Detection and molecular characterization of lumpy skin disease and bovine papular stomatitis viruses in lumpy skin disease-suspected outbreaks in Tanzania

Lumpy skin disease (LSD) is a highly contagious vector-borne viral disease of cattle. LSD has emerged in Bangladesh in 2019, causing significant economic losses due to its high morbidity and mortality. This research was designed to isolate, identify, and assess the immunogenicity of LSD virus (LSDV) using nodular tissue samples obtained from affected cattle during the 2019-20 outbreak across nine districts of Bangladesh. To determine the presence of LSDV in nodular tissues, we initially used iiPCR and PCR, followed by histopathological examination. 151 were positive via iiPCR and PCR among the 180 collected samples. The PCR positive 151 samples were then inoculated into 10-day-old embryonated chicken eggs via the CAM route to isolate LSDV, confirmed through PCR. Subsequently, partial sequencing and phylogenetic analysis of the P32 gene were performed to determine the origin of the circulating LSDV strain. The immunogenicity of selected LSDV strains was assessed through an ELISA test. The PCR results revealed a distinct positive band at 192 bp in both the nodular tissue samples and the LSDV isolated from chicken embryo inoculations. Microscopic analysis of the nodular lesions revealed thickening of the epidermis, ballooning degeneration of keratinocytes, and proliferation of follicular epithelia. Additionally, mononuclear infiltration was observed at the demarcation line between infected and healthy tissue, with necrosis of muscular tissues beneath the epidermis. The LSDV isolate from Bangladesh exhibited a close genetic relationship with LSDV strains isolated from neighboring and other regional countries including India, Myanmar, and Mongolia. This observation strongly suggests the possibility of a transboundary spread of the LSD outbreak in Bangladesh during 2019-2020. The results of the immunogenicity test showed that the serum antibody titer remained at a protective level for up to 18 months following secondary immunization with inactivated LSDV antigen. This finding suggests that the inactivated LSDV antigen could be a potential vaccine candidate to protect cattle in Bangladesh against LSDV. In conclusion, our research successfully isolated, identified, and characterized LSDV in cattle nodular tissues from the 2019-20 outbreak in Bangladesh. Furthermore, it provided insights into the probable origin of the circulating strain and investigated a potential vaccine candidate to protect cattle in the region from LSDV.

Lumpy skin disease (LSD) of cattle is a highly contagious viral skin disease causing sever economic losses. In Egypt, Protection of cattle against LSD was carried out using sheep poxvirus vaccines. In the present study, confirmative identification of previously isolated lumpy skin disease virus (LSDV) and commonly used sheep poxvirus (SPPV) vaccine (RM65 strain) nucleic acids was carried out by conventional polymerase chain reaction (PCR) and dot blot hybridization (DBH) depending on attachment protein gene (192 bp) of capripoxviruses. Differentiation between this LSDV isolate and SPPV vaccine (RM65 strain) was performed via PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and real time-PCR melting curve analysis depending on 390 bp genome fragment in which EcoRV sites is present in case of SPPV and absent in LSDV. Therefore, the amplified 390 bp genome fragment was digested by EcoRV in case of SPPV and not digested in LSDV isolate. Also, real time-PCR melting curve analysis revealed that melting temperature (Tm) of LSDV isolate was 78°C where Tm of SPPV vaccine was 76 °C. from this study, we concluded that PCR-RFLP and real time-PCR melting curve analysis could be used to differentiate between cattle infected with LSDV and those vaccinated with SPPV vaccine.

