L
.9gyA471eL7lie"z. 62.7t 5:192-199, (2002).
1
FIELD AND LABORATORY STUDIES ON LUMPY
SKIN DISEASE
By
I-IAMODA, F.K. ; ABOUL SOUD, E.A*; MAGEDA, M.S**.
SHAHEIN, M, A.*** MICHAEL, A,* and DAOUD, A.M.*.
Dept. of Anim Mcd, Fac
WE
Mod, Zagazig University, Benha Branch, Egypt
*Veterinary serum and vaccine Research Institute, Abbasia, Cairo, Egypt
**Central Laboratory for evaluation of Veterinary products., Abbasia., Cairo, Egypt
***Dept. of Virology., Animal Health Research Institute, Dokki. Cairo, Egypt.
SUMMARY
This study was conducted on a total of of 453 animals representative in
240 cows (g(l cross bred and 160 native breed) and 213 buffaloes, out of them 28 cows and 4 buffaloes were showed clinical signs believed to be LSD. These cases were appeared sporadic during the period from end of April
1998 to June 1999 at Kaluobia governorate. Clinical examination of the ailing animals
(cows&huffalocs) revealed presence of multiple fimt round cutanous nodules either localized or generalized on the skin with or without fever and other systemic signs. The clinical signs exhibited on diseased cows were similar to that observed on diseased buffaloes. ('he incidence of the disease in cows and buffaloes was 11.67 % and 1.88 `7,. respectively and the overall incidence was 7.06%. The higher incidence of the disease was during summer season and lowest during winter.
Trials for virus isolation from skin biopsy or skin scabs collected from diseased cows and buffaloes by inoculation into ECE and MDBK proved detection of LSD virus. Theiso- lated virus was identify and confirmed using IFAT, neutralization teat and histopathological examination.
Further identification was carried out by experimental infection of buffaloes with the virus isolated from naturally infected ones declared appearance of nodular forma- tion at the site of infection onl supportive therapy beside local dressing of the lesion were adopted y without generalinttion or appearance of systemic signs. The therapeutic trials using ancillary and
It can be conclude that, the overall results pointing to that LSD naturally affected cows as well as buffaloes with higher incidence during summer season, so further study should be directed to investigate LSD in buffaloes with special rel crcnce to epizootiological aspects and further laboratory analysis to compare between the LSD virus isolated front buffaloes and that isolated from cows antigenically and biochemically and if buffaloes isolates was similar to that of cow isolates a trial to prepare vaccine from the isolated local LSD virus and the policy of buffaloes vaccination should he go hand by hand with that adopted for cows in Egypt to reduce the economic losses of this disease.
I RnOLICTION
,kin disease (LSD) is an acute, subacute, chronic or inapparent severe systemic
183

r
U
1 e disease of cattle and buffaloes, caused by a member of capripoxvirus (Neeth)ing strain),
I. characterized by fever, salivation, nasal discharge, sudden appearance of multiple
(Asagba firm circumscribed cutaneous nodules and generalized lymphadenitis and Naw- athe., 1982; Prozesky and Barnard.,
1982; John et al., 1988 and
Hungerford., 1990 and Coetzer et a)., 1994). The virus of LSD is virtually identical with Kenyan sheep and goat poxvirus (SPGPV) as reported by
House.,
(1996) but dissimilar to
Middle cast SPGPVB
(Radostits et al., 2000).
The LSDV was isolated for the first time from cattle in Egypt by
House et al., (1990) and from buffaloes by
Ismael.,
(2000). r
M
LSD had the potential for rapid spread with the ability to cause serious economic importance because it reduces production particularly in dairy herds and damage to the
4 hide
(O1E., 1992 and House.,
1996). LSD is a sporadic or epidemic disease of cattle
(01E., 1989), or epizootics interspersed with periods of sporadic occurrence in endemic areas over a period of several years occurs
(Woods., 1988; Davies.,
1991; Moussa., 1.996 and Radostits et al., 2000).
LSD is firstly recognized in Egypt among cattle in
Mn-
13E.S and m­
- ouft v r.3c
-Ae c recorded after that
(Agag et al., 1989.. EL-Ab'y et aL lWi Hasa et ai.. 1fl2; Hassan., 1993; Abou Zeid et al, 1993: DawZ 1 : !J
1ll9 and Abd EL-Rahim et al.. 2002). Moreover clinical LSD a m nd y
noo s ed among Egyp-
M tian buffaloes but the incidence was ioweru! tbzn thc rmacded among cattle (Ali et al.,
1990;Davies., 1991;Hassan., 1993 and Ismael., 2000).
Meanwhile
Dorsey and James., (1973) stated that LSD might occur in buffaloes as well as cattle
And
Rados
tits et al., (2000) published that there was only one report of the natural occurrence of LSD in water buffalo. Also buffaloes might have been inapparent infection as proved by
Davies., (1982, 1991) who detected high levels of antibodies to capripox virus in sera of African buffaloes. On the other hand
Hassan., (1993) cited that the presence of LSD antibodies in buffalo sera

