Pathological, immunological and molecular epidemiological analysis of lumpy skin disease virus in Indian cattle during a high-mortality epidemic.

Lumpy skin disease (LSD) is an economically significant, emerging viral disease of Cattle and Buffaloes. This study aimed to investigate the causes of high mortality in a recent LSD epidemic in India. We examined 1618 animals across seventy outbreaks and conducted post-mortem on 48 cattle out of 513 clinically suspected LSD cases. The morbidity, mortality and case fatality rates recorded were 31.70%, 2.97 and 9.37% respectively. Disease stages were categorized as early (20.81%), mid (42.02%), and late (37.17%) and the distribution of skin lesions was classified as mild (34.14%), moderate (39.39%), and severe (26.47%). Post-mortem findings revealed systemic infection with necrotic and ulcerative nodules on multiple internal organs. Histologically, necrotizing vasculitis and mononuclear cell infiltration with intracytoplasmic inclusions were observed in various organs. The highest viral load was found in skin nodules/scabs, trachea, tongue, and lymph nodes. The viral load was significantly higher in mid- and late-stages of skin nodules and internal organs; whereas, blood from early-stage showed high viral load. The expression of Th1-type and Th2-type cytokines varied significantly across different stages of the disease. The downregulation of the apoptotic intrinsic and upregulation of the extrinsic pathway genes, suggesting that the latter plays a role in LSDV infection. Genetic analysis revealed that the LSD virus (LSDV) isolates were derived from a Kenyan ancestral strain with unique nucleotide changes in RPO30 and P32 gene. In conclusion, the high mortality in the recent Indian LSD epidemic can be attributed to a newly identified, highly virulent strain of LSDV causing systemic infection.

Lumpy skin disease virus isolation, experimental infection, and evaluation of disease development in a calf.

Lumpy skin disease (LSD) is one of the most economically significant viral diseases of cattle caused by the Lumpy Skin Disease Virus (LSDV), classified as a member of the genus Capripoxvirus and belongs to the family Poxviridae. Nodular skin samples were collected from clinically sick cattle in the districts of Amuru and Wara Jarso Ethiopia to isolate LSD virus. The virus was isolated using primary lamb testis and kidney cells. The isolated LSDV was infected into a healthy calf while maintaining the necessary biosecurity measures to generate skin lesions and to assess disease progression using postmortem examinations. On the fourth day after virus inoculation, the calf developed typical LSD skin nodules with increased rectal temperature, which lasted until the 12th day, when they began to decrease. Viral shedding was detected in nasal, oral, and conjunctival swabs from 6 to 14 days after infection using real-time PCR. Post-mortem tissue specimens tested positive for LSD virus using real-time PCR and virus isolation. This study showed that LSDV were responsible for the LSD outbreaks, and the appearance of typical skin nodules accompanied by fever (> 39.5 °C) defined the virus's virulent status. The experimental infection with the isolated infectious LSDV could serve as a platform for future vaccine evaluation study using an LSDV challenge model.

Epidemiology, genetic characterization and population structure analysis of lumpy skin disease virus from North India.

Lumpy skin disease (LSD) is a transboundary, viral disease of cattle with a significant economic impact on the livestock industry. This study describes the epidemiological investigations of outbreaks of LSD that occurred in 2022 in three Indian states viz., Haryana, Himachal Pradesh and Rajasthan and the genetic characterization of Lumpy skin disease viruses (LSDVs). Also, the population structure analysis of LSDVs was carried out. Out of 138 scab samples tested from suspected cattle for LSDV, 106 were found positive. Of these, nine representative scab samples were further genetically characterized. Phylogenetic analysis based on the P32 and EEV gene sequences depicted that the LSDV strains of the present study had nucleotide identity of 100% and 99.10-99.45%, respectively with the LSDV/2019 field strains of India. Multiple sequence alignment of the EEV glycoprotein gene sequences revealed nucleotide polymorphisms at three positions viz., G178A, G253A and A459G in circulating LSDV/2022 field strains. Based on the median joining network analysis of the EEV gene, 12 haplotypes were identified among the LSDV populations. Population structure analysis corresponding to the EEV gene revealed high haplotype (0.8486 ± 0.026) and low nucleotide diversities (0.00636 ± 0.0006) and negative values for neutrality indices, indicating a high number of closely related haplotypes and the studied population may have undergone a recent expansion. The findings will help in understanding the distribution and dynamics of LSDV populations, which will prove pivotal in designing and implementing effective management and control strategies.

