viral associated diseases.6
Lip-
id peroxidation and oxidative DNA damage
are the principal manifestation of AFB1-in-
duced toxicity, which could be mitigated by
antioxidants.7
Small amounts of AFB1 can
cause mild or negligible effects, while large
amounts cause serious damages in the host.
Animals with lowest possible exposure to
aflatoxins are those existing in a free range
system. Low levels of aflatoxin may not be
detected since they are excreted rapidly from
the body.8
The basis of presumptive diagnosis of
aflatoxicosis in sheep relies on observation
of mortality, gross lesions on the mucosal
layers, cyanosis, and petechial hemorrhage
in the liver, associated with symptoms of
weakness and diarrhea. It is of paramount
importance to reach to final reliable diag-
nosis based on the hypothesis of inclusion
of many other observations and analysis in-
cluding hematological profiles, feed analysis
for aflatoxin, serum biochemical and chemi-
cal profiles, gross and microscopic lesions,
and residual level of aflatoxin in the organs
of affected sheep.
The aim of this research is to evaluate a
diagnostic model for aflatoxicosis, based on
clinical findings, feed analysis for aflatoxin,
hemato-chemical and biochemical profiles,
histopathological changes, and residual
aflatoxin in sheep organs of an ASH versus
a CH, in an attempt to adopt the model in
future regional surveillances.
MATERIAL AND METHODS
Herds
Two sheep herds of Awassi breed were
included in this research, located at Bahran
region of the subtropical kingdom of Saudi
Arabia. The first herd consisted of 500
sheep, showing symptoms and gross lesions
of aflatoxicosis, labeled as ”aflatoxicosis
suspected herd” (ASH), and the other herd
consisted of 100 sheep, showing no symp-
toms of any disease, labeled as ”control
herd” (CH). Records of mortality in the two
flocks were documented, and signs of the
suspected condition were recorded.
Blood and Feed Sampling
Blood was collected from the jugular vein
of the sheep in the two herds. The respective
number of sampled sheep from the ASH and
the CH were 50 and 10. Two blood samples
were collected from each sheep, one is
collected over heparin for hematological
profiles while the other sample was collected
for serum collection. Two feed samples were
collected from each of the two herds, one
from the feeder and the other from the stored
feed, totaling to four samples.
Feed Analysis
Each of the four samples of feed was ana-
lyzed in duplicate for humidity, ash %, total
protein %, ether extract % (fat), calcium
(mg/dl), inorganic phosphorus (mg/dl), mag-
nesium (mg/dl), potassium (mEg/l), sodium
(mEg/l), aflatoxin B1 (ppb), and ochratoxin
(ppb). The determination of aflatoxin B1 and
ochratoxin by thin layer chromatography,
and the proximate analysis of nutrients were
accomplished as described by the Associa-
tion of Official Agricultural Chemists9
.
Hemato-biochemical and Chemical
Analyses of Blood
The hematological analysis applied on non-
Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med.123
coagulated blood samples
included the counting of
white blood cells, neutro-
phils, lymphocytes, mono-
cytes, eosinophils, and ba-
sophils.10
In addition, the
RBC count, hemoglobin
%, and PCV were deter-
mined by previously docu-
mented procedures.11
The
biochemical analysis of
the collected sera included
the analysis for alanine
aminotransferase (ALT)
and aspartate-aminotrans-
ferase (AST),12
alkaline
phosphatase (ALP),13
total proteins (TP),14
and
albumin.15
The determina-
tion of the globulin was
calculated by subtracting
the albumin level from the TP of the same
serum sample. The serum creatinine and
uric acid were determined by the respective
previously documented methods.16,17
The
minerals in the blood (Ca, K, Mg, Na, and
P) were determined by graphite furnace
atomic absorption spectrometry,18
while the
vitamins A and vitamin E were determined
by Thin-layer chromatography.
Organ Analysis for Aflatoxin B1 and
Ochratoxin
Three nature of organs were collected from
the ASH and CH sheep namely the liver,
kidney, and muscle. The numbers of each
organ from ASH versus CH were: liver (35
vs. 15), kidney (30 vs. 20), and muscles
(12 vs. 38). The analysis for aflatoxin B1
and ochratoxin were according to the same
procedures used for analysis of both toxins
in the feed.
Histopathological study
The following number of samples were
collected from the ASH vs the CH sheep
namely, lungs (30 vs. 20), hearts (35 vs. 15),
livers (35 vs. 15), kidneys (30 vs. 20), and
intestines (30 vs. 20). Each sample was fixed
in 10% neutral buffer formalin. The samples
were processed by standard paraffin embed-
ding technique, sectioned at a thickness of
5μm, stained with H&E procedure,19, and
examined microscopically for recording the
histopathological changes in these tissues.
Statistical analysis
The comparison of means of different mea-
sured parameters between the ASH and CH
sheep was analyzed by t-student test using
the SPSS statistical computing package
(SPSS 14, 2006). Statistical differences were
reported at P values of <0.001, <0.01, and
<0.05.
RESULTS
Clinical findings
The clinical findings showed a 10% mortal-
ity in the aflatoxicosis-suspected herd (ASH)
versus its absence in the control herd (CH)
(p<0.05). The sheep in the ASH showed
symptoms of weakness, depression, conges-
tion of mucous membranes, and diarrhea.
However, the control herd (CH) had a com-
plete absence of these symptoms.
Feed analysis
The analysis of feed collected from the ASH
versus that of the CH showed a similar com-
position of nutrients and humidity (Table 1).
