Monitoring of Rabbit Hemorrhagic Disease Virus in European Wild Rabbit (Oryctolagus cuniculus) Populations by PCR Analysis of Rabbit Fecal Pellets

Rabbit hemorrhagic disease (RHD), caused by a lagovirus (rabbit hemorrhagic disease virus; RHDV) belonging to the Caliciviridae family, is a fatal, highly infectious disease that affects European rabbits (Oryctolagus cuniculus). Outbreaks of RHD, which cause significant mortality, occur on almost all continents; RHD is endemic in areas with wild populations of European rabbits (Abrantes et al. 2012). Since 2010, a new virus variant RHDV2/b has spread worldwide that is also able to cause disease outbreaks in wild populations of hares (Lepus spp.) in Europe; North American Sylvilagus and Lepus species appear also to be susceptible to fatal RHDV2/b infection, demonstrating a wider spectrum of hosts than for previous RHD viruses (reviewed in Le Pendu et al. 2017; Lankton et al. 2021).

Noninvasive surveys of RHD epidemiology in wild rabbit populations (i.e., surveys in which animals are not trapped) have been mostly based on data obtained from carcasses actively or opportunistically collected in the field (e.g., Wells et al. 2015; Rouco et al. 2018). These data are usually used to derive information regarding the timing of RHD outbreaks or simply to verify circulation of RHD viruses. However, because sick rabbits can be killed by predators, carcasses can be consumed by scavengers, and rabbits can die inside their warrens, searching for rabbit carcasses is highly time consuming and requires regular, frequent, intensive searches. Opportunistic sampling strategies are less time consuming but require networks of volunteers, hunters, or other stakeholders who collaborate in collecting carcasses, thus reducing researchers' control of sampling effort.

Given that feces are an important dissemination route of RHD viruses and viral RNA can be detected in rectal swabs and feces several weeks after infection (Matthaei et al. 2014; Calvete et al. 2021), detection of viral RNA in fecal pellets has been proposed as a less time consuming method to monitor the presence of RHD viruses in wild rabbit populations (Dalton et al. 2018). Furthermore, given the expected direct relationship between infection incidence and the proportion of animals excreting viral RNA in a population, surveillance of viral RNA in feces could be useful to monitor the evolution of infection incidence over time, constituting an alternative to invasive procedures such as animal live trapping or shooting. However, there is a complete lack of information regarding the sampling effort needed and the sensitivity of this approach to monitor RHD in wild populations. This, combined with the economic and laboratory cost of viral RNA detection in a high number of samples, has prevented its evaluation in field surveys.

Our study was a first approach to determining the performance of this procedure to monitor RHD virus infection incidence, as well as to derive information regarding the timing of mortality in RHD outbreaks, by assessing concordance between wild rabbit carcass discovery and viral RNA detection in field-collected fecal pellets. The study was conducted in two enclosed European wild rabbit populations located in Zaragoza Province, northern Spain, in which yearly RHD outbreaks had occurred naturally since their foundation in 2009. These populations, termed population A and B, were placed 2 km apart and confined to 1-ha enclosures that exclude terrestrial predators. Inside each enclosure, rabbits lived in 12 natural warrens surrounded by refuge traps equipped with latching trapdoors, which were blocked to allow rabbit trapping. Such populations enable more controlled population monitoring, rabbit carcass collection and fecal sampling than is possible in free-living populations.

During January to December 2015, a night-trapping session was conducted monthly to estimate population size. All trapped rabbits were individually identified by numbered metallic ear tags and classified into two age groups: 1) breeding adults born during the breeding period of the previous year or before and 2) juvenile rabbits born during the breeding period of 2015 (mainly January–June). Additionally, three consecutive night-trapping sessions were performed in October to capture all rabbits for routine population management in which a random sample of rabbits was removed to balance the number of breeding rabbits. At this time, a 1.5-mL blood sample was obtained from each rabbit to determine the prevalence of anti-RHDV antibodies with a commercial indirect ELISA (INgezim Rabbit, Ingenasa Laboratory, Madrid, Spain).

Enclosures were inspected at least five times per week to collect carcasses of rabbits dying from RHD. Diagnosis of RHD as the cause of death was based on observed lesions and confirmed by analyzing liver or spleen samples with duplex quantitative (q)PCR to detect RNA from RHDV and RHDV2/b lagoviruses in a single analysis (Calvete et al. 2018). Because lagovirus RNA can be detected in surviving rabbit tissues months after infection (Gall et al. 2007; Calvete et al. 2018), only rabbits with quantification cycle (Cq) ≤27 were considered to have died from acute RHD (Calvete et al. 2018).

The animal experiments were performed in compliance with the provisions of Spanish national and European laws and approved by the CITA ethical committee for animal experimentation (protocol 2014-18 and 19).

