Lumpy Skin Disease (LSD): Pathomorphological Features and Molecular Detection in Dairy Cattle of West Coastal India

Lumpy skin disease (LSD) is an emerging pox viral disease affecting cattle population worldwide. In India, the first outbreak of LSD is reported during August 2019 in Odisha state, which then followed by outbreaks in crossbred and indigenous cattle population of other states. Present investigation designed to study the prevalence, pathomorphological changes and molecular detection of LSD virus in naturally infected cattle. The overall morbidity of LSD was 4.48% among 30 dairy farms. Skin nodular biopsy, whole blood and serum samples (n= 66) were collected for the diagnosis of LSD by histopathology, PCR and sequencing. The envelope protein gene (P32), Fusion protein (F) and DNA dependent RNA polymerase 30 kDa subunit (RPO30) genes were targeted for PCR testing. Out of 66, 46 cattle showed generalized skin nodules and papules of various sizes (0.5 - 6.5cm) on the skin particularly at neck, face, nose, tail, perineum and udder. Microscopic examination of the skin nodule biopsy tissue revealed presence of diffuse granulomatous inflammation, hyperkeratosis, focal to diffuse vasculitis and lymphangitis, vacuolar degeneration, spongiosis and acanthosis. The inflammatory cells typically comprised of macrophages, lymphocytes, neutrophils and eosinophils along with diffuse necrosis in dermis in chronic cases. The eosinophilic intracytoplasmic viral inclusions in keratinocytes and epithelial cells were detected in few cases. Gel-PCR assay detected P32 gene in 83%, F gene in 72% and RPO30 gene in 77% of skin biopsy samples. Three blood samples were also found positive for P32 gene by PCR. Whereas TaqMan™ probe Real Time PCR targeting EEV glycoprotein gene (LSDV126) detected LSDV in 94% of biopsy samples and three blood samples which indicated its higher sensitive for the diagnosis of LSDV. Phylogenetic analysis of RPO30 gene sequence showed that the isolates from this study were grouped in same cluster with LSDV isolates of Bangladesh, Kenya and other Indian isolates detected during 2019-20.

Lumpy skin disease outbreaks in Egypt during 2017-2018 among sheeppox vaccinated cattle: Epidemiological, pathological, and molecular findings

The General Organization of the Veterinary Services in Egypt has adopted a sheeppox vaccination policy to control lumpy skin disease (LSD) in cattle. Over the course of the last two years, recurrent outbreaks were reported, with animals showing severe clinical signs and consequentially higher fatalities than that of cases reported in previous LSD outbreaks. A total of 1050 cattle showing typical clinical signs suggestive of LSD were clinically and pathologically investigated during 2017–2018. Skin nodules were collected and lumpy skin disease virus (LSDV) was screened in collected skin samples using PCR for the RPO-30 gene. Furthermore, the entire P32 protein coding gene was sequenced. Histopathology and immunohistochemistry of the skin nodules were also conducted. The obtained results showed an overall mortality rate of 6.86%. LSDV was confirmed in all the examined nodules as evidenced by immunohistochemistry and positive PCR amplification of the RPO30 gene. Sequencing analysis of the P32 gene revealed a highly conserved nature and genetic stability of the LSDV. The results of the present study show that the current vaccination protocol was not effective for a multitude of reasons. These results also serve as evidence for a strong recommendation of an amendment of homologous vaccine use aside from a complete coverage of cattle populations in order to reduce the incidence of LSD among cattle population in Egypt.

Lumpy skin disease (LSD): Pathomorphological features and molecular detection in dairy cattle of west coastal India

Lumpy skin disease (LSD) is an emerging pox viral disease affecting cattle population worldwide. In India, the first outbreak of LSD is reported during August 2019 in Odisha state, which then followed by outbreaks in crossbred and indigenous cattle population of other states. Present investigation designed to study the prevalence, pathomorphological changes and molecular detection of LSD virus in naturally infected cattle. The overall morbidity of LSD was 4.48% among 30 dairy farms. Skin nodular biopsy, whole blood and serum samples (n= 66) were collected for the diagnosis of LSD by histopathology, PCR and sequencing. The envelope protein gene (P32), Fusion protein (F) and DNA dependent RNA polymerase 30 kDa subunit (RPO30) genes were targeted for PCR testing. Out of 66, 46 cattle showed generalized skin nodules and papules of various sizes (0.5 - 6.5cm) on the skin particularly at neck, face, nose, tail, perineum and udder. Microscopic examination of the skin nodule biopsy tissue revealed presence of diffuse granulomatous inflammation, hyperkeratosis, focal to diffuse vasculitis and lymphangitis, vacuolar degeneration, spongiosis and acanthosis. The inflammatory cells typically comprised of macrophages, lymphocytes, neutrophils and eosinophils along with diffuse necrosis in dermis in chronic cases. The eosinophilic intracytoplasmic viral inclusions in keratinocytes and epithelial cells were detected in few cases. Gel-PCR assay detected P32 gene in 83%, F gene in 72% and RPO30 gene in 77% of skin biopsy samples. Three blood samples were also found positive for P32 gene by PCR. Whereas TaqMan™ probe Real Time PCR targeting EEV glycoprotein gene (LSDV126) detected LSDV in 94% of biopsy samples and three blood samples which indicated its higher sensitive for the diagnosis of LSDV. Phylogenetic analysis of RPO30 gene sequence showed that the isolates from this study were grouped in same cluster with LSDV isolates of Bangladesh, Kenya and other Indian isolates detected during 2019-20.

