Eeva Tuppurainen

Mathematical modelling and evaluation of the different routes of transmission of lumpy skin disease virus

Lumpy skin disease (LSD) is a severe viral disease of cattle. Circumstantial evidence suggests that the virus is transmitted mechanically by blood-feeding arthropods. We compared the importance of transmission via direct and indirect contact in field conditions by using mathematical tools. We analyzed a dataset collected during the LSD outbreak in 2006 in a large dairy herd, which included ten separated cattle groups. Outbreak dynamics and risk factors for LSD were assessed by a transmission model. Transmission by three contact modes was modelled; indirect contact between the groups within a herd, direct contact or contact via common drinking water within the groups and transmission by contact during milking procedure. Indirect transmission was the only parameter that could solely explain the entire outbreak dynamics and was estimated to have an overall effect that was over 5 times larger than all other possible routes of transmission, combined. The R0 value induced by indirect transmission per the presence of an infectious cow for 1 day in the herd was 15.7, while the R0 induced by direct transmission was 0.36. Sensitivity analysis showed that this result is robust to a wide range of assumptions regarding mean and standard deviation of incubation period and regarding the existence of sub-clinically infected cattle. These results indicate that LSD virus spread within the affected herd could hardly be attributed to direct contact between cattle or contact through the milking procedure. It is therefore concluded that transmission mostly occurs by indirect contact, probably by flying, blood-sucking insects. This has important implications for control of LSD.

Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses

The genus Capripoxvirus (CaPV) comprises three members namely, sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affecting sheep, goats and cattle, respectively. CaPV infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. Since there are conflicting opinions regarding the host specificity of CaPVs, particularly for goatpox and sheeppox viruses, the development of rapid genotyping tools will facilitate more accurate disease diagnosis and surveillance for better management of capripox outbreaks. This paper describes a species-specific, real time polymerase chain reaction (PCR), based on unique molecular markers that were found in the G-protein-coupled chemokine receptor (GPCR) gene sequences of CaPVs, that uses dual hybridization probes for their simultaneous detection, quantitation and genotyping. The assay can differentiate between CaPV strains based on differences in the melting point temperature (Tm) obtained after fluorescence melting curve analysis (FMCA). It is highly sensitive and presents low intra- and inter-run variation. This real time PCR assay will make a significant contribution to CaPV diagnosis and to the better understanding of the epidemiology of CaPVs by enabling rapid genotyping and gene-based classification of viral strains and unequivocal identification of isolates.

Use of the Capripoxvirus homologue of Vaccinia virus 30 kDa RNA polymerase subunit (RPO30) gene as a novel diagnostic and genotyping target: Development of a classical PCR method to differentiate Goat poxvirus from Sheep poxvirus

Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are Capripoxviruses (CaPVs) responsible for causing severe poxvirus disease in sheep, goats and cattle, respectively. Serological differentiation of CaPVs is not possible and strain identification has relied on the implicitly accepted hypothesis that the viruses show well defined host specificity. However, it is now known that cross infections can occur and authentication of identity based on the host animal species from which the strain was first isolated, is not valid and should be replaced with molecular techniques to allow unequivocal strain differentiation. To identify a diagnostic target for strain genotyping, the CaPV homologue of the Vaccinia virus E4L gene which encodes the 30 kDa DNA-dependent RNA polymerase subunit, RPO30 was analyzed. Forty-six isolates from different hosts and geographical origins were included. Most CaPVs fit into one of the three different groups according to their host origins: the SPPV, the GTPV and the LSDV group. A unique 21-nucleotide deletion was found in all SPPV isolates which was exploited to develop a RPO30-based classical PCR test to differentiate SPPV from GTPV that will allow rapid differential diagnosis of disease during CaPV outbreaks in small ruminants.

Characterization of sheep pox virus vaccine for cattle against lumpy skin disease virus.

Highlights • KSGP O-240 strain was identified as lumpy skin disease virus.• Commercially available KSGP O-240 vaccines should be re-characterized.• The safety of these vaccines in cattle against LSDV should be re-evaluated.• Two GTPV candidates were identified for use as a broad-spectrum capripox vaccine.

Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA

Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries.