However, the aflatoxin B1 was high in the
feed of the ASH (300 ppb) compared to the
undetectable level in the feed of the CH. The
Parameter ASH1
ration CH2
ration
Humidity % 8 7.8
Ash% 9 10
Total protein % 14 13
Ether extract % 4.3 4.1
Calcium (mg/dl) 2.1 2
Inorganic phosphorous
(mg/dl)
1.3 1.2
Magnesium (mg/dl) 2 1.9
Potassium (mEq/l) 95 100
Sodium (Meq/l) 100 105
Aflatoxin B1(ppb) 300 Undetected level
Ochratoxin (ppb) Undetected level Undetected level
Table 1. The means of ration composition
1
ASH = Aflatoxin Suspected Herd
2
CH = Control Herd
Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 124
ochratoxin was undetectable in the feed of
both ASH and CH.
Erythrogram and Leucogram Profiles
The profiles of the erythrogram and the
leucogram are respectively shown in Tables
2 and 3. The mean values ± SE of the differ-
ent parameters of the erythrogram showed
significant lower red blood cells (RBC)
counts, hemoglobin (Hb) %, and packed
cell volume (PCV) in the ASH compared to
controls (p<0.001) (Table 2). In addition, the
mean values ±SE of the parameters included
in the leucogram showed significant drop
in the white blood cell (WBC) count of the
ASH compared to CH (p<0.001) (Table 3).
The other parameters of the erythrogram
and leucogram did not differ significantly
(p>0.05).
Hemato-biochemical and Chemical
Analyses
The data related to hemato-biochemical
analyses of sera, reflecting the liver and
kidney functions of the ASH versus CH
sheep, are shown in Table 4. Six out of the
10 parameters were elevated in the ASH
compared to CH, with significant differ-
ences in ALT (p<0.001), AST (p<0.001), AP
(p<0.001), LDH (p<0.01), urea (p<0.01),
and creatinine (p<0.01). On the contrary,
three other parameters were decreased sig-
nificantly in the ASH compared to controls
namely, the total protein (p<0.01), albumin
(p<0.01), and globulin (p<0.01). It is worth
noting that the ratio of the albumin/globulin
did not differ significantly between the two
herds (p>0.05).
The data related to the serum’s chemical
analysis of the sheep belonging to the two
flocks are shown in Table 5. All chemical
analysis of the seven measured serum pa-
rameters revealed a significant drop in sheep
of the ASH compared to controls including,
Ca (p<0.001), Pi (P<0.01), Mg (p<0.05), Na
(p<0.05), K (p<0.05), Vitamin A (p<0.01),
and Vitamin E (p<0.01).
Residual Aflatoxin B1 and Ochratoxin in
Sheep Organs
The range of aflatoxin B1 levels in liv-
ers, kidneys, and muscles of sheep in the
compared two herds is shown in Table 6.
All three organs of the ASH sheep showed
higher range of aflatoxin B1 compared to
levels in organs of the CH. The respective
aflatoxin B1 ranges in sheep of ASH versus
CH (μg/kg) were: livers (25-50 vs. 10-20),
kidneys (45-70 vs. 30-40) and muscles (55-
Herds RBC(x106
/μl) Hb (g/dl) PCV (%) Mean Corpuscular
Volume
(MCV) (fL)
Mean Corpuscular
Hemoglobin
(MCH) (pg)
Mean Corpuscular
Hemoglobin
Concentration
(MCHC)(g/dl)
CH 9.66 ±0.13 10.24 ±0.14a
39.23 ±1.12a
40.61 ±1.19 10.60 ±0.41 26.10 ±0.84
ASH 8.26 ±0.19 8.80 ±0.15b
31.40 ±0.53b
38.14 ±0.83 10.68 ±0.16 28.06 ±0.54
Table 2. Mean values ± SE of erythrogram parameters of ASH1
(N=50) and CH2 (N=10)
sheep
1
ASH = Aflatoxin Suspected Herd
2
CH = Control Herd
a,b
Means in a column followed by different alphabet superscripts are significantly different at P<0.001
Herds WBCs Neutrophils lymphocytes Monocytes Eosinophils Basophils
CH 8.76±0.18a 2.09 ±0.14 6.26 ±1.12 0.25 ±1.19 0.12 ±0.00 0.04 ±0.02
ASH 6.02±0.56b 1.92 ±0.34 3.74 ±0.53 0.21 ±0.02 0.10 ±0.02 0.04 ±0.02
Table 3. Mean values ± SE of leucogram parameters (absolute values x103
/μl) of ASH1
(N=50) and CH2
(N=10).
1
ASH = Aflatoxin Suspected Herd
2
CH = Control Herd
a,b
Means in a column followed by different alphabet superscripts are significantly different at P<0.001
Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med.125
80 vs. 40-80)
Histopathology
The histopathology of ASH sheep lungs (Fig
1), hearts (Fig 2), liver (Fig 3), kidney (Fig
4), and intestine (Fig 5) showed various mi-
croscopic changes, detailed in their legends,
with predominance of congestion, hemor-
rhage, necrosis, and edema.
DISCUSSION
The clinical findings in the two compared
herds of sheep, based on the presence in
the first herd of 10% mortality and
symptoms conforming with the afla-
toxicosis, and the absence of mortal-
ity and symptoms in the control herd
(CH), helped in the presumptive
diagnosis of the affected animals
as belonging to an ”aflatoxicosis
suspected herd” (ASH). It is worth
noting that the LD50 of AFB1 in
sheep is reported at 1.00-2.00 mg/kg
body weight.20
The acceptance of the hypoth-
esis that the designed diagnostic
model in this research could lead
to a final diagnosis of aflatoxicosis,
depended on comparative analysis
of a wide spectrum of parameters in
the feed and samples collected from
the sheep of the ASH and CH.
