Evaluation of vector potential of Rhipicephalus appendiculatus, Amblyomma hebraeum and Rhipicephalus decoloratus ticks for lumpy skin disease virus

Lumpy skin disease (LSD) is an economically important pox disease of cattle and Asian water buffalo caused by a lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus. The disease occurs in Africa and the Near East, causing substantial economic losses for the whole cattle industry in affected countries. The disease is characterized by skin nodules, high fever, lymphadenopathy and loss of production of infected animals. Transmission of LSDV is known to occur mechanically by a variety of blood-feeding insects and to a lesser extent through contaminated feed and water, semen or via direct contact. The disease is classified as notifiable by the World Animal Health Organization (OIE). Currently, Finland is free of LSD. The general aim of the study was to investigate the vector capacity of common sub-Saharan tick species, Rhipicephalus appendiculatus, Amblyomma hebraeum and Rhipicephalus decoloratus for LSDV in cattle via mechanical, intra/transstadial or vertical routes. The specific aim was to investigate if mechanical transmission occurs by R. appendiculatus males and transovarial by R. decoloratus females. As many of the infected animals become viraemic without showing skin lesions, it was investigated if feeding on healthy looking skin of viraemic animals was sufficient for successful mechanical transmission. The final objective was to investigate if the virus was able to grow in vitro in Rhipicephalus spp. tick cell lines. In addition, the presence of the virus or viral DNA in ticks collected from naturally infected animals was investigated. Two animal experiments, using naïve cattle and laboratory-reared ticks were conducted at the Department of Veterinary Tropical Diseases, University of Pretoria, South Africa and samples were tested at the Pirbright Institute, United Kingdom. For the first time, transmission of LSDV (or any pox virus) by hard ticks was demonstrated to occur mechanically/intrastadially by R. appendiculatus males and vertically by R. decoloratus females. Feeding directly on skin lesions was not necessary for transmission of the virus between infected and naïve cattle. No evidence of viral replication in Rhipicephalus tick cell lines was obtained. The presence of the viral DNA was detected in Rhipicaphalus, Amblyomma and Hyalomma ticks collected during natural LSDV outbreaks in South Africa and Egypt. In 2013 - 2015 LSDV is spreading in the Near East at a scale never seen before, posing a threat to the European Union, Caucasus region, Afghanistan and Pakistan. In order to control and eradicate the disease, it is fundamental to understand the role of different arthropod vectors and their importance in the field. The presence of infected tick eggs or different instars in the environment underlines the importance of effective prophylactic tools and sufficient vaccination coverage. In addition, this study contributes to the recommendations set for the international trade of live cattle from affected countries.Capripoxviruksiin kuuluva Lumpy skin disease (LSD) on taloudellisesti merkittavä Lumpy skin disease- viruksen (LSDV) aiheuttama, Afrikassa ja Lähi-Idässa esiintyva nautakarjan rokkotauti. Väitöskirjassa selvitettiin viruksen tarttumista eläimestä toiseen punkkien välityksellä mekaanisesti ja infektoituneiden naaraspunkkien munien välityksellä jälkeläisiin. Lisäksi työssä tutkittiin onko punkin purema vireemisen eläimen terveellä iholla riittävä virukseen siirtymiseen, vai tarvitaanko tartuntaan purema viruksen aiheuttaman ihomuutoksen päällä. Työssä selvitettiin myös LSDV:n kykyä lisääntyä puutiaissolulinjoissa ja LSDV:n löytymistä luonnollisesti infektoituneesta karjasta kerätyistä punkeista. Tutkimuksessa käytetyt puutiaislajit ovat hyvin yleisiä Afrikan mantereella, mutta Euroopassa ei niitä tällä hetkellä esiinny. Tutkimus on antanut täysin uutta tietoa LSDV:n vektoreista. Aikaisemmin lähinnä hyönteisten on ajateltu toimivan vektoreina LSDV:n leviämisessä. Tässä työssä puutiaisten osuus osoitettiin kiistatta. Työssä osoitettiin ensimmäista kertaa molekuläärimenetelmin Ixodid puutiaisten mahdollisesti toimivan LSDV:n vektorina. Huolimatta lievistä kliinisistä oireista, vastaanottajaeläimet tulivat vireemisiksi ja niiden vasta-aine tasot nousivat. Vaikka vireemisen luovuttajanaudan iholla ei ollut LSDV:n aiheuttamia ihomuutoksia, imiessään verta terveeltä näyttävältä iholta, puutiaisen todettiin myöhemmin voivan infektoida vastaanottajanaudan. Lisäksi osoitettiin LSDV:n siirtuvän vertikaalisesti puutiaisen munan kautta toukkaan, joka puolestaan pystyy infektoimaan taudille alttiin naudan. Yrityksistä huolimatta virusta ei onnistuttu kasvattamaan puutiaissolulinjoissa. Virus kuitenkin säilyi infektiivisena puutiaissoluviljelmissä pitkään, 35 vuorokautta. Lisäksi työssä osoitettiin LSDV-DNA:ta Egyptin ja Etelä-Afrikan taudinpurkauksissa kerätyistä punkeista. Tämä on ensimmäinen kerta, kun on voitu osoittaa potentiaalisesti virulentti LSDV luonnollisesti infektoituneista eläimistä. Johtopäätöksissä painotettiin lisätutkimuksen tarvetta, kattavaa rokoteohjelmaa ja punkkiestolääkityksen käyttöä taudin leviämisen rajoittamisessa. Lisäksi kansainvälinen eläinkauppa infektoituneiden ja tautivapaiden maiden välillä tulee olla äärimmäisen hyvin kontrolloitua. Suomen eläintautiluokituksessa LSD on määritelty helposti leviäviin eläintauteihin, vaikka taudin rantautuminen Suomeen tuntuukin epätodennäköiseltä. Myös Suomessa esiintyvien nautojen, porojen ja pienten märehtijöiden rokkovirusten mahdollista leviämistä puutiaisten välityksellä tulisi tutkia

