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The Isolation of Lumpy Skin Disease Virus and Bovine Herpesvirus- from Cattle in Egypt James A. House, Terrance M. Wilson, Sinan El Nakashly, Ikram A. Karim, Ibrahim Ismail, Nabil El Danaf, Aly M. Moussa and Nazmi N. Ayoub J VET Diagn Invest 1990 2: 111 DOI: 10.1177/104063879000200205 The online version of this article can be found at: http://vdi.sagepub.com/content/2/2/111
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J Vet Diagn Invest 2:111-115 (1990)
The isolation of lumpy skin disease virus and bovine herpesvirus- from cattle in Egypt James A. House, Terrance M. Wilson, Sinan El Nakashly, Ikram A. Karim, Ibrahim Ismail, Nabil El Danaf, Aly M.Moussa, Nazmi N. Ayoub Abstract. Lumpy skin disease (LSD) virus (LSDV) was isolated for the first time from cattle in Egypt in 2 disease outbreaks. Bovine herpesvirus-4 (BHV-4) and LSDV were detected in a pooled sample from the first outbreak (Suez). Only LSDV was isolated from the second outbreak (Ismalia). The capripoxviruses were identified as LSDV by neutralization with specific antiserum and by their ability to produce generalized LSD in experimentally inoculated cattle.
Lumpy skin disease (LSD), a generalized skin disease of cattle caused by a capripoxvirus, was first described in northern Rhodesia in 1929.7 The disease spread in sporadic epizootics, reaching South Africa in 1944 and Kenya in 1957.8 Since 1973, LSD has spread to western and northeastern Africa. l3 The first reports of LSD outside of Africa were from Kuwait in 1986-1988.2 This report describes the first diagnosis of LSD in Egypt, whose geographical location may allow LSD to spread to Europe and further in Asia. Materials and methods Clinical history and sample collection. Suez, Egypt: On June 2, 1988, a disease clinically resembling LSD was reported in Holstein cattle at a government farm near the quarantine station in Suez, Egypt. Fourteen of 194 cattle were affected with fever, increased salivation, generalized deep skin nodules, enlarged peripheral lymph nodes (LN), and in some cases respiratory signs. Clinically normal cattle from Somalia had been moved into the quarantine station in early May 1988. Forty-seven buffalo and 280 sheep in contact with the affected cattle remained clinically healthy. A severely affected animal was slaughtered, and skin, muscle, prescapular LN, and fluid from a skin lesion were collected for virus isolation. A skin biopsy was also collected from another affected bull. Samples were kept frozen at -70 C until used for virus isolation. Approximately 6 mo postexpsoure, serum was collected from blood obtained from 7 cattle present during the June LSD outbreak. Ismalia, Egypt: On October 31, 1988, an LSD-like disease was reported in the village of Tel El Kabir in the govemorate of Ismalia about 50 km from Suez. On November 30, 1988, approximately 50% of 250 native cattle (Balidy) had deep From the Foreign Animal Disease Diagnostic Laboratory (FADDL), US Department of Agriculture, Science and Technology National Veterinary Services Laboratories, Box 848, Greenport, NY 11944 (House, Wilson), Animal Health Research Institute, Dokki, Cairo, Egypt (Nakashly, Karim, Ismail, El Danaf, Ayoub), and General Organization of Veterinary Service, Cairo, Egypt (Moussa). Received for publication September 28, 1989. 111
skin nodules ranging in number from a few up to 300 (Fig. 1). Sitfasts and purulent discharge from lesions with secondary bacterial involvement, fever, increased salivation, and emaciation were observed. Three young calves had severe LSD. One 3-mo-old calf was in lateral recumbency with extensor rigidity of the forelimbs, paddling of the hind legs, and opisthotonus. Four adult water buffalo, each with a calf, and at least 50 sheep were in direct contact with the affected cattle but remained clinically normal. A biopsy was collected from a skin nodule of a 3-mo-old bull calf clinically ill for 3 days. Blood was collected from a calf ill for 4 days and from 6 cattle clinically affected for 1-6 wk. Cultures for virus isolation. Eight-day-old embryonating chicken eggs (ECE) were inoculated with samples from the Suez outbreak via the chorioallantoic membrane (CAM) route. Primary goat kidney (GK) cultures, fetal bovine lung (FBL) cells (first to seventh passage), and newborn lamb testicle (LT) cells (first to fifth passage) propagated with Eagle’s minimum essential medium (EMEM) supplemented with 5% fetal bovine serum (FBS) were used for virus isolation. Viruses. Bovine herpesvirus- 1 a (BHV-1) Colorado strain, bovine herpesvirus- (BHV-2) Minnesota strain, bovine herpesvirus-4a (BHV-4) DN 599 strain, alcelaphine herpesvirus-l (AHV-1) Minnesota strain, and LSD virus (LSDV) Kenyan strain B2490 were used for reference purposes. Antisera. Reference antisera to BHV-1,a BHV-2, BHVa 4, AHV-1, and LSDV were used for virus identification. Virus isolation studies in cell cultures and cattle. Samples from Suez: Field samples of muscle, skin, lymph node, and lesion vesicular fluid were passaged twice in GK. Approximately 1 ml of cell culture fluid from each sample was inoculated intradermally (ID) in individual sites in the neck of 2 5-mo-old mixed-breed calves (numbers 173 and 175) after collecting a 0-day-postinoculation (0 DPI) blood sample. The calves were housed in high containment (> P3) at the Foreign Animal Disease Diagnostic Laboratory (FADDL). These samples were also inoculated into LT cultures, which were observed daily and passaged weekly for up to 3 wk, and examined by histological staining. Cell cultures were propagated on 8-chamber slides.b Cultures exhibiting cytopathogenic effect (CPE) were fixed with absolute methanol for 5 min or with Bouin’s fixative until stained. Fixed cultures
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House et al.