The analysis of the feed col-
lected from the ASH versus that of
the CH (Table 1) showed a similar
composition of nutrients and humid-
ity, thus eliminating differences
in available nutrients offered to
both flocks. However, the level of
aflatoxin B1 in the feed of the ASH
was higher (300 ppb) compared to
the undetected level in the feed of
the CH. The unacceptability of the
300 ppb level is based on standards
reported previously for acceptable
limit of aflatoxin B1 in the feed
of dairy animals, which is set at 5
ppb.21
The non-coagulated blood col-
lected from the sheep of ASH and
CH, and subjected to the eryth-
rogram (Table 2) and leucogram
(Table 3) profiles revealed a significant drop
in the RBC count of the ASH compared
to CH sheep (p<0.001). This observation,
within the diagnostic model, is in agreement
with data presented by other workers,22
sug-
gesting that this decline in RBC is due to the
aflatoxin effect on the myeloid tissue of the
bone marrow.23
In addition, the leucogram
revealed a significant lower count in the
WBC in the sheep of the ASH compared to
that of the CH, which is most likely result-
Fig 1. Lung of ASH sheep showing mild emphysema
of alveoli, dilation of bronchi, with proliferation of
epithelial cells lining bronchi associated with conges-
tion of some pulmonary blood vessels, and presence of
blood corpuscles hemolysis (H&E staining).
Fig 2. Heart of ASH sheep showing Zenker necrosis
of most of cardiac muscles, severe oedema dispersing
the cardiac muscles, mononuclear inflammatory cells,
associated with hemorrhage in between the cardiac
muscles (H&E staining).
Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 126
ing from the immunosuppressive effect of
aflatoxin B1 present in the administered feed
to sheep of the ASH. Previous researchers
proved that a level of aflatoxin B1 equiva-
lent to 7.2 μg/kg can induce leucocytopenia
in rats,24
a condition that is responsible for
immunosuppression by this toxin.25
The compared hemato-biochemical and
chemical analyses of the sheep
sera of the ASH and CH (Table
4) resulted in significant rise of
the four serum enzymes in the
ASH sheep namely, the ALT,
AST, ALP, and LDH, indicat-
ing an injury to the liver.26
The
injury to the kidney was con-
firmed by the significant rise in
urea and creatinine level in the
sera of ASH sheep, a result that
is in agreement with previous
published work on aflatoxin B1
in rabbits.27
On the contrary,
three other parameters in the
serum declined significantly in
the ASH sheep compared to the
CH namely, the total protein,
albumin, and globulin, which
is most likely due to the injury
by the toxin to the liver and
the hemopoetic and lymphoid
tissues.28,29
The consistent drop in
all the analyzed serum lev-
els of minerals and vitamins
in the ASH sheep compared to the CH
(Table 5) reflects the injury on the kidney
by the ingested aflatoxin, resulting in loss
of homeostasis of electrolyte balance,30,31
and even vitamins A and E. The drop in
serum vitamins A and E is most likely due
to the use of these vitamins in their known
Blood biochemical
parameters
ASH (N=50) CH (N=10)
ALT (IU/ml) 95 ±1.6a
31 ±1.7b
AST(IU/ml) 92 ±1.3a
40 ±1.6b
ALP(U/L) 325 ±2.4a
139 ±1.9b
LDH(IU/l) 2500 ±5.6c
730 ±4.3d
Urea (mg/dl) 65 ±1.9c
29 ±1.1d
Creatinine (mg/dl) 1.88 ±0.03c
0.9 ±0.02d
Total protein (g/dl) 5.1 ±0.02c
7.5 ±0.09d
Albumin (g/dl) 2.8 ±0.01c
3.8 ±0.03d
Globulin (g/dl) 2.3 ±0.02c
3.7 ±0.02d
Albumin/Globulin ratio 1.2 ±0.01e
1.02 ±0.03e
Table 4. Means of liver and kidney parameters of ASH1
and CH2 sheep reflecting the two organs functions
1
ASH = Aflatoxin Suspected Herd
2
CH = Control Herd
a,b
Means in a row followed by different alphabet superscripts of ‘a’and ‘b’
are significantly different at P<0.001
c,d
Means in a row followed by different alphabet superscripts of ‘c’and ‘d’
are significantly different at P<0.01
e
Means in a row followed by the same alphabet ‘e’are non-significantly
different at P˃0.05
Minerals & vitamins ASH CH
Total Calcium (mg/dl) 7.4±0.3a
11.2 ±0.92b
Inorganic Phosphorus (mg/dl) 4.1±0.61c
5.7 ±0.31d
Magnesium (mg/dl) 1.8±0.03e
3.1 ±0.11f
Sodium (mEq/l) 115±3.2e
135 ±2.44f
Potassium (mEq/l) 2.7±0.12e
3.8 ±0.09f
Vit. A (IU/dl) 21±0.94c
48 ±1.6d
Vit. E (μg/dl) 450±3.6c
715 ±3.9d
Table 5. Means of serum minerals and vitamins of ASH1 (N=50) and CH2 (N=10) sheep
1
ASH = Aflatoxin Suspected Herd
2
CH = Control Herd
a,b
Means in a row followed by different alphabet superscripts of ‘a’and ‘b’are significantly different at P<0.001
c,d
Means in a row followed by different alphabet superscripts of ‘c’and ‘d’are significantly different at P<0.01
e,f
Means in a row followed by different alphabet of ‘e’and ‘f’are significantly different at P<0.05
Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med.127
Fig 3. Liver of ASH sheep showing necrosis of hepatocytes, vacuolar
degenerative changes, oedema dispersing hepatocytes, and disorga-
nization of liver cell arrangement (H&E staining)
Fig 4. Kidney of ASH sheep showing periglomerular oedema,
dispersing oedema of the renal tubes, vacuolar degeneration of epi-
thelial cells lining the renal tubules, and necrosis (H&E staining).
