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  1. 1. ISRAEL JOURNAL OF VETERINARY MEDICINE LUMPY SKIN DISEASE (LSD) IN A LARGE DAIRY HERD IN ISRAEL, JUNE 2006 1 Brenner, J., 2 Haimovitz, M., 3 Oron E., 1 Stram, Y., 1 Fridgut, O., 1 Bumbarov, V. 1 Kuznetzova, L., 2 Oved, Z., 2 Waserman, A., 2 Garazzi, S., 4 Perl, S., 4 Lahav, D., 4 Edery, N. and 1 Yadin, H. 1 Division of Virology, 2 Veterinary Services, 50250 Bet Dagan 3 “Hacklait” Caesarea, Israel 4 Department of Pathology, Kimron Veterinary Institute Summary This communication provides details on an outbreak of LSD in Israel and describes the clinical and epidemiological aspects of the infection directed at clinicians in particular. This manuscript highlights the rapidity with which diagnostic confirmation was made in the 2006 outbreak, based on the intellectual property and laboratory tools acquired by the Israel Veterinary Services and the diagnostic laboratories of the Kimron Veterinary Institute from the previous LSD outbreak in Israel in 1989. Introduction Lumpy skin disease (LSD) presents as an acute, sub-acute or inapparent infectious disease of cattle caused by a single strain of capripox virus known as Neethling virus. It is characterized by rapid eruption of multiple circumscribed skin nodules, and generalized lymphadenitis and fever and may result in mastitis and orchitis (3, 4). Other lesions visible at post-mortem examination include necrotic plaques in the membranes, chiefly of the upper respiratory tract, the oral cavity and rumen. Historically, the stable fly Stomoxys calcitrans Linnaeus was the arthropod thought most likely to have a role in the epidemiology of LSD. This was based on virus isolation from flies that had fed on infected cattle (5).In a literature search, one report claims that the female mosquito, Aedes aegypti (Diptera: Culicidae) is capable of the mechanical transmission of LSDV from infected to susceptible cattle, but the clinical disease recorded in the animals experimentally exposed to infected mosquitoes, was mild in nature only (6). In addition, an attempt at mechanical transmission of LSDV by biting insects failed (7). There is a report from Israel in 1989 of the probable role of biting insects in the epidemiology of LSD , when the transmission was attributed to infected, wind- borne S. calcitrans from Egypt (1). A further report is cited in the OIE fact-sheet "Transmission of the LSD virus is primarily by biting insects, particularly mosquitoes (e.g. Culex mirificens and Aedes natrionus) and flies (e.g.Stomoxys calcitrans and Biomyia fasciata)"( LSD is an infectious disease primarily confined to the African continent (3). It first appeared outside Africa in Kuwait in 1986. A LSD outbreak also occurred in 1989 in Israel in Peduyim and two neighbouring villages (1). The outbreak was controlled by
  2. 2. culling all the animals in the village and performing ring vaccination with a sheep pox vaccine (1, 8). Additionally, Israeli investigators assessed the efficacy of the sheep- pox vaccine, RM-65 strain, by performing a controlled challenge study with vaccinated and non-vaccinated calves in the high containment unit of the Kimron Veterinary Institute (2). The Israeli wild-type strain that was first isolated by Abraham and Zissman in 1989 (9) served as the challenge virus. Historical and more recent overview In 1929, LSD was described as a clinical manifestation, termed “pseudo-urticaria” in Zambia (10), and was recognized as an infectious disease in 1943 (11). The first report outside Africa was described in Kuwait in 1986, where 642 cases were reported in cattle (12). Since then cases have been confirmed or suspected in the United Arab Emirates, Arab Republic of Yemen, Democratic People’s Republic of Yemen (13), and Israel in 1989. The 1989 LSD episode occurred at Peduyim and was probably an extension of the Egyptian outbreak (1, 8, 14). Since LSD virus was also reported in Saudi Arabia in 1992 (15), and was mentioned in other publications it is possible that LSD virus circulates endemically in the Middle East. Currently an epidemic of LSDV is affrcting 16 provinces in Egypt ( This epidemiological situation is similar to that encountered in Israel during the 1989 outbreak (1), posing the same risks and probably bringing the same consequences to the Israeli dairy industry. Since veterinary practitioners working with cattle are the first to note or become aware of any clinical changes in dairy herds, the information provided in this communication is an essential part of establishing control measures. The rapidity of communication stemming from prompt diagnosis depends very much on diagnosis at the farm. The main aim of this manuscript, therefore, is to give updated epidemiological information about LSD and provide concise clinical information to assist dairy practitioners by identifying lumps and other dermal lesions related to LSD virus infection, and in including a differential diagnosis of all kinds of bovine skin lesions Materials and Methods The 2006 LSD outbreak and its clinical description On the evening of 19.6.06, an urgent phone call massage was made announcing the appearance of lumpy/urticaria-like lesions in 7 to 10 lactating cows in a large dairy herd. The probable reappearance of LSD was immediately suspected. A team of veterinary officers and virologists was convened to be ready for an early morning visit to the affected herd, and on the next day, the clinical diagnosis was confirmed. Two members of the visiting team had been involved in diagnosis of the 1989 outbreak (1,2), and one of them had participated in the controlled clinical trial to assess the efficacy of a sheep-pox vaccine by performing the intradermal titration of the vaccine on vaccinated and naive young calves (2). The most significant clinical features were the presence of the characteristic lumps that covered the entire skin of the affected cows (Fig.1). All the developmental stages of the lumps could be noted, from the initial miliary scattered form, namely, the presence of small localized intradermal lesions, seen as small intradermal swellings (Fig. 2). These progressed to well developed nodules (Fig. 3), very hard on touch, and
