A retrospective evaluation of pooled serum ELISA testing in the frame of the French eradication program for infectious bovine rhinotracheitis.

Pooled serum testing using whole-virus indirect ELISA has been recently recognized as an official method for surveillance of bovine herpesvirus 1 (BoHV1) in cattle herds in Europe. In this study, a retrospective analysis of data from the French BoHV1 surveillance campaign 2018-2019, including 7434 BoHV1-free certified herds and 157 infected herds, was performed in order to evaluate the diagnostic specificity and sensitivity of two pooled serum indirect ELISAs (from IDEXX and IDVet), in comparison with individual testing by blocking ELISAs targeting the gB and gE proteins. Pooled serum testing showed a relative specificity higher than 97.5% and a detection rate of 100% since all gB+/gE+ samples were found in positive pools. At the herd level, no more than one false positive pool was observed in most of BoHV1-free certified herds, leading to a herd relative specificity of 85.1% and 86.0% for the IDEXX and IDVet pooled serum ELISAs, respectively. Among infected herds tested by pool sizes up to 10 sera (n = 122), 46% of herds were detected through pools of size 10 containing a single positive sample, 23% through pools of size 10 containing at least two positive samples, and 31% through pools of smaller sizes. A complementary study based on manually constituted pools revealed that at least one positive sample in 100% and 93.4% of herds could be detected individually by pools of size 10 with the IDEXX and IDVet ELISAs, respectively. However, pooled serum ELISAs were influenced by the level of individual reactivity, since pools composed of either one weak-positive sample or one gB+/gE- sample could yield negative results. Altogether, these results provided the first evidence that pooled serum testing (pool size up to 10) is a suitable strategy for surveillance of BoHV1-free cattle farms.

Improvement of eradication program for infectious bovine rhinotracheitis in France inferred by serological monitoring of singleton reactors in certified BoHV1-free herds.

Within the framework of the national voluntary eradication program for Bovine alphaherpesvirus 1 (BoHV1) in France, the proportion of certified-free herds which experienced no more than two positive animals (termed singleton reactors) steadily increased to reach up to 95% in 2015. The aim of this study was to collate and evaluate serological data to gain insight into these epidemiological questionable BoHV1 seropositive animals. Preliminary evaluation of the performances of BoHV1 ELISA kits using a collection of 997 field sera with well-defined status revealed a relatively low specificity of the two gB blocking ELISAs most used in France for confirmatory testing (93.2% and 97.5% for gB-IDVet and gB-Idexx, respectively). In both ELISAs, the suboptimal specificity was associated with the presence of antibodies against BoHV2. Reassessment of the cut-offs led to a specificity and a sensitivity higher than 99.3%. Consequently, a comprehensive analysis of gB-positive sera from 2551 singleton reactors was performed by using gB ELISAs with optimized cut-offs, combined with viral neutralization test (campaign 2014-2015) or gE ELISA (campaign 2015-2016). Fifty percent of the 728 sera collected in 2014-2015 reacted below the optimized cut-offs in both gB ELISAs. Analysis of new blood samples collected at a minimum 6-week interval showed that these weak-positive reactions did not increase with time and could not be confirmed by confirmatory tests. Among the 1823 sera collected in 2015-2016, only 84 samples tested positive by gE ELISA, most of them corresponding to sera with reactivity above the optimized cut-offs in gB ELISAs. Screening for BoHV2 antibodies revealed a significantly increased prevalence among herds with singleton reactors, compared with the between-herd prevalence in French cattle herds. Altogether, these results provided suitable analytical strategies to limit the occurrence of false-positive BoHV1 reactions and inappropriate withdrawal of the BoHV1-free status, without alteration of diagnostic costs and reliability of eradication programs.