Fig 5. Intestine of ASH sheep showing villar hemorrhage, infiltra-
tion of mononuclear inflammatory cells, associated with complete
necrosis of intestinal glands (H&E staining).
Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 128
protective role against aflatoxin B1-induced
oxidative stress.32
This pattern of significant
differences in the minerals and vitamins
parameters strengthens the reliability on the
diagnostic model put in the hypothesis of
this research.
The sheep in CH had an acceptable
range of the aflatoxin residue in their livers.
However, all examined control sheep have
an unacceptable level of this aflatoxin in
their kidneys and muscles that were very
near to the levels in these two organs of
the ASH. The half-life of aflatoxin B1 in
different organs of the sheep is not yet
determined. Its presence in the CH sheep’s
kidney and muscle could be due to remote
ingestion of aflatoxin.33
Future investiga-
tion should study the half- life of this toxin
in different sheep organs. The liver seems
to reflect the present situation in which the
ASH received aflatoxin contaminated feed,
while the feed offered to controls had an
undetectable level at the same time that this
study was performed. The research in human
body shows that the aflatoxin converts into
AFQ1 by the cytochrome P450 enzyme
(P450IIIAY), a process that is not yet proven
to exist in kidneys and muscle fibers.33
The diagnostic model was further
strengthened by the results of the histopatho-
logic study, showing the absence of micro-
scopic lesions from sheep of the CH and the
presence of the specific aflatoxicosis lesions
in different organs of the ASH. The sheep
of the ASH had lungs with emphysema of
alveoli, dilation of the bronchi and prolif-
eration of their epithelial cells, congestion,
and hemolysis of blood corpuscles (Fig. 1),
observations that are not previously reported
in sheep, but are extensively studied in rat
and mouse models.34
The Zember necrosis in the heart of
the ASH sheep, associated with edema and
hemorrhage, are shown in Fig. 2. The liver
necrosis of the hepatocytes associated with
edema and vaculosis (Fig. 3), the periglo-
merular oedema, necrosis, and degeneration
of the epithelial cells lining of the renal
tubules (Fig. 4), the presence of villous
hemorrhages associated with mononuclear
inflammatory cells infiltration and complete
necrosis of some intestinal glands (Fig. 5)
are collective new microscopic observations,
that could indicate the involvement of other
targeted organs of sheep by the injuries cre-
ated by aflatoxin B1.
CONCLUSION
The implementation of the diagnostic model
on the ASH and CH sheep resulted in data
that confirmed the reliability of the ap-
plicability of this model to reach to a final
diagnosis of aflatoxicosis in sheep. The
many agreements between the obtained
data and those reported in literature and the
significant differences in many analyzed
parameters between the sheep of the ASH
and CH, allows for accepting the hypothesis
of implementing this model for reaching
to a final diagnosis of sheep aflatoxicosis.
It is recommended in the near future to
include this diagnostic model in the national
surveillances of aflatoxicosis in sheep of
the subtropical areas of the Middle Eastern
countries, and other regions of the world
that are experiencing similar signs in their
aflatoxicosis suspected herds.
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Aflatoxicosis paper

  • 1. Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med.121 KEY WORDS: aflatoxicosis, aflatoxin B1, diagnostic model, evaluation, sheep ABSTRACT The aim of this research is to evaluate a diagnostic model for uncovering an afla- toxicosis outbreak in sheep. The diagnostic model is based on differentiation between an aflatoxicosis suspected herd (ASH) and a control herd (CH) including, clinical find- ings, feed analysis for aflatoxin and other nutrients, hemato-chemical and biochemical profiles, histopathologic changes, and re- sidual aflatoxin levels in their organs. There was a significant difference between the sheep of the ASH and CH in relation to mor- tality percent, symptoms, aflatoxin B1 level in their feed and tissues, hematological and biochemical parameters, liver and kidney enzymes and metabolites, serum electro- lytes, and vitamins A and E. The relationship of the histopathological lesions of affected Evaluation of a Diagnostic Model for Aflatoxicosis in Sheep: A Prerequisite for Future Adoption of National Surveillances E.K. Barbour1 ,* M.E. Abou-Alsaud2 N.A. Gheith3 M.A. Abdel-Sadek2,4 H.M.A. Heba2 S. Harakeh5 G.I.A. Karrouf 6 1 Department of Animal and Veterinary Sciences, Faculty of Agricultural and Food Sciences, American University of Beirut, P.O. Box 11-0236, Beirut, Lebanon; Adjunct Professor at the Biochemistry Department, King Abdulaziz University (KAU), Jeddah, Kingdom of Saudi Arabia (KSA) 2 Biological Science Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia. 3 Public Administration Departments, Faculty of Economic and Administration, King Abdulaziz University, Kingdom of Saudi Arabia. 4 Vacsera, Agouza, Giza, Egypt. 5 Special Infectious Agents Unit – Biosafety Level 3, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia 6 King Fahd Medical Research Center King, Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia; Surgery, Anaesthesiology and Radiology Department, Faculty of Veterinary Medicine, Mansoura University, 35516 Mansoura, Egypt *Corresponding author: Elie K. Barbour, Department of Animal and Veterinary Sciences, Faculty of Ag- ricultural and Food Sciences, American University of Beirut, P.O. Box 11-0236, Beirut, Lebanon, Fax: 961-1-744470, Tel: 961-1-350000 Ext. 4460, Email:eb01@aub.edu.lb