1 although there was no vaccination program gave an in- dication for the ability of LSDV to introduced, infected and stimulate immune defense mechanism of buffalo which can overcome and refract the disease or might due to the innate resistance of buffalo (host resist).
On contrary
Young et al., (1966) and Young et aL
(1970) reported that buffalo inoculated subcutanously by the LSD virus did not de-mlop any lesions anti All and
Oheid.,
If
184 j • Egypt.
V et. Med.Assoc. 5,2002
9oeU and
4c &o at u[iea off ....
(1977) found that buffaloes present close to an infected area did not show any pox like disease. j
The disease control depends primarily on vaccination of all cattle population in all governorates of Egypt using local sheep pox vaccine (Michael et al., 1994). The appearance of
LSD in Egypt despite all the control measures was thought due to the accidental invol v ement of other animals species in the maintenance cycle of LSD (Ismael., 2000). This work was aimed to investigate the LSD problem concerning the following aspects:
(-Description of clinical signs exhibited on ailing cows and buffaloes and declaring some epizootiological aspects.
2-Trials for isolation of the causative agent on CAM and MDBK cell culture. 3-Identification and confirmation of the etiological agent by serological techniques namely IFAT, serum neutralization test and histopathological findings.
4-Further identification through car r ying out experimental infection of clinically normal cows and buffaloes.
5-Monitoring serum neutralizing antibodies in sera of diseased cattle and buffaloes and in contact apparently healthy ones.
6-Therapeutic trials of the diseased animals

MATERIAL ANI) METHODS
1-Animals, clinical and epidemiological investigations- This study was conducted on a total of 453 animals representative in 240 cows (80 cross breed and 160 native
0 breed) and 213 buffaloes, out of them 28 cows and 4 buffaloes were showed clinical signs believed to be LSD namely fever and formation of multiple cutanous nodules with or without systemic signs. This study was done during the period f r om end of April 1998 to June 1999 at Kaluobia governorate. Full clinical examination, detailed history and the important epizootiological data were recorded.
2-Reference virus and antiserum: Il was kindly obtained from the foreign animal disease diagnostic laboratory, Plum Island, USA and stored at Pox
Dept, Veterinary serum and vaccine Research Institute, Abbasia, Cairo. These agents were mainly used for application of SNT, IFAT and FLISA.
1992). a
1
J.Eg pt.Vet.Mted.Assoc.¢2,5,2002
3-Santpling and laboratory investigation:
A-Virus isolation: The suspension of skin scrapings and skin biopsy taken from diseased cows and buffaloes were inoculated into 9-day-old embryonating chicken eggs via Chorio-allantoic membrane (CAM) with a dose of 0.2ml/egg (30 eggs were used for each passage and five passage were done) according the method of Van rooyen et al.,
(1969). The 5th egg passages were propagated on monolayer of
MDBK cell line for three passage (OIE .,
B-Virus titration: It was performed in ECE and MDBK cell line. The titration of the isolated virus in ECE was bone according to of Van rooyen et al., (1969) . Ten fold serial dilutions were made in PBS and
0.2 ml of each dilution inoculated into chorio- alantioc membrane of 9-day chicken embryo (at least 5 eggs for each dilution) and the inoculated eggs were candled daily for 7 days and death of the embryos recorded at first 24 h regarded as being non specific. Infective dose 50 end point were calculated by the method of
Reed and Muench., (1938). The titration of the isolated virus in
185

