Studies of molecular detection and histopathological examination of lumpy skin diseased cattle

Introduction

The world was plagued by an abundance of disease outbreaks during the past 20 years, particularly those driven on by re-emerging viruses such as the Zika virus, the Nipah virus, the severe acute respiratory syndrome virus 2 (SARS-CoV-2), and monkeypox virus (MPXV) (Mourya et al., 2019) [21] . These have been linked to the spillovers from different animal species brought about by increased contact (with humans), mutations in genes that finally brought about variations, and species-crossing (Chadha et al., 2022) [6] . The lumpy skin disease (LSD) is experiencing an unprecedented pandemic in various Asian nations. The lumpy skin disease virus (LSDV) is responsible for this highly contagious and re-emerging viral infection in domestic cattle and Asian water buffaloes.It is a member of the family Poxviridae, subfamily Chordopoxviridae, and the genus Capripoxvirus (Tuppurainen et al., 2017) [30] . Like other poxviruses, LSDV replicates in an infected cell's cytoplasm, generating unique perinuclear viral factories. The LSD virus is a sizable, brick-shaped particle with dimensions of 262-273 nm in width and 293-299 nm in length. Similar to other poxviruses, the LSDV genome is double-stranded linear DNA with 25 per cent GC content that is 1,50,000 base pairs (bp) in length and encodes about 156 open reading frames (ORFs). At each end of the linear genome, there is a 2200-2300 bp inverted terminal repeat sequence. This disease is known as 'Neethling virus disease,' 'knopvelsiekte,' 'pseudo-urticaria,' and 'exanthema nodularis bovis' (Tuppurainen et al., 2017; Khan et al., 2021) [30,18] . Moreover, LSDV is closely related to the goat poxvirus (GTPV) and sheep poxvirus (SPPV), as it shares high sequence similarity and antigenic relationships with them (Diallo and Viljoen, 2007) [9] . The first reports of the lumpy skin disease in India was at 2019, and later on spread to various states, like Gujarat, Rajasthan, Haryana, Delhi, Tamilnadu, Tripura, Telangana, Odisha, Kerala, West Bengal, Maharashtra, Assam, Madhya Pradesh, Jharkhand, Andhra Pradesh, and Chhattisgarh (Datten et al., 2023; Yadav et al., 2021) [8,31] . Lumpy skin disease is a transboundary, vector-borne, non-zoonotic disease and become a major hazard to livestock around the world in recent years (Gupta et al., 2020) [20] . Using hematoxylin and eosin staining, histopathology of skin lesions offers a way to identify the intracytoplasmic inclusion bodies of LSD virus-infected cells (Tuppurainen et al., 2011) [29] . The disease can be diagnosed by routinely used serological and molecular tests (OIE, 2017).

Hence, the present study was conducted to diagnose the lumpy skin disease by PCR and histopathological changes occurred in the Skin of affected cattle that were located in and around the Tirupati District of Andhra Pradesh.

Materials and Methods

The present study was carried out at household cattle located in five mandals of Tirupati District, Veterinary Clinical Complex, College of Veterinary Science, Tirupati and SLDL, Tirupati, from December 2022 to September 2023. A total of 582 cattle, which exhibited classical signs of LSD were selected among the 40 skin biopsy and 40 whole blood samples were collected for PCR and five were collected for Histopathological Examination. The LSD virus was available at state level diagnostic laboratory, SLDL,Tirupati was used as a positive DNA control during the present study and the collected whole blood sample was stored at -4 ℃ or at -20 ℃, whereas tissue samples was stored at -70 ℃ untill further processing. These samples were processed as per standed protocol mentioned by OIE, 2017. Isolation of viral DNA from blood samples, tissue homogenate was done using QIAamp® DNA Mini Kit (QIAGEN). The blood samples and tissue samples collected from suspected cases of LSD were confirmed with conventional PCR targeting genus specific P32 gene, species specific GPCR gene, RPO030 gene and Ankyrin repeat gene according to Binepal et al. (2001) [4]: Kumar et al. (2021) [19] . The amplification reactions were carried out in 0.2 ml micro centrifuge tubes using thermal cycler. The extracted DNA from all the collected blood and tissue samples were subjected to conventional PCR to detect the presence of Capri pox virus genome using Capri pox virus genus specific primers targeting the P32 gene. The oligonucleotide primers used for PCR.