  • 2. Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 122 tissues to the aflatoxin B1 is discussed. This diagnostic model resulted in significant dif- ferences among many assigned parameters in ASH compared to the CH, allowing for its future adoption in national surveillances of aflatoxicosis in sheep. INTRODUCTION Aflatoxins are secondary metabolites produced by the fungus Aspergillu.s1-3 The produced aflatoxins are acutely toxic, im- munosuppressive, mutagenic, teratogenic, and carcinogenic compounds, targeting mainly the liver organ, inducing toxicity and carcinogenicity.4 In livestock, the consumption of aflatoxin-contaminated feed causes many health problems. Aflatoxin B1 (AFB1) acts as a hepatotoxicant, hepatocarcinogen and mutagen.5 The acute toxic effects of AFB1 include hemorrhage and death. Chronic exposure to the aflatoxin affects growth rate, feed efficiency and susceptibility towards bacterial and viral associated diseases.6 Lip- id peroxidation and oxidative DNA damage are the principal manifestation of AFB1-in- duced toxicity, which could be mitigated by antioxidants.7 Small amounts of AFB1 can cause mild or negligible effects, while large amounts cause serious damages in the host. Animals with lowest possible exposure to aflatoxins are those existing in a free range system. Low levels of aflatoxin may not be detected since they are excreted rapidly from the body.8 The basis of presumptive diagnosis of aflatoxicosis in sheep relies on observation of mortality, gross lesions on the mucosal layers, cyanosis, and petechial hemorrhage in the liver, associated with symptoms of weakness and diarrhea. It is of paramount importance to reach to final reliable diag- nosis based on the hypothesis of inclusion of many other observations and analysis in- cluding hematological profiles, feed analysis for aflatoxin, serum biochemical and chemi- cal profiles, gross and microscopic lesions, and residual level of aflatoxin in the organs of affected sheep. The aim of this research is to evaluate a diagnostic model for aflatoxicosis, based on clinical findings, feed analysis for aflatoxin, hemato-chemical and biochemical profiles, histopathological changes, and residual aflatoxin in sheep organs of an ASH versus a CH, in an attempt to adopt the model in future regional surveillances. MATERIAL AND METHODS Herds Two sheep herds of Awassi breed were included in this research, located at Bahran region of the subtropical kingdom of Saudi Arabia. The first herd consisted of 500 sheep, showing symptoms and gross lesions of aflatoxicosis, labeled as ”aflatoxicosis suspected herd” (ASH), and the other herd consisted of 100 sheep, showing no symp- toms of any disease, labeled as ”control herd” (CH). Records of mortality in the two flocks were documented, and signs of the suspected condition were recorded. Blood and Feed Sampling Blood was collected from the jugular vein of the sheep in the two herds. The respective number of sampled sheep from the ASH and the CH were 50 and 10. Two blood samples were collected from each sheep, one is collected over heparin for hematological profiles while the other sample was collected for serum collection. Two feed samples were collected from each of the two herds, one from the feeder and the other from the stored feed, totaling to four samples. Feed Analysis Each of the four samples of feed was ana- lyzed in duplicate for humidity, ash %, total protein %, ether extract % (fat), calcium (mg/dl), inorganic phosphorus (mg/dl), mag- nesium (mg/dl), potassium (mEg/l), sodium (mEg/l), aflatoxin B1 (ppb), and ochratoxin (ppb). The determination of aflatoxin B1 and ochratoxin by thin layer chromatography, and the proximate analysis of nutrients were accomplished as described by the Associa- tion of Official Agricultural Chemists9 . Hemato-biochemical and Chemical Analyses of Blood The hematological analysis applied on non-
  • 3. Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med.123 coagulated blood samples included the counting of white blood cells, neutro- phils, lymphocytes, mono- cytes, eosinophils, and ba- sophils.10 In addition, the RBC count, hemoglobin %, and PCV were deter- mined by previously docu- mented procedures.11 The biochemical analysis of the collected sera included the analysis for alanine aminotransferase (ALT) and aspartate-aminotrans- ferase (AST),12 alkaline phosphatase (ALP),13 total proteins (TP),14 and albumin.15 The determina- tion of the globulin was calculated by subtracting the albumin level from the TP of the same serum sample. The serum creatinine and uric acid were determined by the respective previously documented methods.16,17 The minerals in the blood (Ca, K, Mg, Na, and P) were determined by graphite furnace atomic absorption spectrometry,18 while the vitamins A and vitamin E were determined by Thin-layer chromatography. Organ Analysis for Aflatoxin B1 and Ochratoxin Three nature of organs were collected from the ASH and CH sheep namely the liver, kidney, and muscle. The numbers of each organ from ASH versus CH were: liver (35 vs. 15), kidney (30 vs. 20), and muscles (12 vs. 38). The analysis for aflatoxin B1 and ochratoxin were according to the same procedures used for analysis of both toxins in the feed. Histopathological study The following number of samples were collected from the ASH vs the CH sheep namely, lungs (30 vs. 20), hearts (35 vs. 15), livers (35 vs. 15), kidneys (30 vs. 20), and intestines (30 vs. 20). Each sample was fixed in 10% neutral buffer formalin. The