Surveillance for lumpy skin disease and foot and mouth disease in the Kimberley, Western Australia.

We quantified the sensitivity of surveillance for lumpy skin disease (LSD) and foot and mouth disease (FMD) in cattle in the Kimberley region of Western Australia. We monitored producer and veterinary activity with cattle for 3 years commencing January 2020. Each year, ~274,000 cattle of 685,540 present on 92 pastoral leases (stations) were consigned to other stations, live export or slaughter. Veterinarians examined 103,000 cattle on the stations, 177,000 prior to live export, and 10,000 prior to slaughter. Detection probabilities for the disease prior to transport or during veterinary procedures and inspections were elicited by survey of 17 veterinarians working in Northern Australia. The veterinarians estimated the probabilities that they would notice, recognise, and submit samples from clinical cases of LSD and FMD, given a 5% prevalence of clinical signs in the herd. We used scenario tree methodology to estimate monthly surveillance sensitivity of observations made by producers and by veterinarians during herd management visits, pre-export inspections, and ante-mortem inspections. Average monthly combined sensitivities were 0.49 for FMD and 0.37 for LSD. Sensitivity was high for both diseases during the dry season and low in the wet season. We estimated the confidence in freedom from the estimated surveillance sensitivity given one hypothetically infected herd, estimated probability of introduction, and prior confidence in freedom. This study provided assurance that the Kimberley is free of these diseases and that routine producer and veterinary interactions with cattle are adequate for the timely detection of the disease should they be introduced.

Development and Validation of a New DIVA Real-Time PCR Allowing to Differentiate Wild-Type Lumpy Skin Disease Virus Strains, Including the Asian Recombinant Strains, from Neethling-Based Vaccine Strains.

The current epidemic in Asia, driven by LSDV recombinants, poses difficulties to existing DIVA PCR tests, as these do not differentiate between homologous vaccine strains and the recombinant strains. We, therefore, developed and validated a new duplex real-time PCR capable of differentiating Neethling-based vaccine strains from classical and recombinant wild-type strains that are currently circulating in Asia. The DIVA potential of this new assay, seen in the in silico evaluation, was confirmed on samples from LSDV infected and vaccinated animals and on isolates of LSDV recombinants (n = 12), vaccine (n = 5), and classic wild-type strains (n = 6). No cross-reactivity or a-specificity with other capripox viruses was observed under field conditions in non-capripox viral stocks and negative animals. The high analytical sensitivity is translated into a high diagnostic specificity as more than 70 samples were all correctly detected with Ct values very similar to those of a published first-line pan capripox real-time PCR. Finally, the low inter- and intra-run variability observed shows that the new DIVA PCR is very robust which facilitates its implementation in the lab. All validation parameters that are mentioned above indicate the potential of the newly developed test as a promising diagnostic tool which could help to control the current LSDV epidemic in Asia.

An Extensive Examination of the Warning Signs, Symptoms, Diagnosis, Available Therapies, and Prognosis for Lumpy Skin Disease.

The lumpy skin disease virus (LSDV) infects cattle and buffalo and causes lumpy skin disease (LSD). It affects the lymph nodes of the sick animals, causing them to enlarge and appear as lumps (cutaneous nodules) that are 2-5 cm in diameter on their heads, necks, limbs, udders, genitalia, and perinea. A high temperature, a sharp drop in milk supply, discharge from the eyes and nose, salivation, a loss of appetite, depression, damaged hides, and emaciation are further warning signs and symptoms. As per the Food and Agriculture Organization (FAO), the incubation period, or the time between an infection and symptoms, is approximately 28 days. Infected animals can transfer the virus by direct contact with the vectors, direct virus secretion from mouth or nose, shared feeding and watering troughs, and even artificial insemination. The World Organization for Animal Health (WOAH) and the FAO both warn that the spread of illnesses could lead to serious economic losses. This illness reduces cow's milk production because oral ulcers make the animal weak and lead them to lose their appetite. There are many diagnostics available for LSDV. However, very few tests yield accurate findings. The best methods for preventing and controlling the lumpy skin condition include vaccination and movement restrictions. As a specific cure is not available, the only available treatment for this illness is supportive care for cattle. Recently, India has developed a homologous, live-attenuated vaccine, Lumpi-ProVacInd, which is specifically intended to protect animals against the LSD virus. This study's primary goal is to accumulate data on symptoms, the most accurate method of diagnosis, treatments, and controls to stop infections from spreading as well as to explore future possibilities for the management of LSDV.