M monolayer MDBK cell line using the method described by the method of FADDL Diagnostic laboratory Protocol 602 (1985).
C-Virus identification: The isolated virus was identifying mainly by:
I -Indirect fluorescen: antibody technique (IFAT): It was carried on
MDBK cells (Davies et al., 1971)
2-11istopathological examination: It was performed on MDBK cell line and on skin biopsy samples collected from diseased animals (House et al., 1990).
3-Virus neutralization test (VNT): It was conducted using reference serum according to Manual of serological microtitration techniques.,
(1981).
4-Experimental infection: The aim of this is experiment was to clarify and confirm clinical LSD among buffaloes, it was carried out at Pox Dept,
Veterinary serum and vaccine Research Institute. Abbasia. Cairo. The study was applied on 6 adults animals of 2-4 years old, 2 buffaloes and
2 cows. it were injected I/D on the lateral side of the neck with a dose of 0.5 ml of the virus isolated from ailing buffaloes while one cow and one buffalo were kept in contact with the inoculated animals as control.
All animals were clinically normal and their sera proved to be free from
LSD antibodies using serum neutralization test using standard LSD virus and all animals were
186 J•Egypt.Vet.Alcd.Aawoc.6Z,5,2002
9 ieed acid
' eaeara to uy 44cd&,4 Mt .... kept in insect proof stables under obser v ation along the period of the experiment (2.m). Serum samples were collected from all animals every week until 8 WPI for monitoring seroconversion using sentm neutralization and solid phase ELISA tests.
D-Serological tests:
The following tests were carried out to detect specific antibodies against

LSDV in seat of diseased cows & buffaloes as following:
1-Serum neutralization test (SNT):
It was carried out using standard LSD virus according to
FADDL Diagnostic laboratory Protocol 602 (1985).
2-Solid phase enzyme linked immunosorbent assay (ELISA): It was applied accord- ing to the methods of
Voller et al., (1976).
E-Therapeutic trials: Diseased animals were isolated iti insect proof stables whenever possibles with complete rest. Ancillary and symptomatic therapy by antibiotic (oxytetracycline hydrochloride 5%., lnil/lOkg, b/w, I/M, Arab Veterinary Industrial co) for combating secondary infection, antipyretic (analginc, 8ml/100kg, hfw. UV, Memphis co) with cold fomentation, anti-inflammatory (Phenylbutazone, 5ml/calf, 20 ml/ cow, I/V, Virbac.co), this beside local dressing of the lesions using antiseptics as Be- tadine solution (Bovidenc-lodinc.U.S.P 10% w/v, The Nile.,co. Cairo).
RESULTS AND DISCUSSION
LSD is creeping insidious panzootic disease has a potential rapid spread with the ability to causes groat economic losses (Anon., 1985; Woods,, 1988;
House., 1996 and Radostits et al.,
2000). LSD is now considered as enzoobc disease in Egypt (Davies., 1991 and Hassan.,
1993)
With regard to clinical manifestation of LSD on infected cows. The majority of diseased cows revealed fever, sudden development of intrademal nodules (multiple, slightly raised, circumscribed, firm, round, flat-topped) which might be covered the whole body with or without onset of other clinical signs as anorexia, depression, decrease in milk production, salivation, ocular discharges and rapid labored respiration, moderate :o severe loss in condition and few of diseased ones showed enlargement of superficial lymph node (prefentoral or prescapular one).
Such findings were similar to prior findngs of EL-
Kanawaty., (1989).,
All et al.,
(1990);Youssef et al., (1990), Agag et al.,
J.Egypt.Vet \ted.Assoc. 62.5.2002 ! 187
OMr
C (1992); Sedeek., (1992); Hafez et al., (1992); Hassan., (1993); Abou
-Zaid et al (1994);
Castro and Heuschele., (1996); Ismael., (2000) and Abd EL-Rahim et