Background and Aim:Lumpy skin disease (LSD) is a highly infectious viral disease upsetting cattle, caused by LSD virus (LSDV) within the family Poxviridae. Sporadic cases of LSD have been observed in cattle previously vaccinated with the Romanian sheep poxvirus (SPPV) vaccine during the summer of 2016 in Sharkia province, Egypt. The present study was undertaken to perform molecular characterization of LSDV strains which circulated in this period as well as investigate their phylogenetic relatedness with published reference capripoxvirus genome sequences.Materials and Methods:A total of 82 skin nodules, as well as 5 lymph nodes, were collected from suspect LSD cases, and the virus was isolated in embryonated chicken eggs (ECEs). LSD was confirmed by polymerase chain reactions amplification of the partial and full-length sequences of the attachment and G-protein-coupled chemokine receptor (GPCR) genes, respectively, as well as a histopathological examination of the lesions. Molecular characterization of the LSDV isolates was conducted by sequencing the GPCR gene.Results:Characteristic skin nodules that covered the whole intact skin, as well as lymphadenopathy, were significant clinical signs in all suspected cases. LSDV isolation in ECEs revealed the characteristic focal white pock lesions dispersed on the chorioallantoic membranes. Histopathologic examination showed characteristic eosinophilic intracytoplasmic inclusion bodies within inflammatory cell infiltration. Phylogenetic analysis revealed that the LSDV isolates were clustered together with other African and European LSDV strains. In addition, the LSDV isolates have a unique signature of LSDVs (A11, T12, T34, S99, and P199).Conclusion:LSDV infections have been detected in cattle previously vaccinated with Romanian SPPV vaccine during the summer of 2016 and making the evaluation of vaccine efficacy under field conditions necessary.

Lumpy skin disease (LSD) caused by lumpy skin disease virus (LSDV) inflicts significant economic losses in cattle production with impact on livelihoods of smallholders. This study reports the first occurrence of LSD in cattle in India and analyses epidemiological and genetic characterization data from LSD outbreaks in five districts of Odisha state in August 2019. In all, 182 of 2,539 cattle were affected with an apparent morbidity rate of 7.1% and no mortality. Out of 102 samples from 60 LSD suspected and 17 asymptomatic in-contact cattle tested, 29.87% cattle were positive by capripoxvirus generic PCR and 37.66% were positive by LSDV real-time PCR. All the in-contact cattle tested were negative for LSDV. Among affected cattle, LSDV genome was detected more in scabs (79.16%) than blood (31.81%) and frozen bull semen (20.45%). Differential diagnosis by PCR was negative for pseudo-LSD, buffalopox, cowpox, pseudo-cowpox and bovine papular stomatitis. Five selected PCR and real-time PCR-positive LSDV DNA were sequenced in three genomic regions, P32 (LSDV074), F (LSDV117) and RPO30 (LSDV036). Phylogenetic analysis based on partial P32 and F gene sequences and complete RPO30 gene sequences showed that all the five Indian LSDV strains were identical and clustered with other field strains of LSDV circulating globally. However, the F and RPO30 gene sequence analyses revealed that Indian LSDV strains are genetically closer to the South African NI2490/KSGP-like strains than the strains detected in Europe, which was rather surprising. The present study established the existence of LSDV in India and involvement of LSDV field strains in the outbreaks. Additionally, we provided evidence of LSDV shedding in semen of naturally infected bulls. Further studies are required to determine the source of LSD introduction, extent of spread, modes of transmission and impact on dairy cattle production in India and effective control measures must be undertaken urgently.

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), an economically significant disease in cattle caused by lumpy skin disease virus (LSDV), is endemic to nearly all of Africa. Since 2012, LSDV has emerged as a significant epizootic pathogen given its rapid spread into new geographical locations outside Africa, including the Middle East, Eastern Europe, and Asia. To assess the genetic diversity of LSDVs in East Africa, we sequenced and analyzed the RPO30 and GPCR genes of LSDV in twenty-two archive samples collected in Ethiopia, Kenya, and Sudan before the appearance of LSD in the Middle East and its incursion into Europe. We compared them to publicly available sequences of LSDVs from the same region and those collected elsewhere. The results showed that the East African field isolates in this study were remarkably similar to each other and to previously sequenced field isolates of LSDV for the RPO30 and GPCR genes. The only exception was LSDV Embu/B338/2011, a field virus collected in Kenya, which displayed mixed features between the LSDV Neethling vaccine and field isolates. LSDV Embu/B338/2011 had the same 12-nucleotide insertion found in LSDV Neethling and KS-1 vaccines. Further analysis of the partial EEV glycoprotein, B22R, RNA helicase, virion core protein, NTPase, and N1R/p28-like protein genes showed that LSDV Embu/B338/2011 differs from previously described LSDV variants carrying the 12-nucleotide insertion in the GPCR gene. These findings highlight the importance of the constant monitoring of genetic variation among LSDV isolates.