Results

The viral DNA of lumpy skin disease was detected by using polymerase chain reaction using skin biopsy and whole blood samples collected from lumpy skin diseased cattle.

Among 40 skin biopsy and whole blood samples, 40 skin biopsy and 10 whole blood samples were found to be positive for lumpy skin disease and as depicted in Table 01

Discussion

In the present study, primarily lumpy skin disease was diagnosed by clinical signs such as pyrexia, formation of characteristic nodules over the surface of the body, anorexia, enlargement of lymph nodes, edema of limbs, brisket and ventral abdomen, nasal and ocular discharges and it is in accordance with Mulatu and Feyisa (2018) [20] ; Awad et al.

(2010) [3] and Constable et al. (2017) [7] . However, the lumpy [30,7] . In the present study, laboratory confirmation of lumpy skin disease virus was done by polymerase chain reaction by using skin nodular biopsy samples and whole blood samples. Histopathological confirmation was done with skin biopsy samples and it is in agreement with Naby et al. (2021) [22] and Hasib et al. (2021) [17] .

In the present study, from 40 suspected cattle, whole blood and skin biopsy samples were subjected for polymerase chain reaction. Out of them 10 (25%) whole blood samples which were collected from pyrexia cases and 40 (100%) skin biopsy samples which were positive on polymerase chain reaction. The present findings were in agreement with Awad et al.

(2010) [3] . Similarly, Prabhu (2022) [27] found less positivity (2/23) in whole blood samples tested by polymerase chain reaction.

In the initial stage of infection, viremic phase lasts for about three to five days, then the virus is disseminated to subcutaneous and intradermal tissues in later stage of infection (Mulatu and Feyisa, 2018) [20] which might be the reason for less positivity with whole blood. However, 41.02 per cent (32/78) positivity with whole blood samples was recorded by Elhaig et al. (2017) [11] .

In our study, the positivity for skin biopsy samples through polymerase chain reaction was 100 per cent, similarly 90.14 per cent positivity from skin biopsy samples was detected by polymerase chain reaction by targeting P32 gene (Gharban et al., 2019) [14] and 89.7 per cent positivity was recorded earlier by Elhaig et al. (2017) [11] . The higher per cent positivity with polymerase chain reaction in tissue biopsy might be due to its capability to detect even low genomic copies of the lumpy skin disease virus (Al-Salihi, 2014 and Awad et al., 2010) [2,3] . Further, it was documented that the lumpy skin disease virus persists in the nodular tissue for more than 120 days (Gumbe, 2018) [15] , facilitating for greater positivity over longer period of time.

The polymerase chain reaction is considered as a confirmatory test, quick and easy to interpretate the results, also it can detect the viral antigen even in the dead state, which was in agreement with (Al-Salihi, 2014; Awad et al., 2010) [2,3] . Other sample of choice for polymerase chain reaction can be nasal swabs, oral swabs, saliva, lacrimal secretions, milk and semen as the virus is proved to be excreted in these samples (Farag et al., 2020) [12] . The histopathological changes noticed in the skin biopsy samples of the present study included, hyperkeratosis of stratum corneum, edema and vacuolization stratum granulosum, severe hydropic degeneration, hyperchromatosis, and mononuclear cell infiltration. This is in agreement with earlier reports by Salib and Osman (2011) [28] ; Gharban et al.

(2019) [14] . Follicle of sebaceous gland, fibroblast proliferation, keratin nests formation was noticed in the epidermis. Whereas, in dermis mononuclear cell infiltration, presence of intracytoplasmic inclusion bodies, pustule with infiltration of neutrophils, liquefaction necrosis and vasculitis were observed and it is corroborated with the findings of Gharban et al.

(2019) [14] . The confirmation of lumpy skin disease was done by histopathology, based on the presence of eosinophilic intracytoplasmic inclusion bodies in particular in the prickle cell layer.