{ were in contrary t,
al (2002). Clinical signs described on the infected buffaloes revealed presence of charac teristic cutanous nodules (Figl& 2) either localized or generalized on the whole of th animal's body with or without onset of systemic effects. These findings were previous ly observed by Ali et al.,
(1990), Hassan., (1993) and Ismael., (2000) who describe clinical signs of LSD among Egyptian buffaloes. While these results
44 that cited by House et al., (1990) who found that four adult water buffalo, each with calf and at least 50 sheep were in direct contact with the affected cattle but remain
M clinically normal and Mageda et al., (1999) who reported that sheep, goats and buffa loes were not clinically susceptible when it was became in contact with infected cattle and the later believed to he the natural host and usual source of LSD viral infection.
With respect to virus isolation, the inoculation of suspected materials
(suspension iron skin scabs or skin biopsy) on ECE resulted in formation of characteristic pock lesion: (Fig 3) on the 5th passage with a titer of up to 5.8 egg 1D50/ML. While culture of MDBK cell line causes CPE (Fig
4) on the second passage after 5 days post inoculatior and characterized by cell rounding and degeneration and the titer of the virus was up t<
4.2TCID50/mi. From the etiological point of view the above results were identical tc
M that obtained by Woods., (1988);Hassan et al., (1992);EL-Allaway et al., (1992); Abou-
Zaid et al., 1994 and Ismael., (2000). However the previous data were contra. dieted to that reported by Hassan., (1993) who failed to isolated
LSDV from clinically infected buffaloes and he attributed that to infections by several microbial/or non mi. crobial factors including LSDV or to natural resistance of buffaloes to the disease (breed resist).
Accordingly, the isolated virus from both cows and buffaloes were identify and con• firmed by serological technique namely IFAT (Fig 5) to revealed bright greenish yel• low fluorescence of the infected MDBK cells and histopathological findings of the in fected cell culture (Fig 6) to declared characteristic degenerative changes with appearance of round or oval dense acidophilic mass of variable size present close to nu• clefts

r188
it__________ which might be surrounded by clear halo after staining by H&E stain.
These results were supported by that obtained previously (House et al.,
1990; Davies, 1991 ;Hassan., 1993; Aboul-Soud., 1995 and EL-
Bagoury., 1995). Moreover the re-
A.N.gypt.V et.Med.Assoc. 02,5,2002
7e(4 4nd 2a$oatr.q 4&d
Oct suits of histopathological examination of skin biopsy samples revealed ballooning de-. generation, edema, necrosis of all layers of epidermis nearly, numerous neutrophils, lymphocytes and few of eosinophils were seen in the basal and prickle cells and the presence of intracytoplasmic eosinophilic bodies. These findings agreed to that observed by Woods., (1988); Ali et al., (1990); House., (1996) and Ismael., (2000).
The incidence of the disease among cows and buffaloes was 11.67 % and 1.88 % respectively. Such results supported the clinical view of that cows were more susceptible than buffaloes (Ali et al., 1990; Davies., 1.991;Hafez et al., 1992; Hassan., 1993 and
Ismael., 2000). Moreover the obtained results respecting to the incidence of I,SD among buffaloes were nearly similar to that recorded by All et al., (1990)_ who found that the incidence of LSD among buffaloes was 1.6% and lowered to that obtained by
Hassan., (1.993) and Ismael:, (2000) who recorded incidence rates of 3.18% and.
9.64% respectively. The suseptiabilty to infection with LSDV is differed in cattle than in buffaloes (Hassan., 1993).
Table (1): The incidence of LSD among cattle and buffaloes
Animals No. of animals \o. of diseased Percent `9c. at risk animals
240
213
453
28
4
32
11.67
1.88
7.06
Cattle
Buffaloes
Total number
Table (2): Seasonal dynamics of LSD among cattle and buffaloes
Animals
Cattle
No %
Buffaloes
No

