Determination of lumpy skin disease virus in bovine meat and offal products following experimental infection

Lumpy skin disease (LSD) has recently expanded its range northwards to include the Balkans, Turkey and Russia. Because there was no solid evidence conclusively verifying the transmission mechanism in the field and LSDV viraemic animals with overt and asymptomatic presentation of disease and their products may represent a risk as an indirect transmission pathway. In this work, we used PCR positivity and infectivity in clinical and subclinical infection to evaluate the safety of meat and offal products from cows infected with the virulent LSDV strain Russia/Dagestan/2015. At day 21 post infection, seven of the 12 animals developed the generalized disease, and four animals became subclinically infected without apparent clinical signs. Upon examination and necropsy, the animals with the generalized disease had skin lesions; noticeably enlarged lymph nodes; and lesions in the lungs, trachea and testicles; whereas subclinically ill animals exhibited only enlarged lymph nodes and fever. For both disease presentations, testing of skeletal meat by PCR and virus isolation showed that the skeletal meat did not contain live virus or viral genome, whereas in cattle with generalized disease, meat with gross pathology physically connected under the site of a skin lesion was positive for the live virus. In subclinical infection, only enlarged lymph nodes carried the infectious virus, while the other internal organs tested in both types of disease manifestation were negative except for the testicles. Overall, our findings demonstrate that clinically and subclinically infected animals are reservoirs of live LSDV in lymph nodes and testicles, whereas deep skeletal meat in both types of infection do not carry live virus and the risk of transmission through this product seems very low. The detection of LSDV in testicular tissues in subclinically ill animals is concerning because of the potential to spread infection through contaminated semen. This aspect requires reconsideration of surveillance programmes to identify these Trojan horses of LSDV infection.     

Detection of vaccine‐like lumpy skin disease virus in cattle and Musca domestica L. flies in an outbreak of lumpy skin disease in Russia in 2017

Since 2012, lumpy skin disease virus (LSDV) has been spreading from the Middle East to south‐east Europe and Russia. Although vaccination campaigns have managed to contain LSDV outbreaks, the risk of further spread is still high. The most likely route of LSDV transmission in short distance spread is vector‐borne. Several arthropod species have been suggested as potential vectors, but no proven vector has yet been identified. To check whether promiscuous‐landing synanthropic flies such as the common housefly (Musca domestica ) could be involved, we carried out entomological trapping at the site of a recent LSDV outbreak caused by a vaccine‐like LSDV strain. The presence of vaccine‐like LSDV DNA was confirmed by the assay developed herein, the assay by Agianniotaki et al. (2017) and RPO 30 gene sequencing. No evidence of field LSDV strain circulation was revealed. In this study, we discovered that M. domestica flies carried vaccine‐like LSDV DNA (C _t  > 25.5), whereas trapped stable flies from the same collection were negative for both field and vaccine LSDV . To check whether flies were contaminated internally and externally, 50 randomly selected flies from the same collection were washed four times and tested. Viral DNA was mainly detected in the 1st wash fluid, suggesting genome or even viral contamination on the insect cadaver. In this study, internal contamination in the insect bodies without differentiation between the body locations was also revealed; however, the clinical relevance for mechanical transmission is unknown. Further work is needed to clarify a role of M. domestica in the transmission of LSDV . To our knowledge, this is the first report demonstrating that an attenuated LSD vaccine strain has been identified in Russian cattle given the ban on the use of live attenuated vaccines against LSDV.     

Detection of lumpy skin disease virus in skin lesions,blood, nasal swabs and milk following preventive vaccination

Lumpy skin disease caused by Capripoxvirus is at the moment the most important threat to European cattle industry. The only way for successful control of disease is fast and efficient diagnosis and vaccination. According to EU legislation, vaccination against LDS can be conducted only after confirmation of the disease. Croatia has a special position regarding LSD —in 2016, for the first‐time vaccination of the entire cattle population was conducted without an index case. The presence of vaccine viral particles was detected in milk, skin nodules, blood and nasal swabs in seven from total of eight herds. The presence of virus genome was detected in five cows from 10 up to 21‐day post‐vaccination. The virus was successfully isolated on cell culture from 10 up to 21‐day post‐vaccination from three animals. The obtained results support the need for further efforts to develop safer vaccines against LSDV .     

Spread rate of lumpy skin disease in the Balkans, 2015–2016

After its introduction in Turkey in November 2013 and subsequent spread in this country, lumpy skin disease (LSD ) was first reported in the western Turkey in May 2015. It was observed in cattle in Greece and reported to the World Organization for Animal Health (OIE ) in August 2015. From May 2015 to August 2016, 1,092 outbreaks of lumpy skin disease were reported in cattle from western Turkey and eight Balkan countries: Greece, Bulgaria, The Former Yugoslav Republic of Macedonia, Serbia, Kosovo, and Albania. During this period, the median LSD spread rate was 7.3 km/week. The frequency of outbreaks was highly seasonal, with little or no transmission reported during the winter. Also, the skewed distribution of spread rates suggested two distinct underlying epidemiological processes, associating local and distant spread possibly related to vectors and cattle trade movements, respectively.     

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.     

Effect of using frozen–thawed bovine semen contaminated with lumpy skin disease virus on in vitro embryo production

Lumpy skin disease (LSD) is an important transboundary animal disease of cattle with significant economic impact because of the implications for international trade in live animals and animal products. LSD is caused by a Capripoxvirus, LSD virus (LSDV), and results in extensive hide and udder damage, fever and pneumonia. LSDV can be shed in semen of infected bulls for prolonged periods and transmitted venereally to cows at high doses. This study examined the effects of LSDV in frozen‐thawed semen on in vitro embryo production parameters, including viral status of media and resulting embryos. Bovine oocytes were harvested from abattoir‐collected ovaries and split into three experimental groups. After maturation, the oocytes were fertilized in vitro with frozen‐thawed semen spiked with a high (HD) or a lower (LD) dose of LSDV, or with LSDV‐free semen (control). Following day 7 and day 8 blastocyst evaluation, PCR and virus isolation were performed on all embryonic structures. After completing sufficient replicates to reach 1,000 inseminated oocytes, further in vitro fertilization (IVF) runs were performed to provide material for electron microscopy (EM) and embryo washing procedures. Overall, in vitro embryo yield was significantly reduced by the presence of LSDV in frozen‐thawed semen, irrespective of viral dose. When semen with a lower viral dose was used, significantly lower oocyte cleavage rates were observed. LSDV could be detected in fertilization media and all embryo structures, when higher doses of LSDV were present in the frozen‐thawed semen used for IVF. Electron microscopy demonstrated LSDV virions inside blastocysts. Following the International Embryo Transfer Society washing procedure resulted in embryos free of viral DNA; however, this may be attributable to a sampling dilution effect and should be interpreted with caution. Further research is required to better quantify the risk of LSDV transmission via assisted reproductive procedures.