  3. 3. to the ensuing sloughing off process that left a red spot on the surface of the skin. This is the last stage of the lesion and is also the beginning of the healing process (Fig. 4). The stage of this semi-healed lesion can be as long as 2 to 3 weeks (3). Generalized lymphadenopathy could be demonstrated especially of the precrural (Figs. 1,5) and supramammary lymph nodes. Edematous swelling of the lower gluteal muscles could also be noted. At the onset there were only 7 to 10 lactating cows showing the characteristic lumps, while in the successive 10 days an additional 10-12 affected cows were noted. Picture 1. The miliary form. Small circumscribed dermal nodules. At this initial stage, the enlargement of the precrural lymph node is well evident. Picture 2. An aspect of dermal nodules on the udder, these lesions are soft to the touch.
  4. 4. Picture 3. A well circumscribed hard dermal nodule at the beginning of the sloughing off process is present. Picture 4. Two to 3 weeks old lesions are seen in this picture.
  5. 5. Anamnesis taken on the farm referred to unusual abortions that occurred one month before the first cutaneous manifestations, as 4 aborted fetuses has been sent to KVI on 21.05.06. These were probably the first non-specific signs of LSD virus infection (3), and a rectal temperature of 400 - 410C was evident then and during our visit.. Three lumps (dermal nodules) and one lymph node were biopsied for laboratory examinations during the visit. Laboratory confirmation by the virology and molecular virology laboratories of the KVI was completed in less than 24 hours. The skin lumps were fixed in 10% neutral buffered formaldehyde embedded in paraffin, sectioned at 4 micron and stained with hematoxylin-eosin. Interim results indicated that about one third of the dairy herd was affected and therefore was destroyed. The distribution of the affected and destroyed populations until mid-July was as follows: 116 lactating cows, 42 heifers, 32 fattening calves, 5 young female and 10 male calves under 2 months of age (the herd totaled 605 animals at the beginning of the outbreak). One additional confirmed case of LSD occurred in an adult lactating cow on an adjacent dairy farm located within the restricted perimeter imposed by the Veterinary Services. The final numbers of affected animals (morbidity rate) will be evaluated after the belt vaccination is completed, and the disease is considered eradicated or at least under control. Picture 5. A simple clinical procedure is shown for the precrural lymph node. This procedure might help is making a decision regarding a generalized infection.
  6. 6. Results PCR PCR recognized specifically the LSDV using primers developed in the laboratory of molecular virology (Stram, in preparation) (Fig 6). The PCR primers and the positive control were based on the Israeli LSDV isolate LSDI4 (9) and the African strain kindly provided by APHIS NSVL, LSD Ismmaelya strain 3 EBL serial 8903 RGT # V2155, from March 1989. Picture 6. Electron microscopy of the LSDV, 20.06.06.
  7. 7. Electron Microscopy (EM) EM revealed a typical capripoxvirus in one of the dermal biopsies (Fig.7). The EM procedure is described elsewhere and followed that used for LSDV in the 1989 episode (9) Picture 7. PCR product that confirms the LSDV Ein-Zurim strain- 20.06.06 specificity as demonstrated by comparison with two control strains used as positive markers (see details in the M & M) Lane 1. Detection of LSD viral DNA in a skin sample: June 2006 outbreak. Lane 2. Negative control.(H2O)
  8. 8. Lane 3. Positive control (Egyptian isolate Ismaelia 1989). Lane 4. Positive control (Israeli isolate 1989). Lane 5. Negative control (H2O) Histopathology The histopathological examination was aimed to exclude bacterial or fungal involvement in the formation of the characteristic nodules. No such pathogens were observed, while inclusion bodies were present. The epidermis was extensively necrotic, while in the intact areas, some ballooning degeneration of squamous epithelial cells with occasional intra-cytoplasmic inclusions were seen. Some lesions can be seen in the squamous epithelial cells of some of the hair follicles. Prominent lesions of vasculitic necrosis with cell debris and severe diffuse infiltration with inflammatory cells mainly neutrophlis, were seen in the superficial and deep dermis. In some areas they also involved the muscular layers (Fig.8). Pictures 8. (A-C). The epidermis is extensively necrotic (B), ballooning degeneration of squamous epithelial cells with occasional intra-cytoplasmic inclusions are noted (C). Vasculitic necrosis with cell debris and severe diffuse infiltration with inflammatory cells mainly neutrophlis, are seen in the superficial and deep dermis (A).