Figure 1. Skin lesions of LSD in an adult Balidy cow, Tel El Kabir, Egypt.
were stained with either xanthine dye/thiazine dye mixture” or hematoxylin and eosin (HE) stains. Additionally, cells grown on g-chambered slides were fixed with acetone at - 20 C and stained with anticapripoxvirus direct fluorescein isothiocyanate (FITC) conjugate10 prepared from caprine origin convalescent goat pox serum and read with a fluorescence microscope.d An indirect fluorescent antibody (IFA) test was performed on fixed cells, using specific antiserum and then antispecies gamma globulin FITC conjugate. Virus isolates were identified by neutralization index studies. Chorioallantoic fluid from the Suez samples was passaged 3 times on LT cells. The nature of the CPE, i.e., rounding of cells and some stranding, especially during the first passage, suggested a mixed viral infection. Stained cell cultures revealed eosinophilic intranuclear inclusion bodies and some variable staining intracytoplasmic inclusion bodies. Anticapripoxvirus FITC conjugate revealed specific staining in a small percentage of the cells. The original ECE passage fluid was mixed with antiserum to BHV-4 prior to each of 2 passages in FBL cells. Both passages developed CPE similar to that of capripoxviruses. Two 450-cm2 roller bottles of FBL cells were inoculated with passage 2 viral fluid and harvested in 5 days at 90% CPE. The resulting virus isolate was not neutralized by anti-BHV-4 serum but was identified as a capripoxvirus with LSD-specific antiserum (titer reduced by 103) and designated the Suez LSD viral isolate. One milliliter of this virus, with a titer of 106.9 TCID50/ml in FBL cells, was used to inoculate each of 2 5-mo-old mixed-breed calves (numbers XXX and 171) by needle prick in the skin of the neck; 9 ml was given intravenously after obtaining a 0 DPI serum sample. A sample of a nodule from calf XXX was collected at the time of necropsy for virus isolation. A suspension of the original skin lesion from the animal sacrificed at Suez was inoculated into FBL cells and passaged twice. Monolayers were stained by HE and direct FA procedures. Samples from Ismalia. The Ismalia calf skin nodule biopsy sample was passaged twice in FBL cells. An aliquot of the clarified culture fluid was diluted lo-fold, and each of 2 5-mo-old mixed-breed calves (numbers 3 and 4) was inoc-
ulated ID with 0.1 ml in 5 sites for each lo-fold dilution (10-4-10-9) after obtaining a 0 DPI serum sample. The sample used for cattle inoculation was titrated in FBL cells. Microtiter plates were examined by microscopy prior to freezing after 7 days of incubation. After thawing, 0.1 ml of culture fluid was transferred into respective wells of a second plate of FBL cells. A third passage was similarly done. Titers were calculated by the Spearman-Karber method of determining 50% endpoints.5 The animals were observed, and their temperatures were taken daily for 26 days. Heparinized blood and skin nodule biopsies were collected for virus isolation at 20 DPI. The animals were necropsied and tissues taken for histopathological examination. Neutralization index (NI) test. The NI test using serum at a final 1:10 dilution was performed as described5 using 10,000 FBL cells/microtiter well. Neutralization indices ≥ 1.5 were considered positive for antibody.5 An NI using pre- and postimmune LSD serum was used for virus identification. Indirectfluorescent antibody test. Monolayers of FBL cells were infected with BHV-4 to yield approximately 30% of the cells infected at day 2 when culture fluid was decanted. The monolayers were rinsed with phosphate-buffered saline (PBS), fixed with acetone at -20 C for 10 min, air dried, and stored at -70 C until used. Serum diluted 1:10 in PBS was used in a traditional screening IFA test. Titrations were performed using 2-fold serum dilutions; the endpoint titer was the highest dilution showing specific fluorescence. Histological methods. Biopsies and tissues obtained at necropsy were fixed in formalin, sectioned at 4-5 µm, stained with HE, and examined by light microscopy. After visualization by light microscopy, selected areas of formalin-fixed tissues were obtained from the planum nasale and routinely postfixed for ultrastructural examination by transmission electron microscopy (TEM).
Results Calves (numbers 173 and 175) inoculated with Suez samples passaged in GK did not develop signs of illness. No neutralizing antibody was detected in the 28 DPI sera against BHV-1, BHV-2, AHV-1, or LSDV, but IFA titers of 1:80 against BHV-4 were detected. No antibody was detected in the 0 DPI sera to any of the 5 viruses. All inoculated LT cell cultures had eosinophilic intranuclear inclusion bodies, and BHV-4 was identified by virus neutralization studies (Table 1). Infected cells stained specifically by IFA with antiBHV-4 serum but not with anticapripoxvirus FITC conjugate. Calves (numbers XXX and 171) inoculated with the Suez samples passaged first in ECE and subsequently treated with BHV-4 antiserum developed a persistent fever beginning 7 DPI and generalized LSD by 9 DPI. Calf XXX was euthanized 13 DPI for humanitarian reasons. Both calves were seronegative to LSDV at the time of inoculation, and calf 171 developed an NI against LSDV of 3.0 by 28 DPI. The LSDV was isolated from a necropsy skin sample of calf XXX. Foci with rounded cells typical of BHV-4 CPE and
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Table 1. Neutralization studies on the Suez BHK-4 isolate passed twice in goat kidney and 4 times in lamb testicle cells.