Molecular phylogenetics of a recently isolated goat pox virus from Vietnam

Background After a decade of silence, an outbreak of the contagious and Asian endemic disease, goat pox re-emerged in North Vietnam affecting more than 1800 heads with a mortality rate of 6.5%. The inevitable impact of goat pox on hide quality, breeding, chevon and milk production has resulted in a significant economic losses to the developing goat industry of Vietnam. In the act of establishing an effective control of this devastating disease, tracing the source of re-emergence via a phylogenetic study was carried out to reveal their genetic relatedness. Either skin scab or papule from the six affected provinces were collected, cultured into Vero cells followed by restricted enzyme digestion of targeted P32 gene DNA encoding. The P32 gene was then cloned and transformed into E.coli competent cells for further sequencing. Results The isolated sequence is deposited into GenBank under Accession No. MN317561/VNUAGTP1. The phylogenetic tree revealed high similarity of nucleotide and amino acid sequences to references goat pox strains accounting for 99.6 and 99.3, respectively. The Vietnamese strain is clustered together with currently circulating goat pox virus in China, India and Pakistan which suggested the origin of South China. Conclusions This Vietnam isolate is clustered together with other Asian goat pox strains indicating the dissemination of a common goat pox virus within this continent.

Identification and Comparative Phylogeny of Sheep and Goat Pox Isolates from Jammu, India

Background: In view of the limited documentation of sheep and goat pox disease in animals of Jammu region, it was felt necessary to conduct a blanket screening of the small ruminant populations in areas where they are largely reared. Methods: The investigation was conducted to study the occurrence of capripox infection amongst small ruminants through clinical survey and confirmatory laboratory diagnosis. PCR amplification of a part of the P32 core protein gene successfully confirmed Capripoxvirus (CaPV) in clinical scab samples from both sheep and goats and from inoculated CAM. Positive amplified samples yielded a predicted 192 bp product. Result: Sequencing of the partial P32 gene of one isolate each from sheep (Sheeppoxvirus, SPPV) and goat origin (Goatpoxvirus, GTPV) revealed that each isolate were distinct and showed 97.8% identity with each other. The SPPV clustered with respective sheeppox cluster with 100% homology to with other SPPV strains reported within India and abroad and also to vaccine strains Srinagar and Rumanian-Fanar (RF). The GTPV was closely identical (~98-99%) with other strains from India and abroad with some unique residues of its own. Both the SPPV and GTPV were divergent from Lumpy Skin Disease Virus (LSDV) strains. The sequence of SPPV strain being similar to circulating strains and prevalent vaccines in the country favours a common vaccination strategy. However, specific GTPV vaccine is not available in the country. The circulating CaPV isolates were found to be host specific. The possibility of vaccination failures or disease caused by vaccines itself, or cross transmission between hosts cannot be ruled out and in such instances it becomes difficult to distinguish with natural disease.

Phylogenetic analysis of Mongolian sheeppox and goatpox viruses

Sheeppox and goatpox are caused by sheep pox virus (SPPV) and goat pox virus (GTPV), members of Capripoxvirus genus, Poxviridae family. SPPV and GTPV damage host animal’s wool and skin and reduce production of mutton and milk. Because of morbidity and mortality of the diseases, they bring huge economic burden to the country. Main goal was to compare Mongolian sheep pox, goat pox sequences with other strains that were registered in Genebank. In this study, two SPPV and two GTPV field strains from Mongolia and Perego M strain (Biocombinat SOI, Mongolia), Russian and Chinese alive vaccine strains were used. The common DNA extraction method was used and samples were amplified on a nested polymerase chain reaction (nested-PCR) which amplify the full p32 gene of Capripoxvirus. The primers were designed based on the conserved sequences just outside of the p32 gene of SPPV or GTPV. By applying this method to the sheep and goat samples, suspected with SPPV and GTPV infection in Mongolia, the nested-PCR products were obtained from all samples on the predicted size, and the presence of SPPV and GTPV were confirmed via full length sequence analysis of P32 gene. Sequence comparison was performed using the online BLAST program. Sequence identities of nucleotides were analyzed using MUSCLE algorithm. A phylogenetic tree derived from nucleotide sequences was constructed for the Capripoxvirus using the neighbor joining method of MEGA (version X) software. Based on the phylogenetic tree, the Mongolian sheep pox virus, 2017 clustered together with Zabaikalsk strain and Perego strain (Biocombinat SOI, Mongolia). The Mongolian sheep pox virus, 2015 was closer to Tunisian and Chinese Gansu, Shanxi province strains. Chinese vaccine strain AV41, sequenced in this study was clustered with EF522181.1 Chinese Goat pox vaccine strain but Russian sheep pox vaccine strain, sequenced in this study was close to Mongolian goat pox viruses, 2009. The present data provides theoretical references to improve the preventive and control strategy. Based on the phylogenetic tree that we made, we conclude that SPPV and GTPV sequences in Mongolia were closer to Chinese SPPV, GTPV sequences therefore they were most likely imported from China.