  9. 9. A B C Clinical Confirmation As mentioned in M&M, the characteristic circumscribed cutaneous nodules, fever, lymphadenopathy (Figs 1-5) and gluteal edema are all consistent with LSDV infection on clinical examination by an expert. Discussion
  10. 10. The primary step in planning an investigation of an outbreak is to establish the goal, which in the case of an infectious disease, is to identify the causative agent. The entire investigation, planning and gathering of data and sample collection must be designed to minimize economic losses to the farmer on one hand and must not affect the reputation of the national veterinary authority on the other hand. This activity embraces many disciplines since it inherently depends on expertise in clinical medicine, epidemiology, microbiology, pathology, and entomology. The preparedness of a national system, such as a veterinary authority and its field and diagnostic laboratory, depends very much on its expertise in organization a campaign in the face of a national epizootic. A methodical approach to the investigation of an outbreak is the best way to achieve the objective of containment or eradication of an exotic disease, the control of an endemic and to prevent reappearance of an eradicated disease. This approach is further justified if the pathogen is on the list of notifiable diseases of the Office International des Epizooties (OIE). The rationale behind the methodology employed to investigate a reemerging disease is fundamentally different from that applied to an endemic disease. Since LSD was reported in Israel in 1989, and the alert announcement reported by the Egyptian Veterinary Authority on the vast spread of LSD in numerous provinces in Egypt, the awareness and therefore the preparedness was raised regarding the possible reappearance of LSD in Israel. Preparedness passed ultimately from the “dormant” state to a real alert. A specific PCR assay was developed by the laboratory of molecular biology (Stram, in preparation) on the base of the African strain and the Israeli isolate (for both strains see M and M) (9). An alert communication was placed on the website and e-mails were sent to all the veterinary practitioners and all the veterinary officers warning about the possible reappearance of LSD. Indeed, a telephone call made to IVS by the local clinician was the first link in the diagnostic chain. Emerging diseases of farm animal diseases pose a real national economic threat, especially, if the causative agent is listed as an exotic pathogen. In 1989, the main reason for the initial misdiagnosis, and postponement of clinical and laboratory diagnosis was related to the unfamiliarity of all the veterinary staff with the clinical forms of LSDV infection. To some degree, this statement was also true of the diagnostic laboratory personnel. The awareness and preparedness of the regional veterinary officers and the diagnostic laboratories of the KVI in the face of the reappearance of this ongoing LSD outbreak was higher as compared to the preceding outbreak, so that the diagnostic chain of events followed the type of the transitional epidemiological presentation (cyclic intervals) as suggested elsewhere (16). The visiting team included two veterinarians who were able to confirm clinically the reappearance of LSD. Both were involved in the 1989 epidemic, one acting as a regional veterinary officer (1), who orchestrated the eradication campaign, and the other as an investigator in a team that had performed the controlled clinical trial to assess the efficacy of the sheep-pox vaccineFortunately, they had enough clinical experience to confirm clinical diagnosis on the spot so that operational precautions could be applied immediately. The field practitioners erroneously considered initially as non-specific cutaneous hypersensitivity the lesions that appeared in both the 1989 and 2006 epidemics. It is