Histopathology was considered as a precised diagnostic method which provides a useful information about a tissue and its architecture in the diagnosis of lumpy skin disease (Otranto et al., 2004) [26] . The World Organization for Animal Health (OIE) recommends that the following methods be used to identify antibodies against lumpy skin disease virus such as virus neutralization test, agar gel immunodiffusion test, indirect fluorescence test, ELISA, and western blot (OIE, 2016). The virus neutralization test is regarded as "the gold standard" and the most precise way to find antibodies against LSDV, its sensitivity is insufficient to determine the identity of every animal that came into contact with the virus (EFSA, 2015). While, western blot is sensitive and specific enough for routine use, it is difficult to set up and expensive. Standardization of the coated antigen in the microtiter plates is necessary for ELISA. Less specific techniques for detecting antibodies against LSDV include immunodiffusion and immunofluorescence (Bowden et al., 2009 and OIE, 2016) [5,24] . Conversely, the primary benefits of ELISA include its affordability, rapidity, and ability to test a large number of samples on a single plate (Gari et al., 2008) [13] . Using hematoxylin and eosin staining, histopathology of skin lesions offers a way to identify the intracytoplasmic inclusion bodies of LSD virus-infected cells (Tuppurainen et al., 2011) [29] .

Conclusion

In the present study, notably, a positivity rate of 100 per cent was observed in nodular skin biopsy samples, while whole blood samples exhibited 25 per cent positivity and histopathology may show the changes occurred in the skin of the affected cattle.

Acknowledgement

AcknowledgementThe authors are thankful to Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh and the farmers of Tirupati District for supporting my research work.