Lumpy skin disease (LSD) has emerged as a global threat to cattle health and production. Although India has been encountering regular episodes of LSD epidemics on the mainland since 2019, the Andaman and Nicobar Islands remained free of LSD until 2021. In this study, we investigated the first LSD outbreak on Great Nicobar Island in 2022 and examined the genetic characteristics of the LSDV strain associated with this outbreak. The morbidity and mortality rates in cattle were 38.29% and 1.89%, respectively. We screened 123 samples (whole blood, nasal swab, or skin tissue) from 111 cattle by LSDV real-time PCR and sera from 86 cattle by LSDV antibody ELISA, and the results confirmed LSDV infection. Subsequent nucleotide sequencing and phylogenetic analysis of five selected marker genes (GPCR, RPO30, P32, EEV, and B22R) revealed that the LSDV strain from Great Nicobar Island resembled cluster 2.5 LSDV recombinant strains from East and Southeast Asia but was distinct from wild-type LSDV strains (1.2.1, 1.2.2) circulating in mainland India, indicating an exotic source of introduction. Phylogenetic analysis using a concatenated sequence (GPCR-RPO30-P32-EEV-B22R) showed that both the wild-type and recombinant LSDV strains formed well-supported clusters, indicating that this type of analysis may be used as an alternative to whole-genome sequence analysis. We also found that determination of the nucleotide sequence of the C-terminal 717 bp of the B22R gene may be sufficient for reliable assignment of circulating LSDV isolates to a particular cluster. This is the first report of the detection of a recombinant LSDV strain in India, demonstrating the spread of cluster 2.5 recombinant LSDV further into South Asia. Our findings highlight the value of LSDV surveillance and genetic analysis for LSDV epidemiology, which may be helpful for developing effective control strategies.

Lumpy skin disease (LSD) is WOAH listed transboundary disease of cattle with high economic impact which threaten the global cattle industry. The disease was first diagnosed in Zambia in 1929 and the first outbreak in Tanzania was in 1981. LSD is regarded endemic in sub-Saharan countries. However, the community knowledge, attitude and practice (KAP) towards LSD in Tanzania is poorly understood. This cross-sectional study was conducted in Tanga and Pwani region between December 2022 and February 2023 to assess KAP towards LSD. A questionnaire tool was used to collect information from116 herds/households. Questionnaire was administered by face-to-face. Data analysis was done using descriptive statistic and univariate logistic regression model. In this study, it was found that majority of the respondents were aware of LSD occurrence (86.93%, CI=79.40-92.51) and over half of the respondents 54.78% (CI=45.23-64.04) had past LSD experience in their herds. However, our study revealed limited knowledge on LSD sign, associated losses, transmission control and on the role of vectors in LSD epidemiology. Majority of the respondent believed LSD has impact (86.09%, CI=78.39-91.83) and believe cattle are at risk (78.26%, CI=69.60-85.41). Moreover, respondents believed vaccine is important in LSD control (70.26%, CI=69.60-85.41). Nevertheless, majority believed they had limited access to vaccine. Respondent age, herd size, district, role in the households, main source of income, time in livestock farming, cattle type and past experience on LSD occurrence appeared to influence both the knowledge and attitude towards LSD in Tanga and Pwani regions.

Understanding molecular epidemiology is essential for the improvement of lumpy skin disease (LSD) eradication and control strategies. The objective of this study was to perform a molecular characterization and phylogenetic analysis of lumpy skin disease virus (LSDV) isolated from dairy cows presenting LSD-like clinical signs in northern Thailand. The skin nodules were collected from 26 LSD-suspected cows involved in six outbreaks during the period from July to September of 2021. LSDVs were confirmed from clinical samples using the polymerase chain reaction (PCR). The PCR-positive samples were subsequently amplified and sequenced using a G-protein-coupled chemokine receptor (GPCR) gene for molecular characterization and phylogenetic analyses. All 26 samples were positive for LSDV by PCR. A phylogenetic analysis indicated that the 24 LSDV isolates obtained from cattle in northern Thailand were closely related to other LSDV sequences acquired from Asia (China, Hong Kong, and Vietnam). On the other hand, two LSDV isolates of the cows presenting LSD-like clinical signs after vaccination were clustered along with LSDV Neethling-derived vaccines. The outcomes of this research will be beneficial in developing effective control strategies for LSDV.