Detection of lumpy skin disease virus in saliva of ticks fed on lumpy skin disease virus-infected cattle

Lumpy skin disease is an economically important disease of cattle that is caused by the lumpy skin disease virus (LSDV), which belongs to the genus Capripoxvirus. It is endemic in Africa and outbreaks have also been reported in the Middle-East. Transmission has mostly been associated with blood-feeding insects but recently, the authors have demonstrated mechanical transmission by Rhipicephalus appendiculatus as well as mechanical/intrastadial and transstadial transmission by Amblyomma hebraeum. Saliva is the medium of transmission of pathogens transmitted by biting arthropods and, simultaneously, it potentiates infection in the vertebrate host. This study aimed to detect LSDV in saliva of A. hebraeum and R. appendiculatus adult ticks fed, as nymphs or as adults, on LSDV-infected animals, thereby also demonstrating transstadial or mechanical/intrastadial passage of the virus in these ticks. Saliva samples were tested for LSDV by real-time PCR and virus isolation. Supernatants obtained from virus isolation were further tested by real-time PCR to confirm that the cytopathic effects observed were due to LSDV. Lumpy skin disease virus was detected, for the first time, in saliva samples of both A. hebraeum and R. appendiculatus ticks. At the same time, mechanical/intrastadial and transstadial passage of the virus was demonstrated and confirmed in R. appendiculatus and A. hebraeum.

Evidence of transstadial and mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks.

Lumpy skin disease (LSD) is an economically important disease caused by LSD virus (LSDV), a Capripoxvirus, characterized by fever and circumscribed skin lesions. It is suspected to be transmitted mechanically by biting flies. To assess the vector potential of Amblyomma hebraeum in transmission of LSDV, mechanical/intrastadial and transstadial modes of transmission of the virus by this tick species were investigated. Two cattle were artificially infected as sources (donors) of infection to ticks. Ticks were infected as either nymphs or adults. Male A. hebraeum ticks were partially fed on donor animals and transferred to recipient animals to test for mechanical/intrastadial transmission. Nymphal A. hebraeum were fed to repletion on donor animals. The emergent adult ticks were placed on recipient animals to test for transstadial transmission of the virus. Successful transmission of LSDV infection was determined in recipient animals by monitoring development of clinical signs, testing of blood for the presence of LSDV by real-time PCR, virus isolation and the serum neutralization test. This report provides further evidence of mechanical/intrastadial and, for the first time, transstadial transmission of LSDV by A. hebraeum. These findings implicate A. hebraeum as a possible maintenance host in the epidemiology of the disease.

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.

Capripox disease in Ethiopia: Genetic differences between field isolates and vaccine strain, and implications for vaccination failure

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) of the genus Capripoxvirus (CaPV) cause capripox disease in sheep, goats and cattle, respectively. These viruses are not strictly host-specific and their geographical distribution is complex. In Ethiopia, where sheep, goats and cattle are all affected, a live attenuated vaccine strain (KS1-O180) is used for immunization of both small ruminants and cattle. Although occurrences of the disease in vaccinated cattle are frequently reported, information on the circulating isolates and their relation to the vaccine strain in use are still missing. The present study addressed the parameters associated with vaccination failure in Ethiopia. Retrospective outbreak data were compiled and isolates collected from thirteen outbreaks in small ruminants and cattle at various geographical locations and years were analyzed and compared to the vaccine strain. Isolates of GTPV and LSDV genotypes were responsible for the capripox outbreaks in small ruminants and cattle, respectively, while SPPV was absent. Pathogenic isolates collected from vaccinated cattle were identical to those from the non-vaccinated ones. The vaccine strain, genetically distinct from the outbreak isolates, was not responsible for these outbreaks. This study shows capripox to be highly significant in Ethiopia due to low performance of the local vaccine and insufficient vaccination coverage. The development of new, more efficient vaccine strains, a GTPV strain for small ruminants and a LSDV for cattle, is needed to promote the acceptance by farmers, thus contribute to better control of CaPVs in Ethiopia.

Detection of capripoxvirus DNA using a novel loop-mediated isothermal amplification assay

BackgroundSheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA.ResultsA single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/μl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device.ConclusionsThis study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.