Download Clinical and Epidemiological studies on Lumpy Skin Disease

SCVMJ, XIII (1) 2008
69
CLINICOEPIDEMIOLOGICAL STUDIES AND
EVALUATION OF PCR ON DIAGNOSIS OF LUMPY SKIN
DISEASE OUTBREAK, EGYPT 2005
Mahmoud, M. M., Ibrahiem, K. S., Ghaniem, F. M., El-HeeG, M.
Infectious Diseases - Animal Med. Dept., Faculty. of Vet. Med,
Suez Canal University
ABSTRACT
In this study the last outbreak of lumpy skin disease (LSD) was investigated
from June 2005 to April 2006 over two Egyptian governorates (Ismailia and
Damietta). A total number of 954 (103 native and 851 mixed) cattle breed were
examined clinically for LSD. Different clinical forms of the disease were
reported and ranged from acute, mild and inapparent forms. Total morbidity and
mortality rates were 50.5% and 1.1% among examined cattle respectively.
Different field samples were collected (Buffy coat 20, skin biopsy 15 and lymph
nodes biopsy 5) for virus isolation and polymerase chain reaction technique
(PCR) and the isolated virus was identified using virus neutralization test
(VNT) and indirect fluorescent antibody technique (IFAT), while 200 sera
samples were collected from both diseased and contact healthy cattle for
serological investigations. The present study showed that the old age groups,
male and native breeds were more resistant to infection. The results of
serological examinations revealed that the incidence of infection was 58% and
49% by Serum Neutralization Test (SNT), while by Enzyme Linked Immune
Sorbant Assay (ELISA) were 62%, and 55% in Ismailia and Damietta
governorates respectively. ELISA test was more sensitive than SNT in early
detection of infection as it detect the infection by 7 day while SNT by 10 days
after fever. The results of PCR test was more sensitive and rapid for detection of
the virus as compared with isolation and identification technique when applied
on blood and tissue samples therefore we can use it as rapid means for LSD
diagnosis.
tible than females, caused by NeethINTRODUCTION
ling virus which belongs to family
Lumpy skin disease (LSD) is a Poxviridae (Alaa, 2000, Wallace,
cattle disease of all ages specially in 2001 and Tuppurainen et al., 2005).
summer season, males more suscep- The disease usually associated with
70
severe economic losses like emaciation, hide damage, male infertility,
mastitis, loss of milk production, with
mortalities (40%) and morbidities
(100%) (Barnard et al., 1994). The
severity of the clinical manifestations
range from acute to inapparent infection, and characterized by eruption of
skin nodules, pyrexia, anorexia, dysagalactia and pneumonia. Most cattle
were seen with edema of legs and
brisket (Khalafalla et al., 1993;
Younis and Aboul Soud, 2005).
Complications include respiratory
manifestation, mastitis, dehydration,
abortion and later on recumbence
(Abdallah and Gawad, 1992).
In May 1988, LSD was clinically
recognized in the Suez-Egypt, where it
was thought to be arrived at the local
quarantine station via cattle that imported from Africa. The disease locally
spread in summer, 1988 and apparently over wintered with little or no
manifestations of clinical disease. It
reappeared in 1989 along a period of
five to six months, spread to 22 out of
26 governorates of Egypt, (Ali et al.,
1990 and House et al., 1990). In
2005, the disease returned and reappeared in Damietta-Egypt, and affected large number of cattle populations then spread to other provinces.
A presumptive diagnosis of the
disease can be based on clinical signs,
Mahmoud et al.,
however, mild and inapparent cases
may be difficult to be detected.
Therefore Rapid laboratory methods are needed to confirm diagnosis,
which can be done either by isolation
and identification of the virus, or by
detection of antibody using serological
tests (Tuppurainen et al., 2005). AS
virus isolation is very difficult and
time consuming we can use a rapid
and sensitive test to confirm the
diagnosis as PCR (Ireland and
Binepal, 1998). Therefore, the aim of
the present study was to monitor some
epidemiological parameters, clinical
signs, serological diagnosis with comparison between PCR and virus isolation as a diagnostic means for LSD in
the last outbreak in Egypt.