Characterization and molecular identification of the lumpy skin disease virus in cattle in the Mekong Delta of Vietnam.

Background: Lumpy skin disease (LSD) is caused by a virus belonging to the genus Capripoxvirus, exhibiting clinical symptoms ranging from mild signs to the development of nodules. LSD emerged in Asia and Southeast Asia, including Vietnam, in October 2020 and has since spread throughout the region, resulting in productivity and economic losses. Aim: This study aimed to investigate the virus-causing papular dermatitis in cattle from the Mekong Delta region of Vietnam by analyzing its GPCR gene and assessing its evolutionary relationship with sequences in the GenBank database. Methods: Blood samples (n = 180) were collected from cattle farms in Ben Tre, Tien Giang, and Tra Vinh provinces. PCR targeting the P32 antigen gene was utilized to detect LSDV presence, and GPCR gene amplification was performed to assess genetic variability. Results: LSDV was detected in 8.33% (15/180) of the samples using PCR targeting the P32 antigen gene. Each sample that tested positive for LSDV demonstrated complete amplification of the GPCR gene. Sequence alignments and phylogenetic analyses of the GPCR gene revealed that Mekong Delta LSDV isolates shared genetic similarities and possessed a 12-nucleotide insertion comparable to strains from China in 2019 and Northern Vietnam in 2020. Conclusion: This study provides preliminary insights into the molecular characteristics of LSDV in cattle from the Mekong Delta region of Vietnam. The observed genetic relatedness to other LSDV sequences from Asia and Southeast Asia underscores the importance of regional surveillance and control measures. These findings contribute to the development of effective strategies for LSDV control and prevention.

The expanding host range of lumpy skin disease virus in wild and domestic animals.

Clinical lumpy skin disease (LSD) predominantly affects cattle and, to lesser extent domestic water buffalos. Whilst earlier work focussed on the disease in Africa, the recent emergence of LSD virus (LSDV) as a major cause of disease in Asia has led to a widening range of susceptible hosts for the virus. This article lists the wild and domestic ungulates in which LSDV infection has been confirmed and considers the significance of the disease for these species in Asia.

Evaluation of the immune responses in buffaloes vaccinated with a live-attenuated lumpy skin disease vaccine (Lumpi-ProVacInd).

Since 2019, Lumpy skin disease (LSD) has suddenly spread in many Asian countries, including India. LSD primarily occurs in cattle. However, recent LSD outbreaks in India have also revealed significant morbidity and production losses in buffaloes. This has raised concerns about the role of buffaloes in the epidemiology and transmission of LSD and necessitates the inclusion of buffaloes in the mass vaccination program for the prevention and control of the disease in the country. However, there is no significant data on the immune response in buffaloes following vaccination with the LSD vaccine. In this study, we evaluated antibody- and cell-mediated immune responses following vaccination with a newly developed live-attenuated LSD vaccine (Lumpi-ProVacInd). The detectable amount of anti-LSDV antibodies was observed at 1-2 months following vaccination, with a peak antibody titer at 3 months. Upon stimulation of the peripheral blood mononuclear cells (PBMCs) with the UV-inactivated LSDV antigen, there was a significant increase in CD8 + T cell counts in vaccinated animals as compared to the unvaccinated animals. Besides, vaccinated animals also showed a significant increase in IFN-γ levels upon antigenic stimulation of their PBMCs with LSDV antigen. In conclusion, the buffaloes also mount a potent antibody- and cell-mediated immune response following vaccination with Lumpi-ProVacInd.

The first outbreak of Lumpy Skin Disease in Indonesia.

This study describes the first outbreak of Lumpy Skin Disease (LSD) in cattle in the Bengkalis region, Indonesia, and vaccination to control the epidemic. Data on the outbreak and vaccination was obtained from the local veterinary authority of the Bengkalis region, Indonesia. Climatological data was provided by the Meteorological, Climatological, and Geophysical Agency of Riau Province. Over the 5.5 months, the outbreak caused 10.4% (94/906) morbidity and 0.6% (6/906) mortality of cattle on infected farms. Temporally, three epidemic waves occurred during the outbreak period. Villages with cattle populations of > 150 animals (n = 36) were 5.3 times more likely to be infected with LSD compared to villages with smaller cattle populations (n = 107) (CI: 2.56-10.90, P < 0.01). The vaccination campaign covered 43.8% of cattle in villages within a 10 km radius of the cases. However, vaccination in villages with larger cattle populations (n = 29) was 0.63 less likely to cover 50% of the cattle populations compared to villages with smaller cattle populations (n = 41) (CI: 0.39-1.02, P = 0.05). By the time the first two and the major waves ceased, vaccination had covered only 0.0% (n = 6036), 27.8% (n = 6,036) and 9.7% (n = 5,697) of the cattle in the 10 km radius of the respective spatial clusters. The outbreak was statistically associated with rainfall and its interaction with temperature (F(2, 13) = 5.822, R2 = 0.47, P = 0.016). This study indicates that the LSD outbreak had low morbidity and mortality. Despite the low vaccination rate, the outbreak ceased, possibly due to plummeting of the abundance of insect vectors.