Lumpy skin disease virus (LSDV) causes lumpy skin disease in cattle and buffaloes, which is associated with significant animal production and economic losses. Since the 2000s, LSDV has spread from Africa to several countries in the Middle East; Europe; and Asia; including, more recently, several south-east Asian countries. In November 2020, Myanmar reported its first LSD outbreak. This study reports on the first incursion of LSD in Myanmar and the molecular analysis of the LSDV detected. Staff from the Livestock Breeding and Veterinary Department (LBVD) of the Ministry of Agriculture, Livestock, and Irrigation collected samples from cattle with suspected LSD infection. The Food and Agriculture Organization (FAO) of the United Nations’ Emergency Centre for Transboundary Animal Diseases (ECTAD) and the Joint International Atomic Energy Agency (IAEA)/FAO program’s Animal Health and Production laboratory provided LSDV diagnostic support to two regional veterinary diagnostic laboratories in Myanmar. Samples from 13 cattle tested positive by real-time PCR. Selected samples underwent sequence analysis in IAEA laboratories. The results show that the Myanmar LSDV sequences clustered with LSDV isolates from Bangladesh and India, LSDV Kenya, and LSDV NI-2490. Further characterization showed that the Myanmar LSDV is 100% identical to isolates from Bangladesh and India, implying a common source of introduction. These findings inform diagnosis and development of control strategies.

Phylogenetic and pathogenic characterization of lumpy skin disease virus circulating in China

Lumpy skin disease (LSD) is a severe infectious, emerging transboundary disease of cattle, caused by a Pox family DNA virus. Lumpy skin disease virus (LSDV) infection is associated with a febrile response followed by emergence of widespread dermal nodules. In addition to the skin, LSDV resides in multiple internal organs and can be isolated from the blood of infected cattle. LSDV is suggested to be mechanically transmitted by biting arthropods. Live attenuated vaccines are commonly used to control disease and its spread. We have characterized the tropism, replication, and dissemination of a LSDV field isolate and of an attenuated vaccine strain usingin vitrosystems. To follow virus infection and dissemination in living cells, we have generated recombinant viruses expressing green fluorescent protein (GFP) under a synthetic viral promoter. Recombinant, GFP-expressing, LSDVs demonstrated similar replication kinetics to their corresponding parental LSDV strains in a bovine kidney cell line (MDBK). We further demonstrated that LSDV-GFP productively replicated in a bovine macrophage cell line and in primary bovine foreskin cells with no apparent differences between the field isolate and the vaccine strain. When bovine peripheral blood mononuclear cells (PBMCs) were infected with either LSDV recombinant strain, we observed specific viral driven GFP fluorescence as well as significant viral gene expression. However, infected PBMCs failed to support substantial viral DNA replication and release of infectious progeny. Subsequent analysis of the anti-viral response revealed that heat treated (HT) LSDV induced the expression of interferon- stimulated genes (ISGs) in PBMCs, but this response was suppressed by infectious viruses. Finally, we show that despite failed replication, LSDV infected PBMCs transmitted the virus to recipient co-cultured MDBK cells and produced infectious foci, suggesting a potential role of PBMCs in LSDV dissemination.HighlightsVirulent and attenuated LSDV productively replicated in bovine kidney and bovine macrophage cell lines as well as in primary fibroblasts.Adherent white blood cells were susceptible to LSDV field and attenuated vaccine infection.LSDV showed active viral transcription in PBMCs yet no significant viral genome replication or production of infectious progeny.PBMCs infected with heat-treated LSDV but not with fully infectious viruses upregulated ISGs’ RNA.PBMCs transmitted and disseminated LSDV to contacting permissive cells.

Detection of lumpy skin disease (LSD) virus and distribution of blood-sucking insects as potential vector in Indonesia.

Molecular evidence of prolonged shedding of lumpy skin disease virus (LSDV) in the semen of naturally infected cattle bulls.

In this study, we investigated and confirmed natural lumpy skin disease virus (LSDV) infection in Himalayan yaks (Bos grunniens) in Himachal Pradesh, India, based on clinical manifestations and results of genome detection, antibody detection, virus isolation, and nucleotide sequencing. Subsequent phylogenetic analysis based on complete GPCR, RPO30, and EEV gene sequences revealed that the LSDV isolates from these yaks and local cattle belonged to LSDV subcluster 1.2.1 rather than the dominant subcluster 1.2.2, which is currently circulating in India, suggesting a separate recent introduction. This is the first report of natural LSDV infection in yaks in India, expanding the known host range of LSDV. Further investigations are needed to assess the impact of LSDV infection in yaks.