  11. 11. therefore advisable for those that had never been familiar with LSD and other similar lesions such as pseudo-lumpy disease (caused by Allerton strain of bovid herpes virus-2 (4)), to focus their attention on the subcutaneous lymph nodes because their swelling is a helpful sign. If they are visible or palpable, with a generalized disease with an elevated body temperature, an infectious disease must immediately be considered. Such a clinical condition could not be induced by a cutaneous hypersensitivity. Moreover, by observing the characteristic progressive stages of the lumps, only LSD remains as a sole and obvious clinical diagnosis (2). The authors hope that the description of the currently ongoing epidemic, even if no final details are provided, might assist others to be better prepared to face other LSD episodes. By doing so, the elapsed time between the discovery of the outbreak and its termination will be minimized as will consequent economic losses. The mode of LSDV transmission under natural conditions is not fully understood because attempts to infect naive animals by using infected blood-suckling insects, have failed (7). Chiota et al., (6) suggested that mosquito species are competent vectors. They produced only the mild disease form that does not mirror the clinical picture generally seen in the field. According to the data from the previous outbreak in Israel (1, 8), and the outbreak described here, the prevailing disease form in Israel is the “severe generalization” form, as classified by Carn and Kitching (17). Moreover, LSDV was not isolated from secretions of animals with the mild form of LSD (6), so that if the mild form was the result of biting by infected mosquitoes, its epidemiological significance seems to be negligible under field conditions. The mode of field transmission is more complicated; as results of controlled studies suggested that the transmission of LSDV between animals by contagion is extremely inefficient, and that parenteral inoculation of virus is required to establish infection (18). Although the field experiences and the OIE fact sheet (see Introduction) states that the transmission of LSDV virus occurred primarily by biting insects no decisive experimental confirmations could be found in the literature. References 1 . Yeruham, I., Nir, O., Braverman, Y., Davidson, M., Grinstein H., Hymovitch, M. and Zamir, O. Spread of lumpy skin disease in Israel dairy herds. Vet Rec. 137: 91- 93. 1995. 2. Brenner, J., David, D., Avraham, A., Klopfer-Orgad, U., Samina, I. and Peleg, B.A. Experimental infection with local lumpy skin disease virus in cattle vaccinated with sheep pox vaccine. Isr. J. Vet. Med. 47: 17-21. 1992. 3. Coetzer J. A. W. Lumpy skin disease. In: Infectious Diseases of Livestock. 2nd Edition. (Eds). J. A.W. Cioetzer and R.C. Tustin. Oxford University Press. 2004. 1268-1276. 2005. 4. Haig, D.A. Lumpy skin disease. Bull. Epiz. Dis. Afr. 5: 421-430. 1957. 5. Weiss, K.E. Lumpy Skin Disease Virus. Virol. Monographs Vol. 3 New York: Springer Verlag. 1968. 6 . Chihota, C., L.F. Rennie, R.P. Kitching and P.S. Mellor. 2001. Mechanical transmission of lumpy skin disease virus by Aedes aegypti (Diptera: Culicidae). Epidemiol. Infect. 126: 317-321. 7 . Chihota, C. M., Rennie, L. F., Kitching, R. P. and Mellor, P. S. Attempted mechanical transmission of lumpy skin disease virus by biting insects. Med. Vet.
  12. 12. Entmol. 17: 294-300. 2003. 8 . Shimshony, A. Lumpy skin disease. Israeli Veterinary Services Epidemiol. Quart. (3) pp. 5-7 (in Hebrew). 1989. 9 . Abraham, A. and Zissman, A. Isolation of lumpy skin disease virus Israel. Isr. J. Vet. Med. 46: 20-23. 1991. 10. MacDonald, R.S.A. Pseudo-urticaria of cattle. North Rhodesian Dept. Anim. Healt. Ann. Report 1930. 11 . Von Backstromm, U. Ngamiland cattle disease: Preliminary report on a new disease, the aethiological agent being probably of an infectious nature. J. South Afr. Vet. Med. Assoc. 16: 20-35. 1945. 12 . Ordner, G. and Lefervre, P. C. La dermatose nodulaire contagieuse des bovines. Etudes et sytheses de l’Institut d’Elevage et de Medicine Veterinarie Tropicale, Maison-Alfort, Paris, pp 92. 1978. 13. Office International Des Epizooties, World Animal Health 5: 703. 1990. 14. Ali, A. A. Esmat, M., Attia, H., Selim, A. and Abdel-Hamid, M. Clinical and pathological studies on lumpy skin disease in Egypt. Vet Rec. 127: 549-550. 1990. 15 . Greth, A., Gourreau, J. M. Vassart, M. Ba-Uy, N. Wyers, M. and Lefevre, P. C. Capripoxvirus disease in an Arabian Oryx (Oryx leucoryx) from Saudi Arabia. J. Wildlife Dis. 28: 295-300. 1992. 16 . Brenner, J, Malkinson, M. and Yadin, H. Application of various diagnostic procedures to epidemiological situations encountered during arboviral infection. Vet. Ital.40: 567-571. 2004. 17 . Carn, V.M. and R.P. Kitching. The clinical response of cattle following infection with lumpy skin disease (Neethling) virus. Arch. Virol. 140: 503-513. 1995. 18. Carn, V.M. and R.P. Kitching. An investigation of possible routes of transmission of lumpy skin disease virus (Neethling). Epidemiol. Infect. 114: 219-226. 1995.