A new variant of lumpy skin disease virus circulating in Vietnam based on sequencing analysis of GPCR gene.

Background: In 2021, Vietnam experienced an outbreak of Lumpy skin disease (LSD), which infected 207,687 cattle and buffaloes, as officially reported, and resulted in the culling of 29,182 animals. Aim: In this study, samples from cattle that died and showed typical signs of LSD in the Ha Tinh province of Vietnam were confirmed by three World Organization for Animal Health (WOAH)-recommended methods and further studied to compare the Vietnam and China reference strains to the new clinical cases. Methods: Three methods recommended by WOAH for agent detection (PCR, virus isolation, and transmission electron microscopy) were used to confirm this clinical LSD case. The sequence analysis of three well-known markers (P32, RPO30, and GPCR genes) has been utilized in Vietnam to understand this circulating pathogen better. Results: Our findings showed that the CX01 LSDV strain is 100% identical to the Vietnam reference strain HL01 and China reference strains based on P32 and RPO30 genes. Interestingly, analysis of the nucleotide sequence of the GPCR gene showed that the CX01 strain belongs to the same cluster as the reference strains, but it has branches different from those of both the HL01 and China LSDV strains. The nucleotide identification between the CX01 strain and these reference virus strains ranked 99.65%-99.91%, suggesting that it is a new variant of LSDV. Conclusion: This finding is new and indicates that at least two variants of the LSD virus were circulating in Vietnam based on analysis of the GPCR gene. Additionally, these results suggest that the sequence analysis of the GPCR gene is a great tool for subgrouping LSDV circulating in Vietnam.

Lumpy skin disease diagnosis in cattle: A deep learning approach optimized with RMSProp and MobileNetV2.

Lumpy skin disease (LSD) is a critical problem for cattle populations, affecting both individual cows and the entire herd. Given cattle's critical role in meeting human needs, effective management of this disease is essential to prevent significant losses. The study proposes a deep learning approach using the MobileNetV2 model and the RMSprop optimizer to address this challenge. Tests on a dataset of healthy and lumpy cattle images show an impressive accuracy of 95%, outperforming existing benchmarks by 4-10%. These results underline the potential of the proposed methodology to revolutionize the diagnosis and management of skin diseases in cattle farming. Researchers and graduate students are the audience for our paper.

Detection of lumpy skin disease virus reads in the human upper respiratory tract microbiome requires further investigation.

Lumpy skin disease virus (LSDV), a double-stranded DNA virus from the Capripoxvirus genus, primarily affects Bos indicus, Bos taurus breeds, and water buffalo. Arthropod vectors, including mosquitoes and biting flies, are the main LSDV transmitters. Although LSDV is not zoonotic, this study unexpectedly detected LSDV reads in the upper respiratory tract microbiome of humans from rural and urban areas in Maharashtra, India. Nasopharyngeal and oropharyngeal swab samples collected for SARS-CoV-2 surveillance underwent whole-genome metagenomics sequencing, revealing LSDV reads in 25% of samples. Split kmer analysis provided insights into sample relatedness despite the low coverage of LSDV reads with the reference genome. Our findings, which include the detection of LSDV contigs aligning to specific locations on the reference genome, suggest a common source for LSDV reads, potentially shared water sources, or milk/milk products. Further investigation is needed to ascertain the mode of transmission and reason for the detection of LSDV reads in human upper respiratory tract.

Protocol for next-generation sequencing of the LSD virus genome using an amplicon-based approach.

Lumpy skin disease (LSD) is a viral disease predominantly affecting cattle caused by a poxvirus belonging to the capripoxvirus genus. Here, we present a protocol for next-generation sequencing of the LSD virus genome using an amplicon-based approach. We describe steps for DNA extraction, viral DNA enrichment, amplicon pooling and purification, and library preparation and pooling. We then detail procedures for sequencing and computational analysis. This protocol can be adapted to any Illumina sequencing platform as an accelerated and scalable system. For complete details on the use and execution of this protocol, please refer to Bhatt et al.1,2.

