Laboratory Diagnosis of Sheep and Goat Pox: Sample Collection and Diagnostic Approaches

Accurate diagnosis of sheep and goat pox (SGP) is essential for timely disease control and effective outbreak management. Although the disease often presents with characteristic clinical signs, laboratory confirmation remains necessary because sheep pox and goat pox produce similar clinical and pathological manifestations, making differentiation based solely on field observations challenging¹. For practicing veterinarians, selecting the appropriate samples, understanding available diagnostic techniques, and maintaining sample quality during transport are key steps that directly influence diagnostic accuracy. 

Clinical Evaluation: The First Step 

Diagnosis begins with a thorough clinical examination. Animals commonly present with small, round, nodular skin lesions distributed over the head, neck, ears, axillary region, and tail base. During postmortem examination, characteristic white necrotic foci, often described as "gunshot-like" lesions, may be observed in the lungs, liver, and occasionally the rumen. 

While these findings strongly suggest SGP, laboratory confirmation is required because the clinical presentation alone cannot reliably distinguish sheep pox from goat pox. A combination of clinical assessment and laboratory testing provides the most reliable approach for confirming infection^1,2. 

Laboratory Diagnostic Techniques 

Several laboratory methods are available, each serving a specific purpose in disease confirmation. 

Virus isolation in tissue culture remains a highly specific method for detecting the virus. However, because it is labor-intensive and time-consuming, its routine use is limited². 

Electron microscopy can be used as a presumptive diagnostic tool by identifying the characteristic inclusion bodies in infected skin biopsy samples. Direct fluorescent antibody (DFA) testing enables rapid detection of viral antigens in infected body fluids using labelled antibodies. Antigen-capture ELISA has also been used successfully to detect poxvirus antigens in lymph node biopsy samples. 

Among currently available diagnostic methods, polymerase chain reaction (PCR) has emerged as the most rapid and reliable technique. PCR assays targeting conserved genes such as P32 and RPO30 provide high specificity and sensitivity, allowing direct detection of capripoxviral DNA from clinical specimens^1,3. Conventional PCR as well as real-time PCR (qPCR) are available, with qPCR offering advantages such as faster turnaround, greater sensitivity, and real-time detection⁴. 

Sample Collection and Handling 

The quality of laboratory results depends heavily on proper sample selection and handling. Appropriate specimens include skin papules, lung lesions, lymph nodes collected during biopsy or postmortem examination, and buffy coat obtained from anticoagulated blood samples for virus isolation. 

Timing of sample collection is equally important. For antigen detection by ELISA, samples should ideally be collected within the first week after the onset of clinical signs and before neutralizing antibodies develop. PCR can detect viral genomes both before and after antibody production, offering greater flexibility in sample collection. 

Proper preservation is essential to maintain sample integrity. Blood samples should be stored at 4°C before processing and should neither be frozen nor kept at room temperature. Scabs and tissue samples should be maintained at 4°C or –20°C for prolonged storage. During transport, samples should be packed using a maintained cold chain with gel packs to preserve viral integrity. Care should also be taken to ensure tissue samples are sufficiently large so that glycerol does not penetrate the centre of the tissue and inactivate the virus¹. 

Serological Diagnosis 

Serological methods detect antibodies produced against capripoxviruses. The virus neutralization test (VNT) is regarded as the gold standard for antibody detection because of its high specificity. Western blotting offers excellent sensitivity and specificity for detecting antibodies against structural viral proteins but is comparatively complex and expensive. More recently, a double antigen ELISA has shown promise for large-scale screening of capripoxvirus antibodies^5,6. 

Since serological assays detect antibodies at the genus level and cross-reactions may occur among capripoxviruses, molecular techniques remain necessary when specific identification of sheep pox, goat pox, or lumpy skin disease virus is required. 

Key Takeaways 

• Clinical findings provide important initial clues but should always be supported by laboratory confirmation. 

• PCR targeting P32 and RPO30 genes offers rapid and reliable diagnosis of capripoxvirus infection. 

• Appropriate sample selection, correct timing of collection, and maintenance of the cold chain are essential for obtaining accurate laboratory results. 

• Serological tests are valuable for antibody detection and surveillance, while molecular diagnostics remain necessary for specific differentiation of sheep pox, goat pox, and lumpy skin disease virus. 

References 

1. Jadhav S, Veeregowda BM, Tadakod S, Naragund M, GB MR. Sheep and Goat Pox Disease: Epidemiology, Diagnosis, Prevention and Control. InAdvances in Animal Sciences (Volume 1) 2025 Jul 26 (pp. 35-47). Cornous Publications LLP. https://www.researchgate.net/profile/Sunil_Tadakod/publication/394089319 

2. Mirzaie K, Barani SM, Bokaie S. A review of sheep pox and goat pox: perspective of their control and eradication in Iran. Journal of Advanced Veterinary and Animal Research. 2015 Dec 31;2(4):373-81. https://banglajol.info/index.php/JAVAR/article/download/26044/17443 

3. Babu TS, Rathnamma D, Shrikrishna Isloor SI, Chandranaik BM, Veeregowda BM, Reddy GM. Diagnosis of orf virus infection in sheep and goats by virus isolation, polymerase chain reaction and sequencing. https://www.researchgate.net/profile/Veeregowda-B-M/publication/323674653 

4. Hurisa TT, Jing Z, Jia H, Chen G, He XB. A review on Sheeppox and Goatpox: insight of epidemiology, diagnosis, treatment and control measures in Ethiopia. J. Infect. Dis. Epidemiol. 2018;4(3):2474-3658. https://www.researchgate.net/profile/Takele-Tesgera/publication/327975572 

5. Milovanović M, Milićević V, Valčić M, Stević N, Nišavić J, Radojičić S. Detection and phylogenetic analysis of B2L gene of ORF virus from clinical cases of sheep in Serbia. Pakistan Veterinary Journal. 2019;39(3):433-7. https://reponivs.nivs.rs/bitstream/handle/123456789/398/395.pdf?sequence=1 

6. Manjunathareddy GB, Saminathan M, Sanjeevakumar L, Rao S, Dinesh M, Dhama K, Singh KP, Tripathi BN. Pathological, immunological and molecular epidemiological analysis of lumpy skin disease virus in Indian cattle during a high-mortality epidemic. Veterinary Quarterly. 2024 Dec 31;44(1):1-22. https://www.tandfonline.com/doi/pdf/10.1080/01652176.2024.2398211