Molecular characterization of sheeppox virus from outbreaks in Karnataka, India

Aim: This study aimed to characterize sheeppox virus (SPPV) using the P32 gene of the Capripoxvirus (CaPVs). Materials and Methods: Clinical samples of skin, scabs, and nasal swab from suspected outbreaks Horalagallu (n=13) and Gerahalli (n=11) at Ramanagara district in Karnataka were collected. All the samples were initially subjected to genus-specific diagnostic polymerase chain reaction (PCR). The pooled clinical samples from each outbreak were also subjected to virus isolation. The isolates were confirmed by CaPVs genotyping PCR targeting the full-length P32 gene, followed by sequencing and phylogenetic analysis. Results: The clinical signs and lesions varied from mild to severe degree with no specificity between age and sex. Specific cytopathic changes in cell morphology were observed in infected Vero cells from both outbreaks, which were confirmed by PCR. The complete P32 gene from two outbreaks was successfully amplified with the expected amplicon size of 1006bp. The sequencing and phylogenetic analysis revealed that both the outbreaks were due to SPPV and shared high similarity with published SPPVs from Karnataka and other parts of India. Conclusion: The current study showed that complete P32 gene-based genotypic PCR assay can be used for genetic characterization and molecular epidemiology of both sheeppox and goatpox diseases and also to differentiate the causative agents. The sequence analysis revealed 100% similarity among the two outbreak isolates suggesting the same strain of the virus and common source of infection for the outbreaks.

Prevalence and PCR based molecular characterization of goat pox virus from field outbreaks of Multan and Bahawalnagar, Pakistan

This study was designed to check the prevalence and PCR-based molecular characterization of goat pox virus (GTPV) in the Multan and Bahwalnagar regions of Punjab, Pakistan. Capripox virus (CPPV) is the cause of goat pox (GTP) and sheep pox (SPP) disease, it highly affects the morbidity and mortality rate of goats and sheep. In this study, the 80 tissues and blood samples of goats were collected on age basis from the goat farms, slaughter houses, tanneries and domestic animals. The epidemiological data was also collected. The collected samples were processed for DNA extraction. We characterized the goat pox virus (GTPV) with specific primers of P32 gene by PCR. Then each amplified product was analyzed by agarose gel electrophoresis visualized by UV fluorescence light. This study showed that Infants of goats (2-10 months) in Multan showed 25% while adult goat in Multan showed 14.2 % positive results. In Bahawlnagar, the affected infants of goats (2-10 months) found were 31.25% while adult infected goats were 11.1%. Both primers were equally effective for the characterization of unknown samples. The most effected goats were adult female and infants. J. bio-sci. 28: 95-103, 2020

Molecular detection and phylogenetic tree of infectious laryngotracheitis virus in layers in Al-Diwaniyah province, Iraq

Background and Aim: Infectious laryngotracheitis (ILT) of chickens is a substantial issue to be studied in Iraq because this disease is one of the most highly contagious respiratory diseases in the world caused by a herpesvirus. However, in Iraq, the ILT virus (ILTV) infection and disease have yet not been confirmed in layers, so farm owners do not vaccinate these layers. The current study aimed to document the detection and characterization of ILTV in layer hens from Al-Diwaniyah city, for the first time in Iraq, using molecular techniques like polymerase chain reaction (PCR) and sequencing. Materials and Methods: Four layer farms (15,000 unvaccinated layers/farm) in Al-Diwaniyah province, Iraq, suffered a severe ILT outbreak, was diagnosed and reported by clinical and PCR tests. This disease has been reported in Iraq, and more recently, it began to show outbreaks in Al-Diwaniyah city. The current work opted to investigate the ILTV using PCR and DNA sequencing techniques. The study targeted the p32 gene of ILTV using pooled tracheal swabs and organs including the trachea, lung, and kidneys which were collected from dead and clinically infected chickens. Results: The analyses revealed that four of six suspected field samples showed positive results by PCR. The DNA sequencing results showed the homology of the amplified fragments with the studied gene. Conclusion: This study confirmed the presence of ILTV in hens with respiratory signs during the outbreak.