LSDV126 gene based molecular assays for specific detection and characterization of emerging Lumpy Skin Disease virus.

Lumpy skin disease (LSD) is a highly infectious and economically important viral disease, which is currently emerging in the Indian subcontinent. LSD is caused by Lumpy Skin Disease Virus (LSDV) under the genus Capripoxvirus and the family Poxviridae. Since its first incursion in India in the year 2019, the virus is rapidly disseminating through different means like direct contact, fomites and mainly by blood-feeding insects. As the disease has never been reported from India or neighbouring countries, there is a lack of planning and preparatory measures in terms of diagnostics and vaccines to control the disease. In the absence of any homologous vaccine, a live attenuated heterologous goat pox vaccine (Uttarkashi strain) is now being widely used in the country for the prevention of LSDV infection. Use of live attenuated goat pox virus vaccine necessitates the availability of an assay which could specifically detect and differentiate LSDV from goat pox virus. In this study, nucleotide sequences of LSDV126 gene encoding extracellular enveloped virus protein of circulating LSDV and goat pox virus were determined and analyzed. Deletion of 27 nt tandem repeats was observed in LSDV in comparison to goat pox and LSDV vaccine viruses. The deletion region was targeted for designing primers specific to LSDV, but not goat pox virus. A novel isothermal polymerase spiral reaction (PSR) was optimized as pen side diagnostic for prompt and sensitive detection of genomic DNA of LSDV. The assay was found to be highly sensitive and specific when compared to the real-time PCR. The assay was found to be specifically detecting only LSDV but not the goat pox virus. The limit of detection was identified as 9 × 10-6 ng of positive DNA. The assay will provide a point of care tool that will be a boon for the successful control of LSD in India.

A highly adaptable platform powered by CRISPR-Cas12a to diagnose lumpy skin disease in cattle.

Lumpy skin disease (LSD) in cattle, a transboundary viral disease of cattle once restricted to Africa, has been spreading to many European and Asian countries in the past decade with huge economic losses. This emerging worldwide threat to cattle warrants the development of diagnostic methods for accurate disease screening of suspected samples to effectively control the spread of LSD. In this study, we integrated pre-amplification and three kinds of sensor systems with CRISPR and therefore established an LSD diagnosis platform with highly adaptable and ultra-sensitive advantages. It was the first CRISPR-powered platform that could identify lumpy skin disease virus from vaccine strains of goat pox virus and sheep pox virus. Its limit of detection (LOD) was one copy/reaction after introducing PCR or recombinase-aided amplification (RAA). Moreover, this platform achieved a satisfactory overall agreement in clinical diagnoses of 50 samples and its reproducibility and accuracy were superior to other qPCR methods we tested. The whole diagnostic procedure, from DNA extraction to the results, could complete in 5 h with a total cost of 1.7-9.6 $/test. Overall, this CRISPR-powered platform provided a novel diagnostic tool for portable, ultra-sensitive, rapid, and highly adaptable disease screening of LSD and may be an effective method to control this transboundary disease's spread.

Serological, virological and molecular diagnosis of an outbreak of lumpy skin disease among cattle in Butana area, Eastern Sudan.

BACKGROUND: Lumpy skin disease (LSD) is a highly infectious disease of cattle caused by a virus of the Poxviridae family, genus Capripoxvirus. The disease has great economic effects on the global cattle industry. In this study, an outbreak of LSD among cattle which occurred in Nahr Atbara and Halfa El Jadida locations in Butana area, Kassala State in November, 2020 is described. METHODS: Lumpy skin disease virus (LSDV) antibody and nucleic acid were detected in specimens (n = 50) using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). RESULTS: The virus was isolated on the chorioallantoic membrane (CAM) of embryonated chicken eggs and identified by PCR. The number of animals at risk was 295 with a morbidity rate of 4.1% and a mortality rate of 2.4%. CONCLUSIONS: LSDV was diagnosed for the first time in the Butana region, Kassala State, Eastern Sudan in 2020.

Assessing machine learning techniques in forecasting lumpy skin disease occurrence based on meteorological and geospatial features.