Different Neutralizing Antibody Responses of Heterologous Sera on Sheeppox and Lumpy Skin Disease Viruses.

Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.

Design and assessment of a double antigen indirect ELISA for lumpy skin disease surveillance in India.

Lumpy skin disease (LSD), caused by the lumpy skin disease virus of the genus Capripoxvirus, is rapidly emerging across most countries in Asia. Recently, LSD has been linked to very high morbidity and mortality rates. Until 2019, India remained free of LSD, resulting in a lack of locally developed diagnostic kits, biologicals, and other tools necessary for managing the disease in a country with such a large livestock population. Therefore, this study aimed to design and validate an indigenous and cost-effective in-house ELISA for large-scale screening of cattle samples for antibodies to LSDV. The viral major open reading frames ORF 095 and ORF 103 encoding virion core proteins were expressed in a prokaryotic system and the recombinant antigen cocktail was used for optimization and validation of an indirect ELISA (iELISA). The calculated relative diagnostic sensitivity and diagnostic specificity of the iELISA were 96.6 % and 95.1 %, respectively at the cut-off percent positivity (PP≥50 %). The in-house designed double-antigen iELISA was found effective to investigate the seroprevalence of LSDV in various geographical regions of India.

Evaluation of longitudinal passive immunity transfer against lumpy skin disease virus in calves by different serological methods.

To implement effective lumpy skin disease (LSD) control measures, such as timely vaccination, particularly in calves and serological monitoring, it is necessary to evaluate immune response after vaccination, both in adult cattle and in their calves. The aim of this study was to evaluate passive immunity transfer and duration of maternal antibodies against lumpy skin disease virus (LSDV) in calves born to vaccinated cows by two different serological methods. The longitudinal study was carried out on two farms in Serbia where no cases were reported during LSD outbreak in 2016. Fifteen cows on each farm were vaccinated and revaccinated with attenuated vaccine - Neethling strain. A total of 30 cows and 30 calves on both farms were included in the study. Serum samples from cows were collected on calving day and serum samples from their respective calves on days 10, 20, 30, 45, 60, 75, 90, 105 and 120 after birth. Colostrum samples were collected only from 15 cows on one farm. In order to determine the presence of antibodies against LSDV a total of 30 cow sera samples, 15 colostrum samples and 270 calf sera samples were examined by commercial enzyme-linked immunosorbent assay (ELISA) and modified virus neutralization test (VNT). Overall, the performance of both serological tests was very satisfactory. The results of this longitudinal study showed that persistence of passive immunity in calves is less than 4 months, and that most calves are not protected against LSDV at that age. Since the vaccination is the most important control measure against LSDV, the recommended age of six months for vaccination of calves born to vaccinated cows should be reassessed to achieve the most optimal protection against LSD.

Insights into the involvement of male Hyalomma anatolicum ticks in transmitting Anaplasma marginale, lumpy skin disease virus and Theileria annulata.

Ticks can transmit viruses, bacteria, and parasites to humans, livestock, and pet animals causing tick-borne diseases (TBDs) mechanically or biologically in the world. Lumpy skin disease virus, Anaplasma marginale, and Theileria annulata inflict severe infections in cattle, resulting in significant economic losses worldwide. The study investigated the potential transmissions of LSDV, A. marginale, and T. annulata through male Hyalomma anatolicum ticks in cattle calves. Two 6-month-old Holstein crossbred calves designated as A and B were used. On day 1, 15 uninfected female ticks (IIa) and infected batch of 40 male ticks (I) were attached on calf A for 11 days. Filial transmission of the infections was observed in female ticks (IIb) collected from calf A, where 8 female ticks had been co-fed with infected male ticks. The blood sample of calf B was found positive through PCR for the infections. The larvae and egg pools obtained from the infected ticks were also tested positive in PCR. The study confirmed the presence of these mixed pathogens and potential intra-stadial and transovarial transmissions of A. marginale, T. annulata, and LSDV in male and female ticks of H. anatolicum and experimental calves to establish the feasibility of infections through an in vivo approach.

Lumpy Skin Disease.