samples were processed by standard paraffin embed- ding technique, sectioned at a thickness of 5μm, stained with H&E procedure,19, and examined microscopically for recording the histopathological changes in these tissues. Statistical analysis The comparison of means of different mea- sured parameters between the ASH and CH sheep was analyzed by t-student test using the SPSS statistical computing package (SPSS 14, 2006). Statistical differences were reported at P values of <0.001, <0.01, and <0.05. RESULTS Clinical findings The clinical findings showed a 10% mortal- ity in the aflatoxicosis-suspected herd (ASH) versus its absence in the control herd (CH) (p<0.05). The sheep in the ASH showed symptoms of weakness, depression, conges- tion of mucous membranes, and diarrhea. However, the control herd (CH) had a com- plete absence of these symptoms. Feed analysis The analysis of feed collected from the ASH versus that of the CH showed a similar com- position of nutrients and humidity (Table 1). However, the aflatoxin B1 was high in the feed of the ASH (300 ppb) compared to the undetectable level in the feed of the CH. The Parameter ASH1 ration CH2 ration Humidity % 8 7.8 Ash% 9 10 Total protein % 14 13 Ether extract % 4.3 4.1 Calcium (mg/dl) 2.1 2 Inorganic phosphorous (mg/dl) 1.3 1.2 Magnesium (mg/dl) 2 1.9 Potassium (mEq/l) 95 100 Sodium (Meq/l) 100 105 Aflatoxin B1(ppb) 300 Undetected level Ochratoxin (ppb) Undetected level Undetected level Table 1. The means of ration composition 1 ASH = Aflatoxin Suspected Herd 2 CH = Control Herd
  • 4. Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 124 ochratoxin was undetectable in the feed of both ASH and CH. Erythrogram and Leucogram Profiles The profiles of the erythrogram and the leucogram are respectively shown in Tables 2 and 3. The mean values ± SE of the differ- ent parameters of the erythrogram showed significant lower red blood cells (RBC) counts, hemoglobin (Hb) %, and packed cell volume (PCV) in the ASH compared to controls (p<0.001) (Table 2). In addition, the mean values ±SE of the parameters included in the leucogram showed significant drop in the white blood cell (WBC) count of the ASH compared to CH (p<0.001) (Table 3). The other parameters of the erythrogram and leucogram did not differ significantly (p>0.05). Hemato-biochemical and Chemical Analyses The data related to hemato-biochemical analyses of sera, reflecting the liver and kidney functions of the ASH versus CH sheep, are shown in Table 4. Six out of the 10 parameters were elevated in the ASH compared to CH, with significant differ- ences in ALT (p<0.001), AST (p<0.001), AP (p<0.001), LDH (p<0.01), urea (p<0.01), and creatinine (p<0.01). On the contrary, three other parameters were decreased sig- nificantly in the ASH compared to controls namely, the total protein (p<0.01), albumin (p<0.01), and globulin (p<0.01). It is worth noting that the ratio of the albumin/globulin did not differ significantly between the two herds (p>0.05). The data related to the serum’s chemical analysis of the sheep belonging to the two flocks are shown in Table 5. All chemical analysis of the seven measured serum pa- rameters revealed a significant drop in sheep of the ASH compared to controls including, Ca (p<0.001), Pi (P<0.01), Mg (p<0.05), Na (p<0.05), K (p<0.05), Vitamin A (p<0.01), and Vitamin E (p<0.01). Residual Aflatoxin B1 and Ochratoxin in Sheep Organs The range of aflatoxin B1 levels in liv- ers, kidneys, and muscles of sheep in the compared two herds is shown in Table 6. All three organs of the ASH sheep showed higher range of aflatoxin B1 compared to levels in organs of the CH. The respective aflatoxin B1 ranges in sheep of ASH versus CH (μg/kg) were: livers (25-50 vs. 10-20), kidneys (45-70 vs. 30-40) and muscles (55- Herds RBC(x106 /μl) Hb (g/dl) PCV (%) Mean Corpuscular Volume (MCV) (fL) Mean Corpuscular Hemoglobin (MCH) (pg) Mean Corpuscular Hemoglobin Concentration (MCHC)(g/dl) CH 9.66 ±0.13 10.24 ±0.14a 39.23 ±1.12a 40.61 ±1.19 10.60 ±0.41 26.10 ±0.84 ASH 8.26 ±0.19 8.80 ±0.15b 31.40 ±0.53b 38.14 ±0.83 10.68 ±0.16 28.06 ±0.54 Table 2. Mean values ± SE of erythrogram parameters of ASH1 (N=50) and CH2 (N=10) sheep 1 ASH = Aflatoxin Suspected Herd 2 CH = Control Herd a,b Means in a column followed by different alphabet superscripts are significantly different at P<0.001 Herds WBCs Neutrophils lymphocytes Monocytes Eosinophils Basophils CH 8.76±0.18a 2.09 ±0.14 6.26 ±1.12 0.25 ±1.19 0.12 ±0.00 0.04 ±0.02 ASH 6.02±0.56b 1.92 ±0.34 3.74 ±0.53 0.21 ±0.02 0.10 ±0.02 0.04 ±0.02 Table 3. Mean values ± SE of leucogram parameters (absolute values x103 /μl) of ASH1 (N=50) and CH2 (N=10). 1 ASH = Aflatoxin Suspected Herd 2 CH = Control Herd a,b Means in a column followed by different alphabet superscripts are significantly different at P<0.001