Lumpy skin disease virus (LSDV) causes an infectious disease in cattle. Due to its direct relationship with the survival of arthropod vectors, geospatial and climatic features play a vital role in the epidemiology of the disease. The objective of this study was to assess the ability of some machine learning algorithms to forecast the occurrence of LSDV infection based on meteorological and geological attributes. Initially, ExtraTreesClassifier algorithm was used to select the important predictive features in forecasting the disease occurrence in unseen (test) data among meteorological, animal population density, dominant land cover, and elevation attributes. Some machine learning techniques revealed high accuracy in predicting the LSDV occurrence in test data (up to 97%). In terms of area under curve (AUC) and F1 performance metric scores, the artificial neural network (ANN) algorithm outperformed other machine learning methods in predicting the occurrence of LSDV infection in unseen data with the corresponding values of 0.97 and 0.94, respectively. Using this algorithm, the model consisted of all predictive features and the one which only included meteorological attributes as important features showed similar predictive performance. According to the findings of this research, ANN can be used to forecast the occurrence of LSDV infection with high precision using geospatial and meteorological parameters. Applying the forecasting power of these methods could be a great help in conducting screening and awareness programs, as well as taking preventive measures like vaccination in areas where the occurrence of LSDV infection is a high risk.

Observations on presumptive lumpy skin disease in native cattle and Asian water buffaloes around the tiger reserves of the central Indian highlands.

CASE HISTORY: Between August and October 2020, following the monsoon, signs of lumpy skin disease (LSD) were recorded and described in 154 oxen, 34 cows, 13 calves (Bos indicus) and two Asian water buffalo (Bubalus bubalis) cows belonging to smallholder farmers in 32 villages located around the Kanha and Bandhavgarh tiger reserves in the state of Madhya Pradesh, central India. Affected animals were subjected to a full clinical examination and detailed findings were recorded in a clinical register. A semi-structured questionnaire was attached to the existing clinical register format to gather information on the clinical disease history and animal husbandry practices relevant to the spread of LSD virus. CLINICAL FINDINGS: The affected animals were between 4 months and 14 years of age (mean 6.4 (SD 2.5) years). Persistent high temperature, depression, anorexia, and characteristic round nodules (lumps) on the skin were reported. The nodules were 2-5 cm in diameter and spread over the face, ears, neck, back, perineum, scrotum, legs, tail, udder, and nasal and oral mucosa. Secondary complications of myiasis (n = 39), mastitis (n = 16) and ulcerative lesions on legs were noticed. Death was reported for one animal (0.5%). The affected animals' recovery times were variable (mean 18.4 (SD 2.7) days). There was a significant positive correlation between delay in initiating treatment and the duration of sickness. Reduction in milk yield of 30-55% was reported in Bos indicus cows. DIAGNOSIS: Clinical findings and treatment responses consistent with lumpy skin disease and its sequelae. CLINICAL RELEVANCE: The presumptive LSD outbreak caused serious economic loss to the animal keepers. LSD is a new disease for India and in the absence of active immunisation, efficient vector control, animal movement control and stall-feeding practice, it will inevitably become endemic in the country. The severe impacts resulting from the introduction of a new disease to a previously unaffected country highlight the need for iterative improvements in global transboundary disease surveillance. The value of clinical examination and recording of findings is demonstrated in the context of smallholder farming systems with limited access to laboratory diagnosis, which are common around the world. The description of an LSD outbreak in naïve populations of cattle and buffalo illustrates the need for increased awareness of the associated clinical signs and maintenance of high levels of biosecurity in hitherto disease-free countries.

A TaqMan probe-based multiplex real-time PCR method for the specific detection of wild type lumpy skin disease virus with beta-actin as internal amplification control.

Lumpy skin disease (LSD) is a transboundary disease of economic importance affecting cattle and buffaloes. In South-Eastern Europe, immunization of cattle with homologous live attenuated vaccines for LSD control has prevented outbreaks since 2017, but has been associated with adverse reactions resembling disease symptoms. Thus, a diagnostic method suitable for disease surveillance in farms during vaccination campaigns with Neethling (Onderstepoort) and SIS type (Lumpyvax) live attenuated LSDV vaccines in Europe should be able to detect the wild type (WT) LSDV in animals with adverse reactions to the vaccines and samples with potentially high titers of the vaccine LSDV. To this end, a real-time PCR method targeting the EEV gene of LSDV was developed for the specific detection of WT strains, along with the use of beta-actin gene as an internal amplification control (IAC). Amplification efficiency of the WT virus target was 99.0% and 98.6%, in the presence and in the absence of high loads of vaccine LSDV, respectively. In the presence of 105.6 vaccine LSDV DNA copies, the limit of detection for WT LSDV was 12.6 DNA copies per reaction. The inter-assay CV was 0.04% for WT LSDV and 0.13% for beta-actin. The method can confirm diagnosis in suspect cases irrespective of the presence of the vaccine LSDV DNA by overcoming the masking effect of the WT LSDV. The simultaneous amplification of the beta-actin gene further assures the quality of diagnostic testing. The new method is a surveillance tool, complementing the DIVA real-time PCR during vaccination campaigns and can provide rapid insight on the targeted EEV gene in countries with novel and recombinant LSDV strains.