Spring
Summer
Autumn
Winter
Total
6
15
4
3
28
21.43 0.0
53.57 3
14.29
10.71
0.0
1
4
0.0
75
0.0
25
J. Egypt. V et.Nled.Assoc. 62 ,5,2002
I
189
Tale (3): Res*Ss S sc= r r n am = sera of cows and Wffakes fir' :> .z z rA LSD isolated virus. vtP1 sana-.s _ter t ta of sera
0
1
2 f
16
32
-
5
16
-
_
-
3 64*
64
32
64
6 8
4 16 32*
5 32
32
16 32
6 32
32
16 32
7 32 32
16
16
8
32 16
16
The highest liter
16
WFI Weeks post infection
IC:
Control cow
*
113; Control buffaloes
NB : The standard titer of positive sera against LSDV was > 441
(control positive sera)
-
+,
-
-
4
4
4
4
2
2
2
Table (4): Results of indirect solid phase E LISA in sera of cows and buffaloes experimentally infected with
LSD isolated virus.
WPI 'liter to cows sera
'titer in butfa- loos sera
Titer in of sera control animals
2 1
2
1C
LB
0
1
4
5
2
3
6
40
40
160
320*
320
160
160
20 <20
40
20
80 40
160 80*
320
160
80
80
160 >20
<20
20
40
80"
80
80
>20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20

Lkl.,.mn
7
8
80
80
40 20
40
20
The highest titer
20
20
WPI Weeks post infection *
IC : Control cow 18: Control buffaloes
NB : The standard titer of positive sera against LSDV was > 40
(control positive scth)
<20
<20
<20
<20
190
J.Eg) pt.Vet.Med.Assoc. (+2,5,2002

+; canod . et aE
These clinical cases appeared sporadically during the period from end Ap ri l 1998 to
June1999. Similarly Mageda et al., (1999) recorded LSD among ca tt le during May 1998 to
1999 in EL-Minia and EL-Fayoum., Egypt and the range of morbidity was 0.003-0.006% and the disease occurred in a sporadic manner. Also EL-Bagoury et al.. (1995) diagnosed sporadic clinical cases of LSD among cattle in Minia Governorate during 1995.
The appearance of LSD in form of limited epizootics might be attributed to the policy of cattle vaccination using sheep pox living attenuated vaccine (Michael et al., 1994). In this respect, Daoud et al., (1998) concluded that the presence of circulating field LSD virus might elevated antibody titer in susceptible cattle to an abnormal high titer and Davies
(1991) declared that LSD when become endemic in a locality is possible to eradicate.
The study revealed that there was no mortalities among infected cattle or buffaloes. Such finding was highly augmented by the prior work of Mageda et al— (1999) who found that the outbreak of LSD during 1998 was relatively mild without deaths and was quickly brought under control.
The higher incidence of the disease was during sum  % season (53.574% & 75%) and the lowest incidence du ri ng winter season (1071% & 25%) is code and buffaloes respectively.
The most common means of LSD mmsr n on *acs b± insects bite. Such findings has been reported whe re
there has been c!rcz11z
y th1 e.idenx that biting insects played a role in the spread of LSD vines (Losos.. 19 .
36:% oods_ 1988;Davies., 1991; Sedeek., 1992;House., 1996 and Radostitis et aL 2
0
00
)
_ In this respect Ali et al., (1990) Cited that the higher rates of
LSD in`:deax in ~rthn localities in Egypt could be attributed to greater number of insect vectors
The results of serum neutralization test declared detection of variable titer of antibodies in the sera of infected cows and buffaloes and inxrrz-t ones. These results were highly augmented by the prior work of many worke rs
(Davies., 1981 and 1982;House et al., 1990;
Davies., 1991; Agag et al., 1992;A1i., 1992; Hassan., 1993;Daoud et at.,
1
998
194
J.Egypt.Vet.MetAssoc.
1iL
5,2002