other areas with stranded cells typical of LSDV CPE were observed in FBL cells inoculated with the original field sample of skin from the animal sacrificed at Suez. Eosinophilic intranuclear inclusion bodies and variably staining cytoplasmic inclusion bodies were observed in cultures in FBL stained with HE. Fluorescent antibody staining revealed both capripoxvirus and BHV-4 viral antigens. A suspension of the biopsy from the Ismalia calf inoculated into FBL cultures produced typical capripoxvirus stranding beginning 5 DPI. The virus was specifically neutralized by LSD antiserum, and infected cultures showed specific fluorescence with an anticapripoxvirus conjugate but not by IFA with anti-BHV-4 serum. Two calves (numbers 3 and 4) used to titrate the virus by intradermal inoculation became febrile 16 DPI. Generalized LSD occurred 20 DPI. Intradermal titration in the 2 calves gave respective titers of 106.0 and 1O6.3/0.1 ml; the viral titer in passages 1, 2, and 3 5.0 5.5 5.6 in FBL cells was 10 10 , and 10 /0.1 ml, respectively. From 19 DPI until they were sacrificed on 26 DPI, both calves had a persistent fever above 40.5 C. Both calves were seronegative on 0 DPI and had LSDNI values of 2.8 and 3.0 at 26 DPI. Lumpy skin disease virus was isolated from skin biopsies and heparinized blood collected 20 DPI from both calves. Three of the 7 serum samples obtained from the government farm at Suez approximately 6 months after the outbreak of LSD were positive for antibody to LSDV. The acutely ill calf and 1 affected cow from Ismalia were negative for antibody to LSDV; 5 cattle were seropositive for LSD. Necropsy lesions (composite of experimentally inoculated calves XXX, 171, 3, and 4). Multiple skin nodules were present over the entire body surface. Singular oval to circular lesions varied in size from several millimeters to 2-4 cm in diameter, whereas some lesions became confluent and formed nodular plaques. Lesions in the upper respiratory system were often surrounded by a distinct red border, whereas others had a pale gray center. Superficial lymph nodes were
Figure 2. An intraepithelial microvesicle with acantholysis. Acanthocytes are free in the fibrinous debris. Note the severe necrosis and edema of the dermal papilla (HE).
edematous and enlarged 3-8 times normal size. Focal lesions, with a circular red border, were evident around the accessory digits and the coronary band. Internal respiratory tract lesions noted in the turbinates, pharynx, larynx and trachea varied in size but were usually oval to circular and 0.5-3 cm in diameter. The periphery of the grey to white lesions was slightly raised. White, slightly raised plaques were present on the tunica vaginalis with adhesions to the testicle. Histopathology. The skin of the lower lip and chin
Figure 3. Dermal necrosis with a pox cell (arrow) showing central nuclear clearing and an eosinophilic intracytoplasmic inclusion body; at-rector pili muscle bundle in lower right (HE).
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Ultrastructural examination of the epidermis from the planum nasale. Ultrastructural features included swollen epithelial cells and wide separation of epithelial cells stretching the desmosomal connections. Viral factories consisting of masses of electron-dense viroplasm mixed with mature virions were located in the cytoplasm. The mature virions measured approximately 150 x 320 nm by TEM and consisted of dumbbell-shaped cores, outer membranes, intermediate laminae, and lateral bodies. There was marked margination of nuclear chromatin and rarefaction of the central nucleoplasm.
Figure 4. Vasculitis in an arteriole of the deep dermis with extensive dermal necrosis (HE).
showed acanthosis, including elongation of rete-ridges. The acanthotic skin and hair follicles had locally extensive epidermal necrosis with ballooning degeneration coalescing into multiple intraepithelial (Fig. 2) or subcorneal microvesicles with acantholysis. The microvesicles coalesced to form larger vesicles. Individual cells in the epithelium were necrotic, and some epithelial cells had vacuolated nuclei and a round to oval eosinophilic intracytoplasmic inclusion body (EICIB). These latter cells, known as “pox cells,” were found in most affected tissues, including keratinocytes, dermis (Fig. 3), respiratory epithelial cells, and sebaceous gland epithelium. There was both superficial and deep dermatitis. Necrosis and mononuclear inflammation were also present in the sebaceous glands. There was extensive edema of the dermal papillae (Fig. 2) with some hemorrhage. A vasculitis with a mononuclear cell perivascular reaction (Fig. 4) with thrombosis leading to necrosis was evident in the dermis. There was lymphoid hyperplasia and reticuloendothelial hyperplasia. The pseudostratified and stratified squamous epithelial surfaces of the posterior pharynx, planum nasale, leg, abdomen, eyelids, and claws had similar microscopic lesions as described for the skin. The turbinates had severe diffuse necrosis with squamous metaplasia and pox cells in the epithelium. The necrosis included blood vessels, interstitial tissue, and cartilage. The testicular tunics had extensive diffuse necrosis extending into the epididymus, with massive fibrin deposits, and a mixed cellular infiltrate of mononuclear cells with EICIB’s and nuclear vacuolation. The submandibular lymph node had lymphoid hyperplasia and sinusoid reticuloendothelial hyperplasia.
