Sero epidemiological study of Sheeppox and Goatpox disease in small ruminants in Karnataka

Introduction

Sheeppox and goatpox (SGP) are infectious and economically significant viral diseases of sheep and goats caused by sheeppox and goatpox viruses of the family Poxviridae, subfamily Chordopoxvirinae, and genus Capripoxvirus (Manjunatha Reddy et al., 2024) [13] . These are highly contagious diseases that spread mostly by direct contact with lesions or contaminated feed, fodder, water, and wool. The virus is excreted in nasal and ocular secretions, milk, and possibly through urine. The prevention and control of pox disease mainly depend on quick and reliable diagnosis (Manjunatha Reddy et al., 2015; 2017; 2023; Sumana et al., 2020) [15, 14, 21 22] . The diagnosis of SGP depends on clinical signs and laboratory confirmation. The clinical signs of severe sheeppox and goatpox are highly characteristic. However, in their mild form, they can be confused with parapoxvirus infections causing orf, urticaria from multiple insect bites, contagious ecthyma (Babu et al., 2018) [4] , bluetongue, peste des petits ruminants, photosensitization, dermatophilosis, parasitic pneumonia, caseous lymphadenitis, and mange (Hurisa et al., 2018). Hence, confirmatory laboratory diagnostic methods are useful. Laboratory diagnosis is carried out either by identifying the antigen through targeting different genes (Sumana et al., 2020; Manjunatha Reddy et al., 2023) [21,22,16] or by detecting antibodies (Manjunatha Reddy et al., 2025). Among serological tests, virus neutralization test (VNT), indirect fluorescent antibody test (IFAT), and enzyme-linked immunosorbent assay (ELISA) are commonly used. Indirect ELISA has been developed and validated to detect antibodies to CaPV for mass screening of populations to determine the prevalence of infection (Manjunatha Reddy et al., 2025) [17] . Seroprevalence studies of sheep and goatpox infections are crucial to understand disease dynamics, map geographical distribution, and assess disease prevalence and immunity status. Such studies are required for identifying high-risk areas and populations to assess the economic impact and to formulate control and preventive measures by guiding targeted vaccination strategies.

Materials and Methods

Sample collection Sampling strategy and sample size for seroprevalence study

The sample size was calculated by using the stratified random sampling method (Fig 1). Accordingly, 30 serum samples from apparently healthy sheep or goats with a history of not being vaccinated against SGP from each district of Karnataka in two intervals with a gap of 10 months were collected. As per WOAH (OIE) guidelines, the collected samples were labeled and placed in a sterilized universal carrier and transported to virology laboratory, ICAR-NIVEDI, Bengaluru. All collected samples were screened by in house iELISA, kit developed by ICAR-NIVEDI Indirect ELISA for detection of antibodies An in-house developed iELISA was used for the serological study (Manjunatha Reddy et al., 2025) [17] . Briefly, antigencoated ELISA plates (Polysorp) were taken out from the refrigerator and washed three times with wash buffer. To block the unbound sites of the ELISA plate wells, 100 µL of blocking buffer (PBST with 3% skim milk powder) was added to all the wells using a multichannel pipette and incubated at 37 °C for one hour. After one hour of incubation, the plates were washed thrice with wash buffer, and 100 µL of 1:100 diluted test and control sera in blocking buffer was added to all the wells and incubated at 37 °C for one hour. After incubation, the plates were washed thrice using wash buffer, and 100 µL of 1:5000 diluted antisheep/goat conjugate was added to all the wells and incubated at 37 °C for one hour, followed by washing thrice with wash buffer. After washing, 100 µL of OPD substrate solution was added to each well and incubated at 37 °C for 15 minutes. After the optimum color (yellow) developed, 50 µL of stop solution (1 M H₂SO₄) was added. The readings were taken at 492 nm using an ELISA plate reader.

Results

Upon screening of 1800 samples collected from all the districts of Karnataka, by iELISA, 95 samples were found positive for anti SGP antibodies, yielding an overall seroprevalence of 5.28 percent. Among 1800 samples, 1039 were from goats and 761from sheep in which 56 and 39 samples found seropositive with 5.39 and 5.12 percents seroprevalence in goats and sheep respectively (Fig. 2). Statistical analysis of influence of species on the rate of seroprevalence is insignificant (P>0.05). Among the divisions, Belagavi division stood first with a seroprevalence of 11.19 percent followed by Kalaburagi and Bengaluru with 5.55 and 3.52 percents respectively. Mysuru division placed last with 1.88 percent of seroprevalence rate (Fig. 3& Table 1.). Belagavi and Haveri districts reported a high rate of seroprevalence of SGP, with 20.00 percent each, followed by Bagalkot with 15.00 percent. The third position was shared by Gadag and Kalaburagi, with 11.67 percent.

Whereas, Chamarajanagar, Dakshina Kannada, Uttara Kannada, Kodagu, and Udupi districts reported nil seroprevalence (Fig. 4 & Table 2). In the current study, we found that the rate of seroprevalence of SGP in Karnataka between divisions and districts was statistically significant.