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

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.Dissertation (MSc (Veterinary Science))--University of Pretoria, 2004.Veterinary Tropical Diseasesunrestricte

Development of a cost-effective method for capripoxvirus genotyping using snapback primer and dsDNA intercalating dye

Sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV) are very closely related viruses of the Capripoxvirus (CaPV) genus of the Poxviridae family. They are responsible for sheep pox, goat pox and lumpy skin disease which affect sheep, goat and cattle, respectively. The epidemiology of capripox diseases is complex, as some CaPVs are not strictly host-specific. Additionally, the three forms of the disease co-exist in many sub-Saharan countries which complicates the identification of the virus responsible for an outbreak. Genotyping of CaPVs using a low-cost, rapid, highly specific, and easy to perform method allows a swift and accurate identification of the causative agent and significantly assists in selecting appropriate control and eradication measures, such as the most suitable vaccine against the virus during the outbreaks. The objective of this paper is to describe the design and analytical performances of a new molecular assay for CaPV genotyping using unlabelled snapback primers in the presence of dsDNA intercalating EvaGreen dye. This assay was able to simultaneously detect and genotype CaPVs in 63 samples with a sensitivity and specificity of 100%. The genotyping was achieved by observing the melting temperature of snapback stems of the hairpins and those of the full-length amplicons, respectively. Fourteen CaPVs were genotyped as SPPVs, 25 as GTPVs and 24 as LSDVs. The method is highly pathogen specific and cross platform compatible. It is also cost effective as it does not use fluorescently labelled probes, nor require high-resolution melting curve analysis software. Thus it can be easily performed in diagnostic and research laboratories with limited resources. This genotyping method will contribute significantly to the early detection and genotyping of CaPV infection and to epidemiological studies. (Résumé d'auteur

Comparative evaluation of lumpy skin disease virus-based live attenuated vaccines

Vaccines form the cornerstone of any control, eradication and preventative strategy and this is no different for lumpy skin disease. However, the usefulness of a vaccine is determined by a multiplicity of factors which include stability, efficiency, safety and ease of use, to name a few. Although the vaccination campaign in the Balkans against lumpy skin disease virus (LSDV) was successful and has been implemented with success in the past in other countries, data of vaccine failure have also been reported. It was therefore the purpose of this study to compare five homologous live attenuated LSDV vaccines (LSDV LAV) in a standardized setting. All five LSDV LAVs studied were able to protect against a challenge with virulent LSDV. Aside from small differences in serological responses, important differences were seen in side effects such as a local reaction and a Neethling response upon vaccination between the analyzed vaccines. These observations can have important implications in the applicability in the field for some of these LSDV LAVs

Evidence of lumpy skin disease virus over-wintering by transstadial persistence in Amblyomma hebraeum and transovarial persistance in Rhipicephalus decoloratus ticks