Discussion The isolation and identification of LSDV from cattle at Suez and Ismalia, Egypt, represent the first confirmation of LSDV in Egypt. The occurrence of LSD in Kuwait in 1986-1988 indicates that this insidious disease continues to increase its endemic area. Lumpy skin disease and BHV-4 viruses were isolated from samples of a pool of skin and lymph node from 2 animals with clinical LSD in Suez, Egypt. Samples passaged twice in GK cells yielded only BHV-4, whereas the field material passaged once in ECE contained both viruses. Retrospectively, a suspension of original skin lesion from Suez was inoculated into FBL cell cultures and yielded both LSDV and BHV-4. Other workers have reported the apparent overgrowth of LSDV by BHV-4.1,12 Lumpy skin disease virus titrated by intradermal inoculation of cattle was comparable to viral titers in FBL cells after 2 passages. Not enough replicates were done to perform statistical analysis, and the virus used was already passed twice in FBL cells. The presence of a herpesvirus in lesions of focal bovine skin disease suggests BHV-2 (bovine herpes mammilitis). However, in Africa, nonpathogenic “Orphan viruses,” now identified as BHV-4,11 have been isolated from cattle with LSD.1 It may be that BHV-4 accompanies mononuclear cells that “home in” on inflammatory processes. The long incubation in cell culture (5-14 days) of both BHV-4 and LSDV represents a difference from BHV-2, which generally causes CPE in 1-4 days. In contrast to BHV-4 and LSDV, BHV-2 usually induces syncytia. Caution must be taken to prevent cross-contamination in laboratories working with sheep and goat pox virus (SGPV), because polyclonal antibodies cannot distinguish between LSDV and SGPV.4,6,12 In the present study, the isolated viruses were neutralized by LSDV antiserum and caused generalized LSD in cattle. The SGPV virus may cause a nodule at the site of intraderma1 inoculation into cattle but does not cause a generalized disease.4 Restriction endonuclease studies may be useful to identify the host preference of capripox-
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virus isolates3 and appear to place the capripoxviruses Sources and manufacturers into 2 groups: I) SGPV and 2) LSDV. a. National Veterinary Services Laboratories, Ames, IA 50010. In both the Suez and Ismalia LSD outbreaks, only b. Nunc, Inc., 2000 N. Aurora Road, Naperville, IL 60566. cattle, not sheep, goats, or water buffalo (Bulbulis bulc. Dif-Quik, Dade Diagnostic, Inc., Aguada, PR 00602. bulis), developed LSD. Experimental infection of sero- d. Leitz Diaplan, E. Leitz, Inc., Rockleigh, NJ 07647. negative African buffalo (Synecus caffer) failed to pro- The use of a particular manufacturer’s product does not constitute duce any clinical disease. l5 The natural host range of an endorsement on behalf of the USDA. LSDV appears limited to cattle. References Studies on cattle serum samples taken 6 months after 1. Alexander RA, Plowright W, Haig DA: 1957, Cytopathogenic the outbreak of LSD at Suez indicated 3 of 7 samples agents associated with lumpy-skin disease of cattle. Bull Epizoot were positive for antibody. This finding may reflect a Dis Afr 5:489-492. low morbidity. Five of the 6 samples from affected 2. Anonymous: 1988, Lumpy skin disease, vol. 1, no. 1. O.I.E., cattle at Ismalia were positive for antibody to LSDV, Disease Information, Paris, France. whereas the acutely affected calf had not yet developed 3. Black DN, Hammond JM, Kitching RP: 1986, Genomic relationship between capripoxviruses. Virus Res 5:277-292. antibody. 4. Capstick PB: 1959, Lumpy skin disease-experimental infecPathological evaluation of skin lesions at various tion. Bull Epizoot Dis Afr 7:51-62. stages of development is important. Sitfasts may be 5. Cottral GE: 1978, Manual of standardized methods for vetrewarding for routine pathological evaluation. Other erinary microbiology. Cornell University Press, Ithaca, NY. workers have described EICIB in numerous cell types 6. Davies FG, Otema C: 1981, Relationships of capripoxviruses found in Kenya with two Middle Eastern strains and some orin early lesions of LSD. 9,14 In acute lesions, the presence thopoxviruses. Res Vet Sci 31:253-255. of intraepithelial acantholysis with ballooning degen7. MacDonald RAS: 1931, Pseudo-urticaria of cattle. Department eration and vesiculation concurrently with a deep vasof Animal Health, Government of Northern Rhodesia, Annual culitis always indicated a systemic disease (infectious Report 1930. pp. 20-21. or immune mediated). Urticaria, insect and tick bites, 8. MacOwen KDS: 1959, Observation on the epizootiology of lumpy skin disease during the first year of its occurrence in or insect stings may mimic LSD clinically, but the Kenya. Bull Epizoot Dis Afr 7:7-20. absence of eosinophils and the presence of a deep vas9. Prozesky L, Barnard BJH: 1982, A study of the pathology of culitis should rule these conditions out. Herpesvirus lumpy skin disease in cattle. Onderstepoort J Vet Res 49:167infection can be differentiated from LSD by examining 175. early lesions for typical eosinophilic intranuclear in- 10. The TH, Feltkamp TEW: 1970, Conjugation of fluorescein isothiocyanate antibodies. II. A reproducible method. Immuelusion bodies. Cutaneous lymphosarcoma streptonology 18:875-881. thrichosis and tuberculosis can also be differentiated 11. Theodoridis A: 1978, Preliminary characterization of viruses histologically. The extensive replication of LSDV as isolated from cases of epididymitis and vaginitis in cattle. Onindicated by EICIB in keratinocytes and germinal cells derstepoort J Vet Res 45: 187-195. of affected hair follicles and sebaceous glands was note- 12. Weiss KE: 1963, Lumpy skin disease. Emerging diseases of animals. FAO Agric Stud Bull 61:179-201. worthy. Acknowledgements We express our sincere thanks to Marjory E. Llewellyn and Rebecca Carter at the FADDL and the support staff of the Animal Health Research Institute for their excellent technical assistance, the animal caretakers at the FADDL, Mr. Stephen E. Perlman, Librarian at the PIADC, and Antone Dobek, photographer, Dr. Douglas A. Gregg for electron microscopy and helpful suggestions, and Dr. Charles A. Mebus for his helpful discussions.
13. Woods JA: 1988, Lumpy skin disease-a review. Trop Anim Health Prod 20:11-17. 14. Yager JA, Scott DW: 1985, The skin and appendages, In: Pathology of domestic animals, ed. Jubb KVF, Kennedy PC, Palmer N, 3rd ed., vol. 1, pp. 407-549. Academic Press, New York, NY. 15. Young E, Basson PA, Weiss KE: 1970, Experimental infection of game animals with lumpy skin disease virus (prototype strain Neethling). Onderstepoort J Vet Res 37:79-88.