Clinico-molecular diagnosis and characterization of bovine lumpy skin disease virus in Andhra Pradesh, India.

Lumpy skin disease (LSD) is an emerging transboundary disease in India. In the recent past, Andhra Pradesh is experiencing outbreaks of LSD in several pockets with a severe economic impact on the farming community. The affected animals showed nodular lesions all over the body in severely affected cases, whereas the lesions were confined to the face, neck, jowl, back, udder, and scrotum in less affected cases. Young ones are highly susceptible to the disease than adults. The mortality was found to be more in young ones than adult cattle which might be due to subsequent secondary bacterial complications. Clinical samples like blood, serum, and tissues were collected randomly from affected animals from four different pockets of Andhra Pradesh. The tissue samples gave positive amplification in PCR targeting LSDV fusion protein gene (ORF 117) and yielded 472 bp product. Another gene specifically targeting ORF036 of LSDV also gave amplification in tissue samples with a product size of 606 bp. The representative samples from four different regions were sequenced for ORF 117 and 036 genes. The phylogeny of the sequenced products of ORF 117 showed more similarity with Kenya Neethling 2490 strain and Russian isolates of 2019. In addition, the phylogeny of ORF 036 showed the path of entry of the virus into the country and also to Andhra Pradesh. The isolates showed similarity with the isolates of India (Odisha), Bangladesh, Russia, Egypt, and Kenya. These studies paved way for the future perspective of developing a vaccine to control the disease.

Molecular characterization of lumpy skin disease virus (LSDV) emerged in Bangladesh reveals unique genetic features compared to contemporary field strains.

BACKGROUND: Lumpy skin disease (LSD) is a contagious viral disease of cattle caused by lumpy skin disease virus (LSDV). LSD has recently spread in Asia following outbreaks in the Middle East and Europe. The disease emerged in Bangladesh in July 2019 in the Chattogram district, then rapidly spread throughout the entire country. We investigated six LSD outbreaks in Bangladesh to record the clinical signs and collect samples for diagnostic confirmation. Furthermore, we performed the molecular characterization of Bangladesh isolates, analyzing the full RPO30 and GPCR genes and the partial EEV glycoprotein gene. RESULTS: Clinical observations revealed common LSD clinical signs in the affected cattle. PCR and real-time PCR, showed the presence of the LSDV genome in samples from all six districts. Phylogenetic analysis and detailed inspection of multiple sequence alignments revealed that Bangladesh isolates differ from common LSDV field isolates encountered in Africa, the Middle East, and Europe, as well as newly emerged LSDV variants in Russia and China. Instead, they were closely related to LSDV KSGP-0240, LSDV NI2490, and LSDV Kenya. CONCLUSIONS: These results show the importance of continuous monitoring and characterization of circulating strains and the need to continually refine the strategies for differentiating vaccine strains from field viruses.

Performance of the currently available DIVA real-time PCR assays in classical and recombinant lumpy skin disease viruses.

The use of live homologous vaccines to protect against lumpy skin disease virus (LSDV) infection requires the use of molecular tools to differentiate between infected and vaccinated animals (DIVA). In this study, the commercial real-time PCR assays; ID Gene™ LSD DIVA Triplex kit and Bio-T kit® LSD - DIVA, as well as published assays targeting the GPCR gene (Journal of Virological Methods, 249, 48-57) and ORF008 and ORF126 (Sel'skokhozyaistvennaya Biologiya, 54, 347-358) were evaluated. These assays correctly identified classical field isolates (European lineage) and vaccine (Neethling vaccine). In contrast, when tested using vaccine-like recombinant viruses, the commercial and published assays were not able to correctly identify recombinant isolates. At the same time, the recombinant viruses were detected as either field and/or vaccine, or not detected at all depending on the assay. The different gene sequences present in recombinant viruses cause these DIVA assays to incorrectly assign recombinant viruses as either a field or vaccine virus. This observation has implications for using these assays and for identification of LSDV vaccine.

Overview of diagnostic tools for Capripox virus infections.