MATERIAL & METHODS
1. Investigated animals:
A total number of 954 cattle (native
& mixed), aged 6 months to 5 years,
were examined from June, 2005 to
April, 2006 at Ismailia and Damietta
governorates.
2- Samples for virus isolation:
2. A. Blood samples: 20 whole blood
samples from clinically affected cases
were drained on anti-coagulant (EDTA)
in vacationer tubes to separate buffy
coat for virus isolation and PCR test.
2. B. Other samples: 20 skin and
lymph nodes biopsies from diseased
cattle were aseptically collected in
sterile clean MacCarteny bottles cont-
SCVMJ, XIII (1) 2008
aining maintenance medium with antibiotics, transported on ice to the
laboratory and stored at -70ºC until
used for virus isolation and PCR test.
3. Serum samples for serological
examination:
A total 310 serum samples were
collected, 200 sera samples from the
two Egyptian governorates were collected from both clinically and apparently healthy contact animals postappearance of signs according to
Hedger and Hamblin (1983).
Another 110 sera sample
were daily collected from 10 cattle suffered from acute clinical signs (6th till
day16th after fever). All samples were
transported on ice to the laboratory
and stored at -20ºC until used for
serological examination.
4. Virus isolation and identification:
the virus was isolated in fertile eggs
according to Michael et al., (1991)
and in cell lines according to OIE
Manual (1992).
5. Polymerase chain reaction (PCR)
assay:
The extraction of DNA was carried
out according to Sambrook et al.
(1989). The PCR reactions were
carried out in a final volume of 25 µl
in PCR tubes, the reaction mixture
consists of 1 µl (200 ng) of the
extracted DNA template, 5 µl of 10X
PCR buffer (75M Tris-HCL, pH 9.0,
2mM MgCL2, 50mM KCL, 20mM
(NH4)2SO4), 2 µl dNTPs (10mM)
71
(Biotools), 1µl Taq DNA polymerase
(Bio tools) and 0.5 µl (50 pM) from
the forward primer (5'TTTCCTGATTTTTACTAT3') and reverse primer
(5'AAATTATATACGTAAATAAC3')
(Integrated DNA Technologies, Inc.
Coralaville) of the gene for viral attachment protein according to (Ireland
and Binepal, 1998). The volume of
the reaction mixture was completed to
25 µl of DDW. The thermal cycler
(Omni-Gene, USA) was adjusted as
follows; initial denaturation 94oC for
10 minutes, followed by 40 cycles of
1minute denaturation at 94oC, 30 sec.
annealing at 50oC and 1 minute extension at 72oC, followed by final 72°C
extension for 1min, then hold at 4°C.
Five micro liters of PCR products,
negative control, positive control (kindly supplied from microbiology department, Vet Med., Cairo University)
and 100bp DNA marker (Promega,
Madison, WI, USA) were electrophoresed at a constant current of 40 V
for 1hours in 1% agarose gel stained
with 0.5ug of Ethidium bromide/ml to
determine the size of the product. The
gels were visualized under U V illumination (Eagle Eye II, Startagene,
Germany) and thereafter photographed
using digital camera. The sizes of the
amplified product were determined by
comparison to DNA marker.
72
RESULTS & DISCUSSION
Now LSD is considering as enzootic disease in Egypt whereas several
mild outbreaks recorded after 1988
(Radostitis et al., 2007). In the present
study, the total number and percentage
of examined cases in Ismailia and
Damietta governorates during 2005
outbreak were shown in (Table 1).
The clinical observations based on
fever and numbers of skin nodules
classify the disease into acute, mild
and inapparent forms. Acute form
showed signs of fever (41.5ºC), nasal
and ocular discharges. Two days later,
the characteristic skin lumps developed and covered the whole body.
The cattle suffered from mild form
showed a few skin lesions or transient
fever while, inapparent form showed
complete absence of clinical signs and
diagnosis based on serological tests.