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The Isolation of Lumpy Skin Disease Virus and Bovine Herpesvirus- from Cattle in Egypt James A. House, Terrance M. Wilson, Sinan El Nakashly, Ikram A. Karim, Ibrahim Ismail, Nabil El Danaf, Aly M. Moussa and Nazmi N. Ayoub J VET Diagn Invest 1990 2: 111 DOI: 10.1177/104063879000200205 The online version of this article can be found at: http://vdi.sagepub.com/content/2/2/111
Published by: http://www.sagepublications.com
On behalf of:
Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.
Additional services and information for Journal of Veterinary Diagnostic Investigation can be found at: Email Alerts: http://vdi.sagepub.com/cgi/alerts Subscriptions: http://vdi.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://vdi.sagepub.com/content/2/2/111.refs.html
>> Version of Record - Apr 1, 1990 What is This?
Downloaded from vdi.sagepub.com at Bibliotheque de l'Universite Laval on July 5, 2014
J Vet Diagn Invest 2:111-115 (1990)
The isolation of lumpy skin disease virus and bovine herpesvirus- from cattle in Egypt James A. House, Terrance M. Wilson, Sinan El Nakashly, Ikram A. Karim, Ibrahim Ismail, Nabil El Danaf, Aly M.Moussa, Nazmi N. Ayoub Abstract. Lumpy skin disease (LSD) virus (LSDV) was isolated for the first time from cattle in Egypt in 2 disease outbreaks. Bovine herpesvirus-4 (BHV-4) and LSDV were detected in a pooled sample from the first outbreak (Suez). Only LSDV was isolated from the second outbreak (Ismalia). The capripoxviruses were identified as LSDV by neutralization with specific antiserum and by their ability to produce generalized LSD in experimentally inoculated cattle.
Lumpy skin disease (LSD), a generalized skin disease of cattle caused by a capripoxvirus, was first described in northern Rhodesia in 1929.7 The disease spread in sporadic epizootics, reaching South Africa in 1944 and Kenya in 1957.8 Since 1973, LSD has spread to western and northeastern Africa. l3 The first reports of LSD outside of Africa were from Kuwait in 1986-1988.2 This report describes the first diagnosis of LSD in Egypt, whose geographical location may allow LSD to spread to Europe and further in Asia. Materials and methods Clinical history and sample collection. Suez, Egypt: On June 2, 1988, a disease clinically resembling LSD was reported in Holstein cattle at a government farm near the quarantine station in Suez, Egypt. Fourteen of 194 cattle were affected with fever, increased salivation, generalized deep skin nodules, enlarged peripheral lymph nodes (LN), and in some cases respiratory signs. Clinically normal cattle from Somalia had been moved into the quarantine station in early May 1988. Forty-seven buffalo and 280 sheep in contact with the affected cattle remained clinically healthy. A severely affected animal was slaughtered, and skin, muscle, prescapular LN, and fluid from a skin lesion were collected for virus isolation. A skin biopsy was also collected from another affected bull. Samples were kept frozen at -70 C until used for virus isolation. Approximately 6 mo postexpsoure, serum was collected from blood obtained from 7 cattle present during the June LSD outbreak. Ismalia, Egypt: On October 31, 1988, an LSD-like disease was reported in the village of Tel El Kabir in the govemorate of Ismalia about 50 km from Suez. On November 30, 1988, approximately 50% of 250 native cattle (Balidy) had deep From the Foreign Animal Disease Diagnostic Laboratory (FADDL), US Department of Agriculture, Science and Technology National Veterinary Services Laboratories, Box 848, Greenport, NY 11944 (House, Wilson), Animal Health Research Institute, Dokki, Cairo, Egypt (Nakashly, Karim, Ismail, El Danaf, Ayoub), and General Organization of Veterinary Service, Cairo, Egypt (Moussa). Received for publication September 28, 1989. 111
skin nodules ranging in number from a few up to 300 (Fig. 1). Sitfasts and purulent discharge from lesions with secondary bacterial involvement, fever, increased salivation, and emaciation were observed. Three young calves had severe LSD. One 3-mo-old calf was in lateral recumbency with extensor rigidity of the forelimbs, paddling of the hind legs, and opisthotonus. Four adult water buffalo, each with a calf, and at least 50 sheep were in direct contact with the affected cattle but remained clinically normal. A biopsy was collected from a skin nodule of a 3-mo-old bull calf clinically ill for 3 days. Blood was collected from a calf ill for 4 days and from 6 cattle clinically affected for 1-6 wk. Cultures for virus isolation. Eight-day-old embryonating chicken eggs (ECE) were inoculated with samples from the Suez outbreak via the chorioallantoic membrane (CAM) route. Primary goat kidney (GK) cultures, fetal bovine lung (FBL) cells (first to seventh passage), and newborn lamb testicle (LT) cells (first to fifth passage) propagated with Eagle’s minimum essential medium (EMEM) supplemented with 5% fetal bovine serum (FBS) were used for virus isolation. Viruses. Bovine herpesvirus- 1 a (BHV-1) Colorado strain, bovine herpesvirus- (BHV-2) Minnesota strain, bovine herpesvirus-4a (BHV-4) DN 599 strain, alcelaphine herpesvirus-l (AHV-1) Minnesota strain, and LSD virus (LSDV) Kenyan strain B2490 were used for reference purposes. Antisera. Reference antisera to BHV-1,a BHV-2, BHVa 4, AHV-1, and LSDV were used for virus identification. Virus isolation studies in cell cultures and cattle. Samples from Suez: Field samples of muscle, skin, lymph node, and lesion vesicular fluid were passaged twice in GK. Approximately 1 ml of cell culture fluid from each sample was inoculated intradermally (ID) in individual sites in the neck of 2 5-mo-old mixed-breed calves (numbers 173 and 175) after collecting a 0-day-postinoculation (0 DPI) blood sample. The calves were housed in high containment (> P3) at the Foreign Animal Disease Diagnostic Laboratory (FADDL). These samples were also inoculated into LT cultures, which were observed daily and passaged weekly for up to 3 wk, and examined by histological staining. Cell cultures were propagated on 8-chamber slides.b Cultures exhibiting cytopathogenic effect (CPE) were fixed with absolute methanol for 5 min or with Bouin’s fixative until stained. Fixed cultures
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House et al.