Capripox viruses are the causative agents of important animal diseases in cattle (Lumpy Skin Disease), sheep (Sheeppox) and goats (Goatpox) with severe socio-economic impact in case of wide scale outbreaks. Therefore there is a constant need for adequate diagnostic tools. The assays must be fit-for-purpose to identify the virus quickly and correctly and to be useful for surveillance and monitoring at different stages of an epidemic. Different diagnostic performance characteristics are required depending on the situation and the test purpose. The need for high throughput, high specificity/sensitivity and the capability for differentiating field virus strains from vaccine strains drives the development of new and better assays preferably with an advantageous cost-benefit balance. This review aims to look at existing and new virological and serological diagnostic tools used in the control against diseases caused by Capripox viruses.

An Immunoperoxidase Monolayer Assay (IPMA) for the detection of lumpy skin disease antibodies.

During this study a new Immunoperoxidase Monolayer Assay (IPMA) was developed for the detection of antibodies against lumpy skin disease virus (LSDV) in an easy and low tech setting. Using two dilutions (1:50 and 1:300) in a duplicate format, the test was shown to be highly sensitive, specific and repeatable. In comparison to the VNT and a commercial ELISA, the LSDV-IPMA was able to detect the LSDV antibodies earlier in infected, vaccinated and vaccinated/infected animals. The assay is very flexible as it can be easily adapted for the detection of sheeppox or goatpox antibodies and it can be scaled-up to handle medium size sample sets by preparing the IPMA plates in advance. These plates are safe and can be handled in low biosafety level labs.

Experimental lumpy skin disease virus infection of cattle: comparison of a field strain and a vaccine strain.

Lumpy skin disease virus (LSDV) infections can cause massive clinical signs in cattle and have great economic impact due to severe trade restrictions. For LSDV control, only live attenuated vaccines are commercially available, but they currently are not authorized in the European Union. Moreover, these vaccine virus strains can induce substantial side effects with clinical signs similar to infections with virulent LSDV. In our study, we compared clinical symptoms, viremia, and seroconversion of cattle inoculated either with a virulent field strain from North Macedonia isolated from diseased cattle in 2016 or with the attenuated LSDV vaccine strain "Neethling". Using specimens from the field and from experimental inoculation, different diagnostic tools, including a pan-capripox real-time qPCR, newly developed duplex real-time qPCR assays for differentiation between virulent and attenuated LSDV strains, and several serological methods (ELISA, indirect immunofluorescence test and serum neutralization test [SNT]) were evaluated. Our data show a high analytical sensitivity of both tested duplex real-time qPCR systems for the reliable distinction of LSDV field and vaccine strains. Moreover, the commercially available capripox double-antigen ELISA seems to be as specific as the SNT and therefore provides an excellent tool for rapid and simple serological examination of LSDV-vaccinated or infected cattle.

A real-time PCR screening assay for the universal detection of lumpy skin disease virus DNA.

OBJECTIVE: The resurgence of lumpy skin disease virus isolates of different genotypic natures abolishes the accuracy of assays that target either vaccine or field strain genome. The aim of the present study was to develop a universal real-time PCR assay using TaqMan chemistry to cover field, vaccine, and recombinant strains of lumpy skin disease virus isolates. RESULTS: The PCR assay was designed based on a LSDV044 target region that offers a unique identification locus to facilitate the sensitive and specific detection of all isolates known to date. The efficiency of amplification, determined over five orders of magnitude, was 93%, with the standard deviation remaining in the range of 0.11-0.23. Evaluation of the assay repeatability on three different days revealed that the inter-run variability ranged from 0.83 to 1.22 over five repetitions across three runs. This new screening assay is proposed as a fast, efficient, and sensitive tool that can be employed in the basic or applied surveillance studies regardless of the genotype. Moreover, the assay can be used for the routine laboratory testing of animal samples during eradication programs for lumpy skin disease.

Importance of the lumpy skin disease virus (LSDV) LSDV126 gene in differential diagnosis and epidemiology and its possible involvement in attenuation.