References

1. Abdulqa HY, Rahman HS, Dyary HO, Othman HH. Lumpy skin disease. Reproductive Immunology. 2016;1(4):25.
2. Al-Salihi K. Lumpy skin disease: Review of literature. Mirror of Research in Veterinary Sciences and Animals. 2014;3(3):6-23.
3. Awad WS, Ibrahim AK, Mahran K, Fararh KM, Abdel Moniem MI. Evaluation of different diagnostic methods for diagnosis of lumpy skin disease in cows. Tropical Animal Health and Production. 2010;42:777-783.
4. Binepal YS, Ongadi FA, Chepkwony JC. Alternative cell lines for the propagation of lumpy skin disease virus. 2001.
5. Bowden TR, Coupar BE, Babiuk SL, White JR, Boyd V, Duch CJ, et al. Detection of antibodies specific for sheeppox and goatpox viruses using recombinant Capripoxvirus antigens in an indirect enzyme-linked immunosorbent assay. Journal of Virology Methods. 2009;161(1):19-29.
6. Chadha J, Khullar L, Gulati P, Chhibber S, Harjai K. Insights into the monkeypox virus: Making of another pandemic within the pandemic. Environmental Microbiology. 2022;24(10):4547-4560.
7. Constable PD, Hinchcliff KW, Done SH, Grünberg W. Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs, and Goats. 11th ed. Elsevier; 2017. p.1591.
8. Datten B, Chaudhary AA, Sharma S, Singh L, Rawat KD, Ashraf MS, et al. An extensive examination of the warning signs, symptoms, diagnosis, available therapies, and prognosis for lumpy skin disease. Viruses. 2023;15(3):604.
9. Diallo A, Viljoen GJ. Genus Capripoxvirus. In: Mercer AA, Schmidt A, Weber O, editors. Poxviruses. Birkhäuser; 2007. p.167-181.
10. EFSA Panel on Animal Health and Welfare. Scientific opinion on lumpy skin disease. EFSA Journal. 2015;13(1):3986.
11. Elhaig MM, Selim A, Mahmoud M. Lumpy skin disease in cattle: Frequency of occurrence in a dairy farm and possible impact on Egyptian buffaloes. Onderstepoort Journal of Veterinary Research. 2017;84(1):1-6.
12. Farag TK, El-Houssiny AS, Abdel-Rahman EH, Hegazi AG. A new approach to treatment of lumpy skin disease infection in cattle using propolis encapsulated within alginate nanoparticles. Advances in Animal and Veterinary Science. 2020;8(12):1346-1355.
13. Gari G, Biteau-Coroller F, LeGoff C, Caufour P, Roger F. Evaluation of indirect fluorescent antibody test for the diagnosis and screening of lumpy skin disease using Bayesian method. Veterinary Microbiology. 2008;129(5):269-280.
14. Gharban HA, Al-Shaeli SJ, Al-Fattli HH, Altaee MN. Molecular and histopathological confirmation of clinically diagnosed lumpy skin disease in cattle, Baghdad Province of Iraq. Veterinary World. 2019;12(11):1826-1832.
15. Gumbe AA. Review on lumpy skin disease and its economic impacts in Ethiopia. Journal of Dairy Veterinary and Animal Research. 2018;7(2):39-46.
16. Gupta T, Patial V, Bali D, Angaria S, Sharma M, Chahota R. Lumpy skin disease and its emergence in India: A review. Veterinary Research Communications. 2020;62(5):549-554.
17. Hasib FMY, Islam MS, Das T, Rana EA, Uddin MH, Bayzid M, et al. Lumpy skin disease outbreak in cattle population of Chattogram, Bangladesh. Veterinary Medicine and Science. 2021;7(5):1616-1624.
18. Khan YR, Ali A, Hussain K, Ijaz M, Rabbani AH, Khan RL, et al. Surveillance of lumpy skin disease: A growing problem in Asia. Microbial Pathogenesis. 2021;158:105050.
19. Kumar N, Chander Y, Kumar R, Khandelwal N, Riyesh T, Chaudhary K, et al. Isolation and characterization of lumpy skin disease virus from cattle in India. PLoS One. 2021;16(1):e0241022.
20. Mulatu E, Feyisa A. Review: Lumpy skin disease. Journal of Veterinary Science and Technology. 2018;9:18.
21. Mourya D, Yadav P, Ullas PT, Bhardwaj SD, Sahay RR, Chadha MS, et al. Emerging/re-emerging viral diseases and new viruses on the Indian horizon. Indian Journal of Medical Research. 2019;149(4):447-467.
22. Naby SS, Hala K, Omnia MK, Hala AS. Current situation of lumpy skin disease virus in El-Wady El-Gedid Governorate during 2020. Egyptian Journal of Animal Health. 2021;1:9-20.
23. Neamat-Allah AN. Immunological, hematological, biochemical, and histopathological studies on cows naturally infected with lumpy skin disease. Veterinary World. 2015;8(9):1131-1136.
24. OIE. Lumpy skin disease. Terrestrial Manual. 2016. Chapter 2.4.13.
25. OIE (World Organisation for Animal Health). Lumpy skin disease. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Paris: OIE; 2017.
26. Otranto D, Paradies P, Sasanelli M, Spinelli R, Brandonisio O. Rapid immunochromatographic test for serodiagnosis of canine leishmaniasis. Journal of Clinical Microbiology. 2004;42:2769-2770.
27. Prabhu M. Molecular detection of lumpy skin disease virus from Tamil Nadu, India. The Pharma Innovation. 2022;11(8):75-78.
28. Salib FA, Osman AH. Incidence of lumpy skin disease among Egyptian cattle in Giza Governorate, Egypt. Veterinary World. 2011;4(4):162-167.
29. Tuppurainen ES, Stoltsz WH, Troskie M, Wallace DB, Oura CAL, Mellor PS, et al. Potential role of ixodid (hard) tick vectors in transmission of lumpy skin disease virus in cattle. Transboundary and Emerging Diseases. 2011;58(2):93-104.
30. Tuppurainen ESM, Alexandrov T, Beltrán-Alcrudo D. Lumpy Skin Disease Field Manual: A Manual for Veterinarians. FAO; 2017. p.1-40.
31. Yadav SN, Ahmed N, Nath AJ, Thakuria P, Boro PK. Ethno-veterinary approach for lumpy skin disease management -an update. Journal of Agricultural and Veterinary Science. 2021;9(14):57-59.