.~ and Ismael., 2000). However the diseased cows and buffaloes had antibodies higher than that detected in clinically normal ones, such criteria was documented previously by the work of Davies., (1982) and Ismael., (2000). On other hand the previous results were contradicted to that of Young et al., (1970);IIedger and Hamblin., (1983) and Hamblin et al., (1990) who failed to detect antibodies in sera of buffaloes.
As well, the presence of neutralizing antibodies in buffaloes sera indicated that buffaloes were involved as a vertebrate maintenance host for the LSD virus at inter-epidemic periods
(Ismael., 2000), also the presence of these antibodies contributed to the presence of buffaloes in contact with clinicall y or subelinically infected cows
(Davies., 1982
:t.
Gere.-ail) the presence oY aathodies in sera ::, plc. otanz i from cows and buffaloes with different ages and sexes and in different seasons indicated that LSD could affect different breeds of animals, ages and sexes (French and Geering., 1978 and Ali et al., 1990)
The preseance of serum neutralizing antibodies in buffaloes although there is no vaccination programme for buffaloes gave an indication for the ability of LSDV to introduce,infect and stimulate immune defense mechanism of buffaloes(Hassan,.1993)
Regarding to the results of experimental infection, ID itioculation of the virus isolated from buffaloes revealed formation of well circumscribed firm swelling at the site of injection of all inoculated buffaloes from the 3rd days post infection till the 7th days which regress rapidly within one week without generalization or systemic disturbance while in cattle, there was systemic reaction (increase in temperature, respiration and pulse) beside local nodular lesion and there was no generalization . Incontact animals appeared clinically normal during the period of the experiment. These results were coincided with that reported by Carn and [(itching., (1995) who concluded that following VD inoculation of
LSD virus, local lesions were developed at the site of challenge without viremia and generalization of infection. Moreover the above results were in parallel to that noticed by
Hassan., (1993) who observed the characteristic clinical signs on cattle and buffalo after 5-7 days post inoculation at the sites of inoculation only with some respiratory signs, fever and marked increase of skin thickness with appcarancc of characteristic skin nodules anti the cattle showed clear signs than buffalo. also these findings resemble those obtained by
Young et al., (1968) who reported that
J.Egypt. Vet.Mcd.Assoc- 61,5.2002 195
'{~a<rn , et a4 1
LSD might occur in buffaloes as well as in cattle. On the other hand Yager and Scott.,

(1985) failed to produced any clinical disease after experimental infection of seronegalive African buffalo.
Data obtained in table (3) declared that, the highes t
titer of SNT antibodies achieved at 3WPI and 4\VPl in sera of experimentally infected cows (64) and buffaloes (32) respectively. In the cur re nt study, the levels of antibodies as re vealed by solid phase BLISA (table 4) reached to maximum at 3WPI in sera of both experimentally infected cows (320) and buffaloes (80). The above data cleared that the titer of antibodies (SNT & F,LISA) showed low level in sera of expe ri mentally infected buffaloes and had short period of persist e nc y thn it in seta of experimentally infected cows. These re sults were agreeabl y to tl c react S Da•ies
(1950) and (1982); Hamblin et al., (1990) and Hassai., (1 fl
Young et aL i 19'S) site ft r naiar a A n s tz
W& s az war
eed to that obtained by in young buf- falo calves follow iag saa: results supported the -tOi a y es a t
sn • at& L D is
?ter.. x:s_ tlae abo% a d Asa as cr - tle for LSD_
. specific an tibodies against LSDV, this data agreed to the work of Kurstak and Marusyk., (1984); Sedeek., (1992) and Daoud et al., (1998). The re su lts of
ELBA proved that the test was mo re
sensitive than SNT in detection of
The results or therapeutic trials revealed that majority of the ailing animals were recovered within 1-2 months where the cutanous nodules became necrotic in the center giving rise to the "sit fast" and usually forni hard dark scabs, this lesion detached f ro m the underlying tissues leaving an ulcer which healed by granulation with formation of scar tissues, in few one some of that cutaonus nodules persisted as hard lumps for several weeks. Such findings were similar to that of Ayre-Smith.,
(1960); Hungerford., (1990) and Radostits et al., (2000).
Insect control programs should be applied beside regular and accurate vaccination of susceptible animals with effective vaccine (Hungerford., 1990; Mageda et al.,
1999 and Radostits et al., 2000). Further studies still needed in future to clarify the different hidden points concerning buffalo as a highly resistant animal against many of the enzootic diseases of economic importance (Hassan., 1993).
196 J. Eg y pt. Vet.Med. A ssoc. l2.5,2M12
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