Examination of lumpy skin disease virus (LSDV) isolates from different geographic regions and times revealed that assays developed in our laboratory for differentiating between virulent Israeli viruses and Neethling vaccine virus (NVV) are generally useful in most, if not all, endemic areas in which NVV-based vaccines are used. Recently it was revealed that the LSDV126 gene of field isolates contains a duplicated region of 27 bp (9 aa), while the vaccine viruses have only one copy. Phylogenetic analysis of a 532-bp segment carrying the LSDV126 gene and whole virus genome sequences revealed that LSDV isolates formed two groups: virulent and vaccine viruses. In this analysis, all of the capripox viruses that lack the ability to efficiently infect cattle were found to carry only one copy of the 27-bp fragment, suggesting that the LSDV126 gene plays an important role in the ability of capripox viruses to infect cattle. In silico analysis of potential antigenic sites in LSDV126 revealed that LSDV126 variants with only one copy of the repeat lack a potentially important antigenic epitope, supporting its possible significance in cattle infection. This study provides new information about the nature of the LSDV126 gene and its possible role in the life cycle of LSDV.

Validation of a diagnostic tool for the diagnosis of lumpy skin disease.

BACKGROUND: Lumpy skin disease (LSD) is caused by LSD virus which is a member of the Capripoxvirus (CaPV) genus. Although PCR provides for a rapid and sensitive diagnosis, it has limited use due to its complexity in terms of cost, time and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, specific and cost-effective method with a diagnostic accuracy similar to PCR. OBJECTIVES/HYPOTHESIS: To compare the detection rate (DR) of two LAMP assays versus PCR for the detection of CaPV. ANIMALS: This study used 105 apparently health animals (AHA) and 59 clinically sick animals (CSA). METHODS AND MATERIALS: PCR and LAMP assays (LAMP1 and LAMP 2) were compared for detection of CaPV from AHA and CSA using blood and tissue samples. The detection was confirmed by sequencing of PCR positive samples. Analytical sensitivity and specificity of LAMP assays also were assessed. RESULTS: The DR in CSA was 13.6% for PCR whereas for LAMP it was 39.0% and 25.4% for LAMP 1 and 2 methods, respectively. In AHA, the LAMP assay DR was 14.3% and 1.9% for LAMP 1 and 2, respectively. Phylogenetic tree analysis confirmed the identity of CaPV. Analytic sensitivity showed a detection limit of 8 copies/µL. The analytic specificity test showed no cross detection with other infectious agents. CONCLUSION AND CLINICAL IMPORTANCE: Good sensitivity and specificity results for LAMP assay support its application in the routine diagnosis of LSD, whereas its ability to detect LSDV in apparently healthy animals shows its usefulness in identifying populations at risk of LSD.

Suitability of group-level oral fluid sampling in ruminant populations for lumpy skin disease virus detection.

The geographic expansion of Lumpy skin disease (LSD) from the near East into the European Union highlighted again the need for appropriate disease detection tools applicable to animal host populations where access to individual animals is difficult. This is of particular importance considering that the clinical manifestation of LSD is often mild making early disease detection challenging under the above-mentioned conditions. Building on positive experiences of group-level oral fluid sampling for pathogen detection as it is known to work for swine herds and wild boar, the concept was transferred to ruminants. Two groups of six cattle were infected experimentally with Lumpy skin disease virus (LSDV) under controlled conditions. Blood as well as oropharyngeal and nasal swab samples were collected at regular intervals. Group samples were obtained by placing cotton gauze around a salt lick block provided commonly as dietary supplement. Pieces of the gauze with visible signs of manipulation were tested in parallel to samples obtained from individual animals. Genome load analysis by qPCR technology revealed LSDV detection window starting from day 2 post infection until day 28 post infection, the end of the animal trial. At the individual level, detection periods varied between animals and type of sample and included intermitted detection. The accumulative character of the alternative sampling method makes it suitable to detect LSDV DNA at group-level even at times of the infection where a selective sampling of individuals from a group - as normally done in LSD surveillance - would have most likely failed in the detection.

Identification and characterization of lumpy skin disease virus isolated from cattle in the Republic of North Ossetia-Alania in 2015.

The first notifications of the unknown disease of cattle appeared in September-October 2015 in North Caucasus region of Russia (Republic of North Ossetia-Alania). The clinical signs included watery discharge from eyes, apathy, loss of appetite, salivation, lameness and nodular skin lesions. Capripoxvirus genome was detected by real-time PCR in the tissue samples of sick animals. The aetiological agent was isolated in the primary cell cultures of lamb testis and goat testis, as well as in the continuous MDBK cell culture. Further sequencing of the GPCR gene and phylogenetic analysis showed the close genetic relationship of isolated capripoxvirus with a group of lumpy skin disease virus. Koch's postulates were fulfilled by the experimental infection of four calves with a suspension of tissue samples from sick animals.