  • 5. Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med.125 80 vs. 40-80) Histopathology The histopathology of ASH sheep lungs (Fig 1), hearts (Fig 2), liver (Fig 3), kidney (Fig 4), and intestine (Fig 5) showed various mi- croscopic changes, detailed in their legends, with predominance of congestion, hemor- rhage, necrosis, and edema. DISCUSSION The clinical findings in the two compared herds of sheep, based on the presence in the first herd of 10% mortality and symptoms conforming with the afla- toxicosis, and the absence of mortal- ity and symptoms in the control herd (CH), helped in the presumptive diagnosis of the affected animals as belonging to an ”aflatoxicosis suspected herd” (ASH). It is worth noting that the LD50 of AFB1 in sheep is reported at 1.00-2.00 mg/kg body weight.20 The acceptance of the hypoth- esis that the designed diagnostic model in this research could lead to a final diagnosis of aflatoxicosis, depended on comparative analysis of a wide spectrum of parameters in the feed and samples collected from the sheep of the ASH and CH. The analysis of the feed col- lected from the ASH versus that of the CH (Table 1) showed a similar composition of nutrients and humid- ity, thus eliminating differences in available nutrients offered to both flocks. However, the level of aflatoxin B1 in the feed of the ASH was higher (300 ppb) compared to the undetected level in the feed of the CH. The unacceptability of the 300 ppb level is based on standards reported previously for acceptable limit of aflatoxin B1 in the feed of dairy animals, which is set at 5 ppb.21 The non-coagulated blood col- lected from the sheep of ASH and CH, and subjected to the eryth- rogram (Table 2) and leucogram (Table 3) profiles revealed a significant drop in the RBC count of the ASH compared to CH sheep (p<0.001). This observation, within the diagnostic model, is in agreement with data presented by other workers,22 sug- gesting that this decline in RBC is due to the aflatoxin effect on the myeloid tissue of the bone marrow.23 In addition, the leucogram revealed a significant lower count in the WBC in the sheep of the ASH compared to that of the CH, which is most likely result- Fig 1. Lung of ASH sheep showing mild emphysema of alveoli, dilation of bronchi, with proliferation of epithelial cells lining bronchi associated with conges- tion of some pulmonary blood vessels, and presence of blood corpuscles hemolysis (H&E staining). Fig 2. Heart of ASH sheep showing Zenker necrosis of most of cardiac muscles, severe oedema dispersing the cardiac muscles, mononuclear inflammatory cells, associated with hemorrhage in between the cardiac muscles (H&E staining).
  • 6. Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 126 ing from the immunosuppressive effect of aflatoxin B1 present in the administered feed to sheep of the ASH. Previous researchers proved that a level of aflatoxin B1 equiva- lent to 7.2 μg/kg can induce leucocytopenia in rats,24 a condition that is responsible for immunosuppression by this toxin.25 The compared hemato-biochemical and chemical analyses of the sheep sera of the ASH and CH (Table 4) resulted in significant rise of the four serum enzymes in the ASH sheep namely, the ALT, AST, ALP, and LDH, indicat- ing an injury to the liver.26 The injury to the kidney was con- firmed by the significant rise in urea and creatinine level in the sera of ASH sheep, a result that is in agreement with previous published work on aflatoxin B1 in rabbits.27 On the contrary, three other parameters in the serum declined significantly in the ASH sheep compared to the CH namely, the total protein, albumin, and globulin, which is most likely due to the injury by the toxin to the liver and the hemopoetic and lymphoid tissues.28,29 The consistent drop in all the analyzed serum lev- els of minerals and vitamins in the ASH sheep compared to the CH (Table 5) reflects the injury on the kidney by the ingested aflatoxin, resulting in loss of homeostasis of electrolyte balance,30,31 and even vitamins A and E. The drop in serum vitamins A and E is most likely due to the use of these vitamins in their known Blood biochemical parameters ASH (N=50) CH (N=10) ALT (IU/ml) 95 ±1.6a 31 ±1.7b AST(IU/ml) 92 ±1.3a 40 ±1.6b ALP(U/L) 325 ±2.4a 139 ±1.9b LDH(IU/l) 2500 ±5.6c 730 ±4.3d Urea (mg/dl) 65 ±1.9c 29 ±1.1d Creatinine (mg/dl) 1.88 ±0.03c 0.9 ±0.02d Total protein (g/dl) 5.1 ±0.02c 7.5 ±0.09d Albumin (g/dl) 2.8 ±0.01c 3.8 ±0.03d Globulin (g/dl) 2.3 ±0.02c 3.7 ±0.02d Albumin/Globulin ratio 1.2 ±0.01e 1.02 ±0.03e Table 4. Means of liver and kidney parameters of ASH1 and CH2 sheep reflecting the two organs functions 1 ASH = Aflatoxin Suspected Herd 2 CH = Control Herd a,b Means in a row followed by different alphabet superscripts of ‘a’and ‘b’ are significantly different at P<0.001 c,d Means in a row followed by different alphabet superscripts of ‘c’and ‘d’ are significantly different at P<0.01 e Means in a row followed by the same alphabet ‘e’are non-significantly different at P˃0.05 Minerals & vitamins ASH CH Total Calcium (mg/dl) 7.4±0.3a 11.2 ±0.92b Inorganic Phosphorus (mg/dl) 4.1±0.61c 5.7 ±0.31d Magnesium (mg/dl) 1.8±0.03e 3.1 ±0.11f Sodium (mEq/l) 115±3.2e 135 ±2.44f Potassium (mEq/l) 2.7±0.12e 3.8 ±0.09f Vit. A (IU/dl) 21±0.94c 48 ±1.6d Vit. E (μg/dl) 450±3.6c 715 ±3.9d Table 5. Means of serum minerals and vitamins of ASH1 (N=50) and CH2 (N=10) sheep 1 ASH = Aflatoxin Suspected Herd 2 CH = Control Herd a,b Means in a row followed by different alphabet superscripts of ‘a’and ‘b’are significantly different at P<0.001 c,d Means in a row followed by different alphabet superscripts of ‘c’and ‘d’are significantly different at P<0.01 e,f Means in a row followed by different alphabet of ‘e’and ‘f’are significantly different at P<0.05
  • 7. Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med.127 Fig 3. Liver of ASH sheep showing necrosis of hepatocytes, vacuolar degenerative changes, oedema dispersing hepatocytes, and disorga- nization of liver cell arrangement (H&E staining) Fig 4. Kidney of ASH sheep showing periglomerular oedema, dispersing oedema of the renal tubes, vacuolar degeneration of epi- thelial cells lining the renal tubules, and necrosis (H&E staining). Fig 5. Intestine of ASH sheep showing villar hemorrhage, infiltra- tion of mononuclear inflammatory cells, associated with complete necrosis of intestinal glands (H&E staining).