Discussion

In the current investigation, we used an in-house iELISA kit developed by NIVEDI, Bengaluru, to screen 1800 serum samples collected from all 30 districts of Karnataka and found an overall seroprevalence of 5.28 percent for sheep and goatpox in Karnataka.

In a study conducted in sheep with 772 serum samples, the lowest seroprevalence was observed in Chamarajanagar (3%). Whereas, irrespective of population density, the other three districts (Tumakuru, Koppal, and Kolar) revealed high seropositivity for sheeppox disease, ranging from 29 to 31% (Manjunatha Reddy et al., 2024) [13] .

Seroprevalence and associated risk factors in the occurrence of pox infection among sheep and goats in selected districts (Asayita and Mille) of the Afar region, Ethiopia, during the study period from February to April 2020 were reported by Dubie et al. (2022) [8] using VNT. The study revealed an overall seroprevalence of 15.36 percent, of which 14.5 percent of sheep and 15.69 percent of goats were exposed to poxvirus infection, indicating that both species were equally exposed.

Whereas, Elshafie and Ali (2008) [9] reported a higher seroprevalence in the Kassala, Algash, Nahr Atbara, and Seitit areas during the year 2006, which was attributed to varied animal movement and the introduction of new animals into the herd. Further, Kassa et al. (2024) [12] reported the seroprevalence of SGP in selected districts of Wolaita Zone, Southern Ethiopia, to be 4.95 percent, with five times higher prevalence in sheep than goats, implying that sheep are more likely to contract poxvirus infection than goats. This finding contradicts the report by Assefa et al. (2019) [2] , who opined that capripoxvirus is not host-specific and that the majority of strains cause disease in both species. However, in the present study, we found that both species are equally affected (goats: 5.39% and sheep: 5.12%), and species-specific seroprevalence of SGP was statistically insignificant (P>0.05). The higher seroprevalence of 71.36 percent of goatpox in selected districts of Assam reported by Pariat et al. (2020) [18] using iELISA was attributed to preferential sampling in districts where clinical goatpox was reported.

A cross-sectional seroprevalence study by Adedeji et al.

(2021) [1] elucidated the household animal factors operating in high-risk areas in northern Nigeria using a multi-stage sampling method. The apparent weighted seroprevalence was 2 percent (N = 1800) using a commercial iELISA kit. They also opined that iELISA detects antibodies against specific immunogenic capripoxvirus (CaPV) antigens, whereas VNT detects antibodies with the ability to neutralize CaPV and is more sensitive than iELISA. However, Fenti et al. (2017) attributed the higher seroprevalence of 15.5 percent of SGP detected using VNT to its greater sensitivity. Whereas, Sajid et al. (2012) [20] reported an SGP prevalence of 5.67 percent in Punjab based on the observation of typical pox lesions on the body and internal organs of carcasses. The seroprevalence of 5.28 percent observed in our study of sheep and goatpox, based on samples collected from all 31 districts of Karnataka, aligns with moderate levels of exposure in the region. The recombinant protein antigen-based iELISA used in the present study was found to have the advantage of minimal batch-to-batch variation in the antigen, besides obviating the need for maintenance of live virus strains. This finding is in agreement with Bowden et al. (2008) [7] , who also reported the superiority of recombinant protein antigen-based iELISA for the serodiagnosis of SGP. Whereas, Babiuk et al. (2009) [3] opined that although VNT could detect seroconversion by both virulent and nonvirulent strains, whole antigen-based iELISA detected seroconversion only by virulent strains. However, a recent study has shown that serum neutralization test (SNT) and iELISA can complement each other in measuring seroconversion and immune response against capripoxviruses (Bayyappa et al., 2025) [5] . We found a significant difference in the seroprevalence of SGP among different divisions and districts of Karnataka, without any significant difference between species. The varied seroprevalence of 8.88 percent in sheep and 31.47 percent in goats reported by Hota et al. (2018) [10] in different agroclimatic zones of Odisha was found to be significant (P<0.05) for goats but not for sheep. The higher seroprevalence was attributed to the lack of vaccination against SGP. Similar observations were also reported by Bhanuprakash et al. (2008), who documented a seroprevalence of 68.3 percent of SGP across northern states of India. Additionally, comorbid chronic diseases such as paratuberculosis may interfere with the outcome of other diseases in small ruminants (Reddy et al., 2012) [19] . It can be concluded that these infections are endemic in Karnataka State. Therefore, control measures should be designed to reduce the occurrence of these infections through mass vaccination. There is also a need for frequent serosurveillance, epidemiological investigations, and molecular characterization of sheep and goatpox viruses.

Acknowledgement

AcknowledgmentsThe authors acknowledge the support given by Dean, Veterinary College, Hebbal, Head, Department of Veterinary Microbiology, and Director of ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Bengaluru, Karnataka, for providing the facilities to carry out this work. The authors are also grateful to the field veterinary officers and animal owners for their help in collecting the samples.

References

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