The observed clinical findings
were in similar to results obtained by
Alaa, (2000); Hamoda et al., (2002);
Younis and Aboul soud, (2005) and
Khadr et al., (2006).
The field investigation revealed the
total morbidity and mortality rates
among examined cattle were 50.5 %
and 1.1% respectively (Table 2). The
finding came in the same line of
results recorded by Castro and
Heuschele (1996). The highest rate
recorded for the morbidity among
cattle populations in Ismailia and
Damietta governorates indicated that
Mahmoud et al.,
the outbreak was severe. This observation could be related to decrease of
immunity or vaccination with inadequate dose, or improper route of
vaccination (Younis and Aboul soud,
2005). Table (2) also showed high
morbidity in Ismailia than Damietta
that could be explained by different
breed and climatic conditions between
the two governorates or related to the
affected farms.
The observed high mortalities rate
among calves less than six months
(Tables 3) may be attributed to
insufficient active and passive
immunity (Ali et al., 1990). It was
confirmed from the obtained result
that the age, sex, season and breeds
play an important role in epidemiology of the disease (Tables 3, 4 and 5).
Old age, male and local breeds
were more resistant to infection than
others and these results could be
referred to a previous exposure of
these animal groups via routine vaccination or emergency vaccinations during the outbreak, or natural resistant
among native breeds (Younis and
Aboul soud, 2005).
A tentative diagnosis of the disease
is usually based on clinical signs but,
animals with few skin lesions or
transient fever may be not detected or
confused with other diseases (Barnard
et al., 1994). The results of serological
examinations (Tables 6), revealed that
the incidence of infection was 58%
SCVMJ, XIII (1) 2008
and 49% in Ismailia and Damietta
governorates respectively by SNT,
while by ELISA were 62%, and 55%
respectively, and the ELISA was more
sensitive than SNT and this agree with
Hamoda et al., (2002) and Ibrahim et
al., (2006).
In study of the efficiency of SNT
and ELISA in detection of early infection (Table 7), ELISA was more
sensitive as it detect the infection by
7- 13 days post fever while SNT
started to detect increased antibody by
10 – 15 days and this indicate that the
sensitivity of ELISA in detection of
early infection.This observation supported the findings of (Tuppurainen et
al., 2005) who stated that SNT started
to detect increased antibody titers 13 –
18 days post infection after onset of
fever but, ELISA demonstrated a rise
in antibody titers as early as 8 days
post infection after onset of fever.
Indirect diagnosis of LSD by virus
isolation on embryonated chicken egg
(Table 8) the virus was isolated from
buffy coat 50% and 40% and by 60%
and 50% from tissue samples in
Ismailia and Damietta governorates
respectively, the failure of virus isolation from buufy coat may be due to
the absence of viraemia during collection of samples or low number of virus
in the blood, the observations supported byin accordance with Tuppurainen et al., (2005) who stated that
length of viraemia varies from 1-12
73
days and not correlate with severity of
clinical signs. the highest rate of virus
isolation from tissue than buffy coat
may be due to long persistence of
virus in tissue Tuppurainen et al.,
(2005) who reported that virus can be
isolated up to 39 days post infection.
The virus detection by PCR on both
blood and tissue samples (Tab. 8 and
Fig. 1) revealed positive cases 60%
and 60% in Ismailia and Damietta
governorates respectively, while in
tissue samples positive cases were
80% and 70% respectively The results
is in the same line with Ibrahim et al.,
(2006), the highest percent positive
cases in tissue samples than blood
samples may be attributed to the long
persistence of the virus in tissue than
blood (Tuppurainen et al., 2005). In
comparison between virus isolation
and PCR as shown in (Table 8) there
was high positive cases in PCR than
isolation and this can be explained due
to the sensitivity of PCR as the ability
of the PCR to detect small numbers
and / or inactive virus.
It is recommended that all animals
should be vaccinated intradermal with
recommended dose of vaccine and the
new cattle comers to the herd should
be examined using the PCR assay as
adequate control measures of the
disease and we can advice to use
ELISA and PCR for the rapid
diagnosis.