Figure 1. Skin lesions of LSD in an adult Balidy cow, Tel El Kabir, Egypt.
were stained with either xanthine dye/thiazine dye mixture” or hematoxylin and eosin (HE) stains. Additionally, cells grown on g-chambered slides were fixed with acetone at - 20 C and stained with anticapripoxvirus direct fluorescein isothiocyanate (FITC) conjugate10 prepared from caprine origin convalescent goat pox serum and read with a fluorescence microscope.d An indirect fluorescent antibody (IFA) test was performed on fixed cells, using specific antiserum and then antispecies gamma globulin FITC conjugate. Virus isolates were identified by neutralization index studies. Chorioallantoic fluid from the Suez samples was passaged 3 times on LT cells. The nature of the CPE, i.e., rounding of cells and some stranding, especially during the first passage, suggested a mixed viral infection. Stained cell cultures revealed eosinophilic intranuclear inclusion bodies and some variable staining intracytoplasmic inclusion bodies. Anticapripoxvirus FITC conjugate revealed specific staining in a small percentage of the cells. The original ECE passage fluid was mixed with antiserum to BHV-4 prior to each of 2 passages in FBL cells. Both passages developed CPE similar to that of capripoxviruses. Two 450-cm2 roller bottles of FBL cells were inoculated with passage 2 viral fluid and harvested in 5 days at 90% CPE. The resulting virus isolate was not neutralized by anti-BHV-4 serum but was identified as a capripoxvirus with LSD-specific antiserum (titer reduced by 103) and designated the Suez LSD viral isolate. One milliliter of this virus, with a titer of 106.9 TCID50/ml in FBL cells, was used to inoculate each of 2 5-mo-old mixed-breed calves (numbers XXX and 171) by needle prick in the skin of the neck; 9 ml was given intravenously after obtaining a 0 DPI serum sample. A sample of a nodule from calf XXX was collected at the time of necropsy for virus isolation. A suspension of the original skin lesion from the animal sacrificed at Suez was inoculated into FBL cells and passaged twice. Monolayers were stained by HE and direct FA procedures. Samples from Ismalia. The Ismalia calf skin nodule biopsy sample was passaged twice in FBL cells. An aliquot of the clarified culture fluid was diluted lo-fold, and each of 2 5-mo-old mixed-breed calves (numbers 3 and 4) was inoc-
ulated ID with 0.1 ml in 5 sites for each lo-fold dilution (10-4-10-9) after obtaining a 0 DPI serum sample. The sample used for cattle inoculation was titrated in FBL cells. Microtiter plates were examined by microscopy prior to freezing after 7 days of incubation. After thawing, 0.1 ml of culture fluid was transferred into respective wells of a second plate of FBL cells. A third passage was similarly done. Titers were calculated by the Spearman-Karber method of determining 50% endpoints.5 The animals were observed, and their temperatures were taken daily for 26 days. Heparinized blood and skin nodule biopsies were collected for virus isolation at 20 DPI. The animals were necropsied and tissues taken for histopathological examination. Neutralization index (NI) test. The NI test using serum at a final 1:10 dilution was performed as described5 using 10,000 FBL cells/microtiter well. Neutralization indices ≥ 1.5 were considered positive for antibody.5 An NI using pre- and postimmune LSD serum was used for virus identification. Indirectfluorescent antibody test. Monolayers of FBL cells were infected with BHV-4 to yield approximately 30% of the cells infected at day 2 when culture fluid was decanted. The monolayers were rinsed with phosphate-buffered saline (PBS), fixed with acetone at -20 C for 10 min, air dried, and stored at -70 C until used. Serum diluted 1:10 in PBS was used in a traditional screening IFA test. Titrations were performed using 2-fold serum dilutions; the endpoint titer was the highest dilution showing specific fluorescence. Histological methods. Biopsies and tissues obtained at necropsy were fixed in formalin, sectioned at 4-5 µm, stained with HE, and examined by light microscopy. After visualization by light microscopy, selected areas of formalin-fixed tissues were obtained from the planum nasale and routinely postfixed for ultrastructural examination by transmission electron microscopy (TEM).
Results Calves (numbers 173 and 175) inoculated with Suez samples passaged in GK did not develop signs of illness. No neutralizing antibody was detected in the 28 DPI sera against BHV-1, BHV-2, AHV-1, or LSDV, but IFA titers of 1:80 against BHV-4 were detected. No antibody was detected in the 0 DPI sera to any of the 5 viruses. All inoculated LT cell cultures had eosinophilic intranuclear inclusion bodies, and BHV-4 was identified by virus neutralization studies (Table 1). Infected cells stained specifically by IFA with antiBHV-4 serum but not with anticapripoxvirus FITC conjugate. Calves (numbers XXX and 171) inoculated with the Suez samples passaged first in ECE and subsequently treated with BHV-4 antiserum developed a persistent fever beginning 7 DPI and generalized LSD by 9 DPI. Calf XXX was euthanized 13 DPI for humanitarian reasons. Both calves were seronegative to LSDV at the time of inoculation, and calf 171 developed an NI against LSDV of 3.0 by 28 DPI. The LSDV was isolated from a necropsy skin sample of calf XXX. Foci with rounded cells typical of BHV-4 CPE and
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Table 1. Neutralization studies on the Suez BHK-4 isolate passed twice in goat kidney and 4 times in lamb testicle cells.