Lumpy skin disease (LSD) is a contagious non-zoonotic viral disease of cattle. The disease raises great concern due to the recent rapid spread toward free countries and reoccurrence in countries where control and preventive measures had achieved eradication. Deep nodules involving skin, subcutaneous tissue, and occasionally muscles are localized mostly in the head, neck, perineum, genitalia, udder, and limbs. LSD can cause large economic losses mainly because of the decline in milk production and the decrease in hide value, in addition to the ban of movement of animals and animal products.

Deciphering the genetic landscape of lumpy skin disease: Unraveling variable virulence through comprehensive genome sequence analysis in India.

Lumpy Skin Disease (LSD), a poxvirus disease affecting cattle, emerged in India in 2019 and intensified in 2022, resulting in significant economic losses for dairy farmers. There was unusual shift in mortality and morbidity patterns during the second wave. A comprehensive genetic study conducted, analyzing samples from 2019 to 2022 revealed circulation of two distinct subclades (subclade 1.2a and 1.2b) in India, with the latter showing a different pattern in morbidity and mortality. Notably, the Ankyrin repeats gene-based analysis could differentiate animals with varying clinical scores. Genetic variations were significant, with unique deletions identified, including a 12-nucleotide deletion in the GPCR gene in virus isolates collected during 2022 outbreaks, not reported earlier in Indian LSDV strains. A crucial finding was a significant 95-nucleotide deletion in the Functional Resolution Sequence (FRS) repeats of LSDV genomes from 2022 outbreaks, absent in 2019 samples. These deletions may have influenced the virus's virulence in India.

Genetic Characterization of Lumpy Skin Disease Viruses Circulating in Lesotho Cattle.

Lumpy skin disease is one of the fast-spreading viral diseases of cattle and buffalo that can potentially cause severe economic impact. Lesotho experienced LSD for the first time in 1947 and episodes of outbreaks occurred throughout the decades. In this study, eighteen specimens were collected from LSD-clinically diseased cattle between 2020 and 2022 from Mafeteng, Leribe, Maseru, Berea, and Mohales' Hoek districts of Lesotho. A total of 11 DNA samples were analyzed by PCR and sequencing of the extracellular enveloped virus (EEV) glycoprotein, G-protein-coupled chemokine receptor (GPCR), 30 kDa RNA polymerase subunit (RPO30), and B22R genes. All nucleotide sequences of the above-mentioned genes confirmed that the PCR amplicons of clinical samples are truly LSDV, as they were identical to respective LSDV isolates on the NCBI GenBank. Two of the elevem samples were further characterized by whole-genome sequencing. The analysis, based on both CaPV marker genes and complete genome sequences, revealed that the LSDV isolates from Lesotho cluster with the NW-like LSDVs, which includes the commonly circulating LSDV field isolates from Africa, the Middle East, the Balkans, Turkey, and Eastern Europe.

An insight into emergence of lumpy skin disease virus: a threat to Indian cattle.

Lumpy skin disease (LSD) is a highly infectious and economically devastating viral disease of cattle. It is caused by Lumpy Skin Disease Virus (LSDV) belonging to the genus Capripoxvirus and family Poxviridae. The origin of lumpy skin disease has been traced to Zambia, (an African nation) in Southern part during the year 1929. The first reported case of LSD besides Africa was from Israel, a Middle Eastern nation, thus proving inter-continental spread. Subsequently, the disease entered Middle East, Eastern Europe and Asia with numerous outbreaks in the recent years. LSD has emerged as a significant concern in the Indian sub-continent, due to outbreaks reported in countries such as Bangladesh, India, China in 2019. In the following years, other South and East Asian countries like Taipei, Nepal, Sri Lanka, Myanmar, Bhutan, Vietnam, Hong Kong, Thailand, Malaysia, Laos, Cambodia, Pakistan, Indonesia and Singapore also faced severe outbreaks. At present, LSD is considered to be an emerging disease in the Indian sub-continent due to the recent status of disease. Considering the global scenario, LSDV is changing its transmission dynamics as evidenced by a shift in its epidemiology. As a result of high morbidity and mortality rate among cattle, the current outbreaks have been a major cause of socio-economic catastrophe. This contagious viral disease has eminent repercussions as the estimated monetary damage incurred is quite high. Despite having networked surveillance and comprehensive databases, the recurring outbreaks have raised major concern among researchers. Therefore, this review offers brief insights into the emergence of LSDV by amalgamating the newest literature related to its biology, transmission, clinico-pathology, epidemiology, prevention strategies, and economic consequences. Additionally, we have also provided the epidemiological insights of the recent outbreaks with detailed state wise studies.