74
Mahmoud et al.,
Fig (1): Results of PCR product for detection of Lumby Skin Disease virus
M: 100 bp marker, 1,2: Control positive and negative, 3-7: test samples.
SCVMJ, XIII (1) 2008
75
Table (1): Number and percentage of examined animals.
Locality
Ismailia
Sex
Age /month
No of
cattle
Native
Mixed
male
female
>6
6-30
<30
Sum.
Wint.
Aug.
2005
Sep.
2005
Oct.
2005
Dec.
2005
Jua.
2006
80
-
80
32
48
-
30
50
80
-
120
20
100
35
85
3
67
50
120
-
75
15
60
22
53
4
32
39
-
75
50
15
35
10
40
1
15
34
-
50
25
10
15
7
18
-
20
5
-
25
350
60
290
106
244
8
164
178
200
150
85
5
80
20
65
4
50
31
85
-
200
-
200
69
131
10
80
110
200
-
110
18
92
37
73
9
60
31
110
-
100
-
100
33
67
6
52
52
-
100
65
20
45
20
45
-
35
30
-
65
44
-
44
19
25
-
22
22
-
44
604
43
561
198
406
29
299
276
395
209
954
103
851
304
650
37
463
354
595
359
Total
Damietta
Species
Field
trip
Jul.
2005
Aug.
2005
Sep.
2005
Oct.
2005
Mar
2006
Apr.
2006
Total
Total per two
provinces
Table (2): Morbidity and Mortality rates among examined cattle.
Locality
No of examined cattle
Ismailia
Damietta
Total
350
604
954
Morbidity rate
No
%
205
58.6
277
49.9
482
50.5
Mortality rate
No
%
3
0.9
7
1.2
10
1.1
76
Mahmoud et al.,
Table (3): LSD forms in relation to age groups.
locality
Age/month
Ismailia
Damietta
>6
Ismailia
Damietta
6-30
Ismailia
Damietta
<30
Acute
Mild
Inapparent
deaths
No
%
No
%
No
%
No
%
6
75
2
25
-
-
2
25
18
62.1
8
27.6
10.3
5
17.2
40
24.4
60
36.6
64
39
1
0.6
43
14.4
55
18.4
201
67.2
1
0.3
34
19.1
63
35.4
81
45.5
-
-
42
15.2
111 40.2
123
44.6
1
0.4
3
Table (4): LSD' forms in relation to sex groups.
LSD' forms
sex
Female
Male
Locality/NO
Acute
Mild
Inapparent
No
%
No
No
%
Ismailia (244)
54
22.1
90
36.9
%
100
41
Damietta(406)
66
16.3
108
26.6
232
57.1
Ismailia (106)
26
24.5
35
33
45
42.5
Damietta(198)
37
18.7
66
33.3
95
48
Table (5): LSD' forms in relation to breed groups.
LSD' forms
Breed
Locality/NO
Acute
No
Mix
Native
Mild
%
No
%
Inapparent
No
%
113
39
Ismailia (290)
72
24.8
105 36.2
Damietta (561)
98
17.5
161 28.7
302
53.8
Ismailia (60)
8
13.3
20
33.3
32
53.4
Damietta (43)
5
11.6
13
30.2
25
58.1
SCVMJ, XIII (1) 2008
77
Table (6): Result of antibodies against LSD in collected sera samples using
ELISA and SNT.
Positive sera
Total sera
Locality
ELISA
No.
SNT
%
No.
%
Ismailia
100
62
62
58
58
Damietta
100
55
55
49
49
Table (7): comparison between of means days.
Days after onset of fever
Test
No of
ELISA
mean
7
8
9
10
11
12
13
14
15
1
3
2
1
1
1
1
-
-
9.3
-
-
-
1
1
3
1
3
1
12.7
positive
animals
SNT
(10)
Table (8): Comparison between the results of virus isolation and PCR.
isolation
PCR
Locality
samples
Ismailia
Blood (10)
5
50
5
50
6
60
4
40
Tissue (10)
6
60
4
40
8
80
2
20
Blood (10)
4
40
6
60
6
60
4
40
Tissue (10)
5
50
5
50
7
70
3
30
Damietta
+ ve
%
- ve
%
+ ve
%
- ve
%
78
REFERENCES
Abdalla, M.A. and Gawad S.M. (1992):
Characterization of serum lysosomal
enzymatic activities II. Dutch Tierarztl
Wochenschr. Aug; 99(8): 347-9.