other areas with stranded cells typical of LSDV CPE were observed in FBL cells inoculated with the original field sample of skin from the animal sacrificed at Suez. Eosinophilic intranuclear inclusion bodies and variably staining cytoplasmic inclusion bodies were observed in cultures in FBL stained with HE. Fluorescent antibody staining revealed both capripoxvirus and BHV-4 viral antigens. A suspension of the biopsy from the Ismalia calf inoculated into FBL cultures produced typical capripoxvirus stranding beginning 5 DPI. The virus was specifically neutralized by LSD antiserum, and infected cultures showed specific fluorescence with an anticapripoxvirus conjugate but not by IFA with anti-BHV-4 serum. Two calves (numbers 3 and 4) used to titrate the virus by intradermal inoculation became febrile 16 DPI. Generalized LSD occurred 20 DPI. Intradermal titration in the 2 calves gave respective titers of 106.0 and 1O6.3/0.1 ml; the viral titer in passages 1, 2, and 3 5.0 5.5 5.6 in FBL cells was 10 10 , and 10 /0.1 ml, respectively. From 19 DPI until they were sacrificed on 26 DPI, both calves had a persistent fever above 40.5 C. Both calves were seronegative on 0 DPI and had LSDNI values of 2.8 and 3.0 at 26 DPI. Lumpy skin disease virus was isolated from skin biopsies and heparinized blood collected 20 DPI from both calves. Three of the 7 serum samples obtained from the government farm at Suez approximately 6 months after the outbreak of LSD were positive for antibody to LSDV. The acutely ill calf and 1 affected cow from Ismalia were negative for antibody to LSDV; 5 cattle were seropositive for LSD. Necropsy lesions (composite of experimentally inoculated calves XXX, 171, 3, and 4). Multiple skin nodules were present over the entire body surface. Singular oval to circular lesions varied in size from several millimeters to 2-4 cm in diameter, whereas some lesions became confluent and formed nodular plaques. Lesions in the upper respiratory system were often surrounded by a distinct red border, whereas others had a pale gray center. Superficial lymph nodes were
Figure 2. An intraepithelial microvesicle with acantholysis. Acanthocytes are free in the fibrinous debris. Note the severe necrosis and edema of the dermal papilla (HE).
edematous and enlarged 3-8 times normal size. Focal lesions, with a circular red border, were evident around the accessory digits and the coronary band. Internal respiratory tract lesions noted in the turbinates, pharynx, larynx and trachea varied in size but were usually oval to circular and 0.5-3 cm in diameter. The periphery of the grey to white lesions was slightly raised. White, slightly raised plaques were present on the tunica vaginalis with adhesions to the testicle. Histopathology. The skin of the lower lip and chin
Figure 3. Dermal necrosis with a pox cell (arrow) showing central nuclear clearing and an eosinophilic intracytoplasmic inclusion body; at-rector pili muscle bundle in lower right (HE).
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House et al.
Ultrastructural examination of the epidermis from the planum nasale. Ultrastructural features included swollen epithelial cells and wide separation of epithelial cells stretching the desmosomal connections. Viral factories consisting of masses of electron-dense viroplasm mixed with mature virions were located in the cytoplasm. The mature virions measured approximately 150 x 320 nm by TEM and consisted of dumbbell-shaped cores, outer membranes, intermediate laminae, and lateral bodies. There was marked margination of nuclear chromatin and rarefaction of the central nucleoplasm.
Figure 4. Vasculitis in an arteriole of the deep dermis with extensive dermal necrosis (HE).
showed acanthosis, including elongation of rete-ridges. The acanthotic skin and hair follicles had locally extensive epidermal necrosis with ballooning degeneration coalescing into multiple intraepithelial (Fig. 2) or subcorneal microvesicles with acantholysis. The microvesicles coalesced to form larger vesicles. Individual cells in the epithelium were necrotic, and some epithelial cells had vacuolated nuclei and a round to oval eosinophilic intracytoplasmic inclusion body (EICIB). These latter cells, known as “pox cells,” were found in most affected tissues, including keratinocytes, dermis (Fig. 3), respiratory epithelial cells, and sebaceous gland epithelium. There was both superficial and deep dermatitis. Necrosis and mononuclear inflammation were also present in the sebaceous glands. There was extensive edema of the dermal papillae (Fig. 2) with some hemorrhage. A vasculitis with a mononuclear cell perivascular reaction (Fig. 4) with thrombosis leading to necrosis was evident in the dermis. There was lymphoid hyperplasia and reticuloendothelial hyperplasia. The pseudostratified and stratified squamous epithelial surfaces of the posterior pharynx, planum nasale, leg, abdomen, eyelids, and claws had similar microscopic lesions as described for the skin. The turbinates had severe diffuse necrosis with squamous metaplasia and pox cells in the epithelium. The necrosis included blood vessels, interstitial tissue, and cartilage. The testicular tunics had extensive diffuse necrosis extending into the epididymus, with massive fibrin deposits, and a mixed cellular infiltrate of mononuclear cells with EICIB’s and nuclear vacuolation. The submandibular lymph node had lymphoid hyperplasia and sinusoid reticuloendothelial hyperplasia.