  • 8. Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 128 protective role against aflatoxin B1-induced oxidative stress.32 This pattern of significant differences in the minerals and vitamins parameters strengthens the reliability on the diagnostic model put in the hypothesis of this research. The sheep in CH had an acceptable range of the aflatoxin residue in their livers. However, all examined control sheep have an unacceptable level of this aflatoxin in their kidneys and muscles that were very near to the levels in these two organs of the ASH. The half-life of aflatoxin B1 in different organs of the sheep is not yet determined. Its presence in the CH sheep’s kidney and muscle could be due to remote ingestion of aflatoxin.33 Future investiga- tion should study the half- life of this toxin in different sheep organs. The liver seems to reflect the present situation in which the ASH received aflatoxin contaminated feed, while the feed offered to controls had an undetectable level at the same time that this study was performed. The research in human body shows that the aflatoxin converts into AFQ1 by the cytochrome P450 enzyme (P450IIIAY), a process that is not yet proven to exist in kidneys and muscle fibers.33 The diagnostic model was further strengthened by the results of the histopatho- logic study, showing the absence of micro- scopic lesions from sheep of the CH and the presence of the specific aflatoxicosis lesions in different organs of the ASH. The sheep of the ASH had lungs with emphysema of alveoli, dilation of the bronchi and prolif- eration of their epithelial cells, congestion, and hemolysis of blood corpuscles (Fig. 1), observations that are not previously reported in sheep, but are extensively studied in rat and mouse models.34 The Zember necrosis in the heart of the ASH sheep, associated with edema and hemorrhage, are shown in Fig. 2. The liver necrosis of the hepatocytes associated with edema and vaculosis (Fig. 3), the periglo- merular oedema, necrosis, and degeneration of the epithelial cells lining of the renal tubules (Fig. 4), the presence of villous hemorrhages associated with mononuclear inflammatory cells infiltration and complete necrosis of some intestinal glands (Fig. 5) are collective new microscopic observations, that could indicate the involvement of other targeted organs of sheep by the injuries cre- ated by aflatoxin B1. CONCLUSION The implementation of the diagnostic model on the ASH and CH sheep resulted in data that confirmed the reliability of the ap- plicability of this model to reach to a final diagnosis of aflatoxicosis in sheep. The many agreements between the obtained data and those reported in literature and the significant differences in many analyzed parameters between the sheep of the ASH and CH, allows for accepting the hypothesis of implementing this model for reaching to a final diagnosis of sheep aflatoxicosis. It is recommended in the near future to include this diagnostic model in the national surveillances of aflatoxicosis in sheep of the subtropical areas of the Middle Eastern countries, and other regions of the world that are experiencing similar signs in their aflatoxicosis suspected herds. REFERENCES 1- Kaaya AN, Kyamuhangire W, Kyamanywd S. Fac- tors affecting aflatoxin contamination of har- vested maize in the three agro-ecological zones of Uganda. J Applied Sci. 2006:52:2401-2407 2- Mohamed AM, Metwally NS. Antiaflatoxigenic activities of some plant aqueous extracts against aflatoxin B1 included renal and cardiac damage. J Pharmacol Toxicol. 2009:4:1-16. 3- Probakaran, JJ, Dhanapal, S. Analysis of proximate composition of aflatoxins of some poultry feeds. Asian J Biotechnol. 2009:1:104-110. 4- Peraica M, Radic B, Lucic, A, Pavolvic M. Diseases caused by Molds in Humans. Bulletin of the World Health Organization 1999:77(9):754-766. 5- Alwakel SS. The effect of mycotoxins found in some herbal plants on biochemical parameters in blood of female albino mice. Pak J Biol Sci. 2009:12:637-642. 6- Youssef MI, Salem MH, Kamel KI, Hassan GA, El- Nouty FD. Influence of ascorbic acid supplementa- tion on the haematological and clinical biochemis- try parameters of male rabbit exposed to aflatoxin B1. J Environ Sci Health B. 2003:38:193-209. 7- Patel V, Sail S. Beta-carotene protects the physi- ological antioxidants against aflatoxin B1 induced carcinogenesis in albino rats. Pak J Biol Sci.
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