A field study evaluating the humoral immune response in Mongolian sheep vaccinated against sheeppox virus.

Sheeppox is a transboundary disease of small ruminants caused by infection with the capripoxvirus sheeppox virus (SPPV). Sheeppox is found in Africa, the Middle East and Asia and is characterised by fever, multifocal cutaneous raised lesions, and death. Vaccination with live attenuated capripoxvirus (CPPV) strains is an effective and widely used strategy to contol sheeppox outbreaks, however there are few reports of post-vaccination field surveillance studies. This study used a commercially available ELISA to examine quantitative and temporal features of the humoral response of sheep vaccinated with a live attenuated CPPV strain in Mongolia. 400 samples were tested using the ELISA commercial kit, and a subset of 45 samples were also tested with a virus neutralisation test (VNT). There was substantial agreement between the VNT and ELISA tests. Antibodies to CPPV were detected between 40 and 262 days post vaccination. There was no significant difference between serological status (positive / negative) and sex or age, however an inverse correlation was found between the length of time since vaccination and serological status. Animals between 90 and 180 days post-vaccination were more likely to be positive than animals greater than 180 days post vaccination. Our results show that a commercial CPPV ELISA kit is a robust and reliable assay for post CPPV vaccination surveillance in resource-restricted settings and provide temporal parameters to be considered when planning sheeppox post-vaccination monitoring programmes. This article is protected by copyright. All rights reserved.

while SPPV and GTPV cause disease in sheep and goats. The host preference of SPPV and GTPV varies with some isolates of SPPV causing disease only in sheep, some isolates of GTPV causing disease only in goats and some isolates of SPPV and GTPV causing disease in both sheep and goats . SP and GP are high consequence diseases. They reduce production of meat, milk, wool and cashmere and decrease the value of affected animals, therefore having a substantial negative effect on farmers' livelihoods (Babiuk et al., 2008;Bolajoko et al., 2019;Garner et al., 2000;Limon et al., 2020). Furthermore, countries with endemic SP or GP face restrictions on trade of live animals and animal products (Tuppurainen et al., 2017). Vaccination is considered one of the most effective methods for control of CPPVs (Tuppurainen et al., 2017). Live-attenuated strains of SPPV and GTPV are the most common type of vaccine used against SP and GP; however, the duration of the humoral immune response following vaccination with live attenuated CPPV vaccines is poorly understood. Manufacturers often recommend annual vaccination regimes to maintain herd immunity; however, these recommendations are often based on research conducted for LSD in cattle and/or under controlled conditions (Boumart et al., 2016;Kali et al., 2019;Klement et al., 2018;Milovanovic et al., 2019). There is very little published research describing the humoral immune response following regional or national vaccination programmes to protect against SP or GP.
In this study, we used a subset of serum samples collected from sheep during post-vaccination surveillance in Mongolia to investigate the humoral immune response of vaccinated sheep following a riskbased SP vaccination campaign. We used the results to identify potential factors that might play a role in the detection of seroconversion and to assess the suitability of a commercial enzyme-linked immunosorbent assay (ELISA) test for post-vaccination monitoring in a nonendemic resource restricted setting.

Vaccination
All animals were vaccinated with a live-attenuated capripoxvirus vac-

Sample collection
Blood samples from sheep were collected as part of the postvaccination surveillance programme for SP implemented by the Mongolian General Authority for Veterinary Services in 2016. All provinces that were part of the vaccination programme in the country (n = 8; Multistage sampling was used to select animals for testing. Briefly, in each province between one and three soums (or districts) were ran-

Fluorescent virus neutralization test
The recombinant EGFP-095-LSDV Neethling virus was generated through insertion of the enhanced green fluorescent protein (EGFP) marker at the N terminus of the LSDV ORF 095, encoding for a putative core protein of the LSDV Neethling strain (GenBank: AF409138.1).   (Table 4; Supplementary material Tables S1 and S2). There was no significant difference between serological status (positive/negative) and sex or age (Table 4). Differences were found between serological status and province (Table 4); however, length of time since vaccination and province exhibited strong collinearity (Supplementary material Table   S3) and therefore the univariate models were kept. The same patterns were found using ELISA results from both labs (Table 4).  (Haegeman et al., 2019). This difference in Dsp estimate may be attributed to the origin of the samples used (field versus experimental), the level of antibodies present in the sample and the sample quality (e.g. haemolysis) Haegeman et al., 2020). Correlating antibody levels with protection against viral challenge requires further study. Antibodies alone are known to provide protection against poxviral disease including SP (Kempe et al., 1961;Law et al., 2005;Kitching, 1986), and levels of poxvirus antibodies, measured either by ELISA or neutralization assay, are often used as a correlate of protective immunity in people and animals. However, the level of antibodies that confers protection against poxviruses is unknown. One study found people with pre-existing neutralizing titres <1:32 against vaccinia virus were more susceptible to smallpox infection than those with antibody titers ≥1:32 (Mack et al., 1972). A neutralization index (calculated as the log titre difference between the titre of the virus in the negative serum and in the test serum) of ≥1.5 is considered positive for LSDV, SPPV and GTPV (OIE, 2019 ) although there is no data to link this index with protection from challenge. Importantly, a low antibody response by ELISA or neutralization tests post-vaccination may not necessarily mean absence of protection as (i) there may be sufficient memory B cells present to provide a rapid anamnestic antibody response and therefore protection post challenge (Crotty et al., 2003),

DISCUSSION
(ii) it is likely only a low amount of neutralizing antibodies is required for protection (Boshra et al., 2015;Hammarlund et al., 2003) and (iii)

CONCLUSIONS
Our results show that the use of a commercial CPPV ELISA kit provides a robust and reliable assay for post-vaccination surveillance on a regional or national level for SP in low resource settings. Our work builds on previous studies investigating the humoral immune response to CPPV vaccination and addresses particularly the limited number of studies assessing SP vaccination under field conditions. Our results have indicated that the timing of a post-vaccination SP testing survey is an important factor to consider when planning post-vaccination monitoring. None of the funding bodies played a role in the design of the study, collection, analysis or interpretation of the data, or in writing the manuscript.

ETHICAL APPROVAL
The animal samples used in this study were archived sera that had

AVAILABILITY OF DATA AND MATERIALS
The dataset supporting the conclusions of this article are included within this article, its additional files, and available in a public repository.