Discussion The isolation and identification of LSDV from cattle at Suez and Ismalia, Egypt, represent the first confirmation of LSDV in Egypt. The occurrence of LSD in Kuwait in 1986-1988 indicates that this insidious disease continues to increase its endemic area. Lumpy skin disease and BHV-4 viruses were isolated from samples of a pool of skin and lymph node from 2 animals with clinical LSD in Suez, Egypt. Samples passaged twice in GK cells yielded only BHV-4, whereas the field material passaged once in ECE contained both viruses. Retrospectively, a suspension of original skin lesion from Suez was inoculated into FBL cell cultures and yielded both LSDV and BHV-4. Other workers have reported the apparent overgrowth of LSDV by BHV-4.1,12 Lumpy skin disease virus titrated by intradermal inoculation of cattle was comparable to viral titers in FBL cells after 2 passages. Not enough replicates were done to perform statistical analysis, and the virus used was already passed twice in FBL cells. The presence of a herpesvirus in lesions of focal bovine skin disease suggests BHV-2 (bovine herpes mammilitis). However, in Africa, nonpathogenic “Orphan viruses,” now identified as BHV-4,11 have been isolated from cattle with LSD.1 It may be that BHV-4 accompanies mononuclear cells that “home in” on inflammatory processes. The long incubation in cell culture (5-14 days) of both BHV-4 and LSDV represents a difference from BHV-2, which generally causes CPE in 1-4 days. In contrast to BHV-4 and LSDV, BHV-2 usually induces syncytia. Caution must be taken to prevent cross-contamination in laboratories working with sheep and goat pox virus (SGPV), because polyclonal antibodies cannot distinguish between LSDV and SGPV.4,6,12 In the present study, the isolated viruses were neutralized by LSDV antiserum and caused generalized LSD in cattle. The SGPV virus may cause a nodule at the site of intraderma1 inoculation into cattle but does not cause a generalized disease.4 Restriction endonuclease studies may be useful to identify the host preference of capripox-
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virus isolates3 and appear to place the capripoxviruses Sources and manufacturers into 2 groups: I) SGPV and 2) LSDV. a. National Veterinary Services Laboratories, Ames, IA 50010. In both the Suez and Ismalia LSD outbreaks, only b. Nunc, Inc., 2000 N. Aurora Road, Naperville, IL 60566. cattle, not sheep, goats, or water buffalo (Bulbulis bulc. Dif-Quik, Dade Diagnostic, Inc., Aguada, PR 00602. bulis), developed LSD. Experimental infection of sero- d. Leitz Diaplan, E. Leitz, Inc., Rockleigh, NJ 07647. negative African buffalo (Synecus caffer) failed to pro- The use of a particular manufacturer’s product does not constitute duce any clinical disease. l5 The natural host range of an endorsement on behalf of the USDA. LSDV appears limited to cattle. References Studies on cattle serum samples taken 6 months after 1. Alexander RA, Plowright W, Haig DA: 1957, Cytopathogenic the outbreak of LSD at Suez indicated 3 of 7 samples agents associated with lumpy-skin disease of cattle. Bull Epizoot were positive for antibody. This finding may reflect a Dis Afr 5:489-492. low morbidity. Five of the 6 samples from affected 2. Anonymous: 1988, Lumpy skin disease, vol. 1, no. 1. O.I.E., cattle at Ismalia were positive for antibody to LSDV, Disease Information, Paris, France. whereas the acutely affected calf had not yet developed 3. Black DN, Hammond JM, Kitching RP: 1986, Genomic relationship between capripoxviruses. Virus Res 5:277-292. antibody. 4. Capstick PB: 1959, Lumpy skin disease-experimental infecPathological evaluation of skin lesions at various tion. Bull Epizoot Dis Afr 7:51-62. stages of development is important. Sitfasts may be 5. Cottral GE: 1978, Manual of standardized methods for vetrewarding for routine pathological evaluation. Other erinary microbiology. Cornell University Press, Ithaca, NY. workers have described EICIB in numerous cell types 6. Davies FG, Otema C: 1981, Relationships of capripoxviruses found in Kenya with two Middle Eastern strains and some orin early lesions of LSD. 9,14 In acute lesions, the presence thopoxviruses. Res Vet Sci 31:253-255. of intraepithelial acantholysis with ballooning degen7. MacDonald RAS: 1931, Pseudo-urticaria of cattle. Department eration and vesiculation concurrently with a deep vasof Animal Health, Government of Northern Rhodesia, Annual culitis always indicated a systemic disease (infectious Report 1930. pp. 20-21. or immune mediated). Urticaria, insect and tick bites, 8. MacOwen KDS: 1959, Observation on the epizootiology of lumpy skin disease during the first year of its occurrence in or insect stings may mimic LSD clinically, but the Kenya. Bull Epizoot Dis Afr 7:7-20. absence of eosinophils and the presence of a deep vas9. Prozesky L, Barnard BJH: 1982, A study of the pathology of culitis should rule these conditions out. Herpesvirus lumpy skin disease in cattle. Onderstepoort J Vet Res 49:167infection can be differentiated from LSD by examining 175. early lesions for typical eosinophilic intranuclear in- 10. The TH, Feltkamp TEW: 1970, Conjugation of fluorescein isothiocyanate antibodies. II. A reproducible method. Immuelusion bodies. Cutaneous lymphosarcoma streptonology 18:875-881. thrichosis and tuberculosis can also be differentiated 11. Theodoridis A: 1978, Preliminary characterization of viruses histologically. The extensive replication of LSDV as isolated from cases of epididymitis and vaginitis in cattle. Onindicated by EICIB in keratinocytes and germinal cells derstepoort J Vet Res 45: 187-195. of affected hair follicles and sebaceous glands was note- 12. Weiss KE: 1963, Lumpy skin disease. Emerging diseases of animals. FAO Agric Stud Bull 61:179-201. worthy. Acknowledgements We express our sincere thanks to Marjory E. Llewellyn and Rebecca Carter at the FADDL and the support staff of the Animal Health Research Institute for their excellent technical assistance, the animal caretakers at the FADDL, Mr. Stephen E. Perlman, Librarian at the PIADC, and Antone Dobek, photographer, Dr. Douglas A. Gregg for electron microscopy and helpful suggestions, and Dr. Charles A. Mebus for his helpful discussions.
13. Woods JA: 1988, Lumpy skin disease-a review. Trop Anim Health Prod 20:11-17. 14. Yager JA, Scott DW: 1985, The skin and appendages, In: Pathology of domestic animals, ed. Jubb KVF, Kennedy PC, Palmer N, 3rd ed., vol. 1, pp. 407-549. Academic Press, New York, NY. 15. Young E, Basson PA, Weiss KE: 1970, Experimental infection of game animals with lumpy skin disease virus (prototype strain Neethling). Onderstepoort J Vet Res 37:79-88.
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