The detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques

Abstract

Lumpy skin disease (LSD), affecting cattle in Africa, Madagascar and the Middle East, is caused by a capripox virus that belongs to the family Poxviridae. The disease is of economical importance in endemic areas and the Office International des Epizooties classifies it as a “List A”- disease. Effective control of LSD requires accurate and rapid laboratory techniques to confirm a tentative clinical diagnosis. Comparative studies on different diagnostic tests used at different stages of the disease have not been done. The aim of this study was to compare the different tests that are available and to provide data to assist in the selection of a rapid and sensitive laboratory test for the diagnosis of LSD. Six seronegative, prepubertal bulls were infected via the intravenous route and kept in an insect-free facility. The course of infection was monitored. During a three months’ period blood and semen samples were collected for virus isolation and polymerase chain reaction (PCR), and skin biopsies for the PCR, virus isolation, transmission electron microscopy (TEM), histopathological examination and immunoperoxidase staining of tissue sections. Antibody titres were assessed using the serum virus neutralization test (SNT) and indirect immunofluorescence test (IFAT). The incubation period in infected animals varied from 4 to 5 days. The length of viraemia did not correlate with the severity of clinical disease. By using virus isolation the duration of viraemia was determined to be from 1 to 12 days and by PCR from 4 to 11 days, which is longer than has previously been stated. Virus was isolated from semen until day 43 post-infection (p.i.) whereas the PCR could detect LSD virus nucleic acid until day 161 p.i. Virus was isolated from skin biopsies until day 39 p.i. and PCR could demonstrate viral DNA in them until day 92 p.i. The PCR was a fast and sensitive method to demonstrate viral DNA in blood, skin and semen samples. It could detect viral nucleic acid for significantly longer periods than any of the conventional methods. Virus isolation from blood, skin and semen samples was sensitive and reliable, but as a single test it may be too time-consuming although this depends how rapidly the diagnosis must be confirmed. The IFAT can be used for rapid confirmation of a clinical diagnosis but it needs careful standardization due to non-specific staining. The SNT showed positive results later in the course of the clinical disease than IFAT but it was however, sensitive and reliable in detecting antibodies from all the animals in this experiment. Transmission electron microscopy of skin biopsies detected LSD virus only in one of the four bulls that developed skin lesions. This indicated that even though TEM is usually considered to be a fast and reliable method, a negative result must still be confirmed using another method. Histopathological changes of the skin lesions in sections stained with haematoxylin and eosin were typical for the disease. It was not possible to make a reliable diagnosis of LSD based only on immunoperoxidase staining of tissue sections. In conclusion, this study indicated the PCR to be superior in detecting LSD virus from blood, skin and semen samples. However, virus isolation is still required when the infectivity of the LSD virus is to be investigated. Even though the IFAT has been used for several decades, it is still a valuable tool in detecting antibodies against LSD virus. Both the SNT and IFAT are useful and reliable in retrospective, epidemiological studies.