Twice per month, five samples of fecal pellets were collected in every enclosure. Sampling was performed just after sunrise to collect the freshest pellets laid at night. Every sample comprised six pellets (approximately 2 g total weight), each picked from a different group of pellets located throughout the enclosure, at least 5 m apart, to reduce the probability of sampling feces of the same individual. Feces were stored at 20 C until analysis by a modified procedure for the same duplex qPCR (Calvete et al. 2021).

Given the relatively high number of samples to be analyzed, two strategies were followed: in population A, each sample was analyzed individually (120 samples in all); in population B, to evaluate whether reducing the number of qPCR analyses affected the performance of the procedure, the 10 monthly samples were randomly pooled two-by-two between biweekly samplings, obtaining five samples (about 4 g) per month (60 samples in all). Each pooled sample was homogenized, and 2 g from the homogenate was analyzed by qPCR.

During 2015, RHD caused by RHDV2/b typically coursed in high–rabbit density populations, causing mortality only in juvenile rabbits, all of them being infected throughout the year, such that RHD antibody prevalence in October was 100% both in adults (n=45) and juvenile rabbits (n=42). In all, 34 carcasses of juvenile rabbits were found during the study, with 7 of 16 and 11 of 18 carcasses belonging to individuals that died from acute RHD in populations A and B, respectively. Typically, most rabbits dying from RHD were found in the middle of the breeding period (March–May), when the number of juvenile rabbits was high, followed by a quiet period in June–July and a second peak of deaths in August–September (Fig. 1).

We detected RHDV2/b RNA in fecal samples collected in both populations. For population A, viral RNA was detected in 4 mo (Fig. 1), but only one of the 10 monthly samples was positive in each month at high Cq values ranging from 36.8 to 38.6, except in August, when the sample was positive at 40.5 Cq, beyond the lowest limit of detection of qPCR (38.1–39.2; Calvete et al. 2018). In population B, qPCR was positive in only 2 mo (April and May), with two of five monthly samples positive at Cq values ranging from 38.1 to 38.7. Probably, given the high Cq values at which viral RNA is detected in the feces of individual rabbits (Calvete et al. 2021), pooling the feces of several individuals decreased sensitivity to detect viral RNA in field samples, especially those collected when RHD-susceptible rabbit abundance (mostly juvenile rabbits) was lower. Consequently, this yielded qPCR-positive results only in samples collected when infection incidence was highest (spring months). The proportion of positive monthly samples did not show a pattern closely resembling the expected variation of infection incidence over time (Fig. 1).

Furthermore, the monthly concordance between qPCR and the discovery of rabbits that died from RHD was far from perfect: qPCR results were negative in months in which rabbits died by RHD, and vice versa (Fig. 1). Weak concordance probably occurred because the number of rabbits found dead from RHD is a nonideal surrogate for infection incidence, because the number of juvenile rabbits dying from RHD is affected by rabbit abundance and by lethality after contact with the virus, with lethality being affected by maternal antibodies levels and, therefore, by rabbit age at infection (Baratelli et al. 2020). Additionally, some rabbits surviving infection may excrete lagovirus RNA in feces weeks after infection (Calvete et al. 2021), which may have yielded positive qPCR results in periods in which active viral circulation was low or even nonexistent.

Despite the procedure being implemented in near-ideal field conditions (enclosed dense-rabbit populations with high viral transmission, in which both carcass discovery and fecal sampling could be carried out efficiently and systematically), it lacked sufficient sensitivity to monitor RHDV infection incidence and timing of mortality from RHD. Improvement of qPCR sensitivity and, especially, increasing sampling effort, the analysis of single pellets, or both would enhance the quality of information provided but would significantly increase laboratory costs. Moreover, the design of field samplings covering spatial and temporal dimensions of infection incidence in heterogeneously distributed wild populations would probably be complex. Additional research is needed to enable this procedure to monitor RHD, optimizing the trade-off between sensitivity to monitor infection incidence and field and laboratory effort required.

Conversely, detection of viral RNA in fecal pellets may be a useful noninvasive strategy to determine the presence of RHD viruses in populations, especially if sampling is conducted when juvenile rabbit density and, therefore, viral transmission is probably highest. Because fecal sampling can be carried out simultaneously with active carcass collection, a combined approach may increase the probability of detecting RHD viruses without significantly increasing noninvasive field sampling effort. This methodology could probably also be applied in studies carried out in other lagomorph species, including native North American lagomorphs in which RHDV2 is spreading (Lankton et al. 2021). Nevertheless, additional research is needed on the persistence of viral RNA in feces (to avoid having to collect the freshest pellets) and the excretion dynamics of viral RNA in feces of lagomorphs, especially in species other than O. cuniculus.

The study was funded by a Research and Development collaboration project between Agrifood Research and Technology Centre of Aragon (CITA) and the Management of Health, Food Safety, and Public Health of TRAGSATEC (INMUNIZADOS 1316), the E-RTA2014-00009-00-00 project from the National Institute for Agricultural and Food Research and Technology (INIA), and the Research Group Funds of the Aragón Government (A05-17R and A14-20R). The authors declare that they have no conflicts of interest.