Lumpy skin disease is a debilitating cattle disease caused by the lumpy skin disease virus (LSDV), belonging to the genus Capripoxvirus. Epidemics of the disease usually occur in summer, when insect activity is high. Limited information is available on how LSDV persists during inter-epidemic periods. Transmission of LSDV by mosquitoes such as Aedes aegypti has been shown to be mechanical, there is no carrier state in cattle and the role of wildlife in the epidemiology of the disease seems to be of minor importance. Recent studies in ticks have shown transstadial persistence of LSDV in Rhipicephalus appendiculatus and Amblyomma hebraeum as well as transovarial persistence of the virus in Rhipicephalus decoloratus, R. appendiculatus and A. hebraeum. The overwintering of ticks off the host as part of their life cycles is well known: A. hebraeum and R. appendiculatus over-winter, for example, on the ground as engorged nymphs/unfed (emergent) adults while R. decoloratus over-winters on the ground as engorged females. In this study, transstadial and transovarial persistence of LSDV from experimentally infected A. hebraeum nymphs and R. decoloratus females after exposure to cold temperatures of 5 C at night and 20 C during the day for 2 months was reported. This observation suggests possible over-wintering of the virus in these tick species.Combating Infectious Diseases of Livestock for International Development (CIDLID) research programme, the Department of International Biotechnology and Biological Sciences Research Council (BBSRC), the UK government, the Department for International Development (DFID) and the Scottish Government (CIDLID Project Number BB/H009361/1).http://link.springer.com/journal/10493hb201

Demonstration of lumpy skin disease virus infection in Amblyomma hebraeum and Rhipicephalus appendiculatus ticks using immunohistochemistry

Lumpy skin disease (LSD) is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus. Transmission of the virus has been associated with haematophagous insects such as Stomoxys calcitrans as well as Aedes and Culex species of mosquitoes. Recent studies have reported the transmission of the virus by Amblyomma hebraeum, Rhipicephalus appendiculatus, and Rhipicephalus decoloratus ticks and the presence of LSDV in saliva of A. hebraeum and R. appendiculatus ticks. The aim of this study was to determine which tick organs become infected by LSDV following intrastadial infection and transstadial persistence of the virus in A. hebraeum and R. appendiculatus ticks. Nymphal and adult ticks were orally infected by feeding them on LSDV-infected cattle. Partially fed adult ticks were processed for testing while nymphs were fed to repletion and allowed to moult to adults before being processed for testing. The infection in tick organs was determined by testing for the presence of the viral antigen using monoclonal antibodies with immunohistochemical staining. The viral antigen was detected in salivary glands, haemocytes, synganglia, ovaries, testes, fat bodies, and midgut. Since the virus was shown to be able to cross the midgut wall and infect various tick organs, this may indicate potential for biological development and transmission of LSDV in ticks. This study strengthens the previously reported evidence of the occurrence of LSDV in tick saliva.http://www.elsevier.com/locate/ttbdishb2014ab201

Comparative evaluation of lumpy skin disease virus-based live atenuated vaccines

Vaccines form the cornerstone of any control, eradication and preventative strategy and this is no different for lumpy skin disease. However, the usefulness of a vaccine is determined by a multiplicity of factors which include stability, efficiency, safety and ease of use, to name a few. Although the vaccination campaign in the Balkans against lumpy skin disease virus (LSDV) was successful and has been implemented with success in the past in other countries, data of vaccine failure have also been reported. It was therefore the purpose of this study to compare five homologous live attenuated LSDV vaccines (LSDV LAV) in a standardized setting. All five LSDV LAVs studied were able to protect against a challenge with virulent LSDV. Aside from small differences in serological responses, important differences were seen in side effects such as a local reaction and a Neethling response upon vaccination between the analyzed vaccines. These observations can have important implications in the applicability in the field for some of these LSDV LAVs.The Bill & Melinda Gates Foundation, the GALVmed project Nr CAO-R34A0856 on lumpy skin disease and the Belgian Federal Public Service of Health, Food Chain Safety and Environment through the contract RT 15/3 (LUMPY SKIN 1).http://www.mdpi.com/journal/vaccinespm2022Veterinary Tropical Disease

Review: Vaccines and Vaccination against Lumpy Skin Disease

The geographical distribution of lumpy skin disease (LSD), an economically important cattle disease caused by a capripoxvirus, has reached an unprecedented extent. Vaccination is the only way to prevent the spread of the infection in endemic and newly affected regions. Yet, in the event of an outbreak, selection of the best vaccine is a major challenge for veterinary authorities and farmers. Decision makers need sound scientific information to support their decisions and subsequent actions. The available vaccine products vary in terms of quality, efficacy, safety, side effects, and price. The pros and cons of different types of live attenuated and inactivated vaccines, vaccination strategies, and associated risks are discussed. Seroconversion, which typically follows vaccination, places specific demands on the tools and methods used to evaluate the effectiveness of the LSD vaccination campaigns in the field. We aimed to give a comprehensive update on available vaccines and vaccination against LSD, to better prepare affected and at-risk countries to control LSD and ensure the safe trade of cattle