Alaa, (2000): Some studies on LSD in
cows and buffaloes. PhD, thesis (infectious diseases), Zagazig University.
Ali, A. A.; Esmat, M.; Attia, H.; Selim,
A. and Abdel-Hamid, Y. M. (1990):
Clinical and pathological studies on
lumpy skin disease in Egypt. Vet Rec.
1990 Dec 1; 127(22): 549-550.
Barnard, B. J. H.; Munz, E.; Dumbell,
K. and Prozesky, L. (1994): LSD in
Coetzer JAW, Thomson GR, Tustin
RC, Kriek NPJ. Infectious diseases of
livestock with special references to
southern African cape town Oxford
University Press p: 604-612.
Castro, A. E. and Heuschele, W. P.
(1996): Veterinary diagnostic virology
(LSD). A practitioner guide. Mosby
Year Book, Inc. pp. 106-108.
Hamoda, F. K.; Aboul Soud, E. A.;
Magda, M. S.; Shahein, M. A.; Michael,
A. and Daoud, A. M. (2002): Field and
laboratory studies on lumpy skin
disease. j. Egypt. Vet. Med. Assoc.
62(5): 183-199.
Hedger, R. S. and Hamblin, C. (1983):
Neutralizing antibodies to lumpy skin
disease virus in African wildlife.Comp
Mahmoud et al.,
Immunol Microbiol Infect Dis., 6(3):
209-13.
House, J.A.; Wilson, T.M.; El Nakashly,
S.; Karim, I.A.; Ismail, I.; El Danaf, N.;
Moussa, A.M. and Ayoub, N.N. (1990):
The isolation of lumpy skin disease
virus and bovine herpesvirus-4 from
cattle in Egypt. J Vet Dig. Invest.; 2(2):
111-115.
Ibrahiem, A. K; Ahmed, S. A.; Darderi,
M. A. and Amin, A. S. (2006): Comparative studies on conventional and
molecular assays used for diagnosis of
Lumpy skin disease. Egypt. J. Comp.
Path. & clinic. Path, 19(3): 227-239.
Ireland, D. C. and Binepal, Y. S. (1998):
Improved detection of capripox virus in
biopsy samples by PCR. J. Virol. Meth.,
74: 1-7.
Khalafalla, A. I.; Gaffar Elamin, M.
A. and Abbas, Z. (1993): Lumpy skin
disease: observations on the recent
outbreaks of the disease in the Sudan.
Rev Elev Med Vet Pays Trop.; 46(4):
548-50.
Khadr, A. M.; E-Fayoumy, M. M.;
Abou-Elsoud, E.; El-Ballal, S. and
Zaghawa, A. (2006): An outbreak of
lumpy skin disease in Egyptian farm
during 2005-2006. Clinical, epidemiological, laboratory and electron microscopic investigations. Menofia Vet. J,
4(1): 1-13.
SCVMJ, XIII (1) 2008
79
Michael, A; Salama, s. A.; Soliman, S.
M.; Mousa, A. A.; Bachoum, M. E.;
Barsoum, G. W.; Osman, A. O.and
Nassar, M. I. (1991): The determination of the immune state of cattle to
LSD in Egypt. J. Egypt Vet. Med.
Assoc., 51(2): 427-434.
Office International Des Epizooties
(OIE) (1992): Manual of recommended
diagnostic techniques and requirements
for biological products for lists A and B
diseases. Vol.1:1-5. OIE 12, reu du
Prony, 75017 Pairs- france.
Radostits, O. M.; Gay, C. C.; Blood,
D. C. and Hinchxliff, K. W. (2007):
Veterinary medicine A textbook of the
disease of cattle, sheep, pigs, goats and
horse., 10th Ed. W. B. Saunders Company Ltd.
Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989): Molecular cloning: A
laboratory manual 2nd Ed Cold Spring
Harbor Laboratory Press, Cold Spring
Harbor, N. Y.
Tuppurainen, E. S.; Venter, E. H. and
Coetzer, J. A. (2005): The detection of
lumpy skin disease virus in samples of
experimentally infected cattle using
different diagnostic techniques. Onderstepoort J. Vet. Res.; 72(2): 153-64.
Wallace, H. P. D. (2001): LSD in
southern Africa, a review of the disease
and aspects of control. J. Afr. Vet.
Assoc., 72: 68-71.
Younis, E and Aboul Soud, E. (2005):
Some studies on outbreak of LSD,
Egypt 2005. Mansoura Vet. Med. J.,
VII(2): 37-59.
‫الملخص العربي‬
‫دراسات إكلينيكية وبائية وتقييم اختبار تفاعل البلمرة المتسلسل في تشخيص وباء التهاب الجلد العقدي‬
‫مـ‬5002 ‫مصر عام‬
‫مي في محافظيات‬5002 ‫أجريت الدراسة على الوباء األخير لمرض التهاب الجلد العقدي في مرير عيا‬
( 429 ‫عييدد‬
‫ وقييد ت ي فح ي‬5002 ‫ إل ي رييهر أبري ي‬5002 ‫( اإلسييماعيلية ودميييا ) مييش رييهر يوني ي‬
‫ في‬%303 ‫ ونسيبة الوفييات‬%2002 ‫ خليي ) ميش األبقيار وقيد اانيت نسيبة اإلريابة‬123 ‫محل و‬301
‫ وقييد ت ي تسييجي عييدم رييور للمييرض منهييا الرييورم الحييادم والرييورم ال ي يية‬.‫األبقييار الت ي ت ي فحرييها‬
‫ عينية‬32‫ مسيحة افيية ميش ايرات اليد البي ياء و‬50 ( ‫والرورم الخ ية وقد ت تجميع عينيات مختل ية‬
‫وقيد تي‬
‫ عينات ميش الديدد الليم اويية) لعيل ال ييروس وإجيراء اختبيار ت اعي البلميرم المتسلسي‬2‫جلد و‬
‫علي ي عييش ريييد اختبييار تعيياد ال يييروس واختبييار المري ال لورسييينتى ييير‬
‫عييل ال يييروس والتعيير‬
‫‪80‬‬
‫‪Mahmoud et al.,‬‬
‫المباريير و قييد ت ي تجميييع ‪ 500‬عينيية مييش مر ي الييد للحيوانييات المرييابة والسييليمة المخال يية لل ح ي‬
‫السيييرولوج ‪ .‬وقييد أرييارت النتييا‬
‫للمرض وقد أظهرت نتيا‬
‫ال حي‬
‫إل ي‬
‫أش األعمييار الابيييرم والييااور والسييليت المحلييية أاثيير مقاوميية‬
‫السييرولوج أش نسيبة اإلريابة ي ‪ 21‬و ‪ %94‬باسيتخدا اختبيار‬
‫التعاد المرل ‪ 25‬و‪ % 22‬باستخدا اختبار اإلليلا ف محيافظت اإلسيماعيلية ودمييا عليى التيوال و‬
‫االك إل أش اختبار اإلليلا أاثر حساسية مش اختبار التعاد المرل للار‬
‫أش اختبار اإلليلا قد أظهر نتا‬
‫إيجابية للمرض ف اليو السابع بينميا اختبيار التعياد المريل في الييو‬
‫العارر بعد ظهور الحمى وقد اانت نتا‬
‫بييال رد التقليدييية لعييل وترييني‬
‫سريعة لترخي‬
‫المرض‪.‬‬
‫المباير عيش الميرض حيي‬
‫اختبار ت اع البلميرم المتسلسي أاثير سيرعة وحساسيية مقارنية‬
‫ال يييروس وبالتييال نسييت يع إجييراء اختبييار البلمييرم المتسلس ي اوسيييلة‬