The present study was the first to conduct a large cross-sectional survey of EEHV seroprevalence among captive elephants in Thailand. Using an EEHV-1A gB protein antigen ELISA [23], over 40% of elephants tested were found to be seropositive. Although animals were healthy at the time of blood collection, a significant number appeared to have been exposed to EEHV based on antibody seroprevelance, most likely maintaining this virus within the population. Because it was not possible to sample every elephant at each camp, we could not determine if there were any 100% seropositive or seronegative camps in Thailand. However, the vast majority of seronegative elephants resided at camps with seropositive ones and so could be susceptible to infection in the future. In the study of van den Doel [23], some elephants maintained significant titers for prolonged periods, while others were intermittently seropositive. One seropositive elephant was categorized as healthy at the time of blood collection, but had presented with EEHV-like symptoms a few weeks before. This finding may indicate a prior EEHV infection, but that could not be confirmed. Results suggest that routine serological surveys may help identify prior viral exposure, which would otherwise go undetected as many exposed elephants are asymptomatic.
One of the characteristics of herpes viruses is their ability to go into latency. By certain unknown stimuli these latent viruses may be reactivated [26]. If reactivation does not occur over a long period of time, antibodies may drop to levels near to or below the detection limit of the ELISA. This makes it difficult to conclude that inconclusive or seronegative elephants are actually free of EEHV. The elephants in this study were all over 1 year of age, so maternal antibodies were not likely present to influence the outcome of the ELISA. As there is no vaccine against EEHV available, all antibody titers that were detected are assumed to be the result of previous exposure to EEHV. Each elephant with antibodies against EEHV should be considered as latently infected and a potential periodical shedder [26, 27]. Camps that consist of only seronegative animals are at risk of infection if a seropositive elephant is added to that camp; however, a false seronegative status may be the result of the absence of virus reactivation over a prolonged period, or insensitivity of the ELISA to detect a significant titer. As a consequence animals newly introduced into a camp are at risk of infection depending on the presence of even only one animal classified as EEHV infected. Model building initiated by submitting sex, age, regions, camp cluster size and sampling period (without management type) to multivariable analyses gave rise to the final multiple logistic regression model that identified “regions” as the most potent risk factor to EEHV in Thailand. More specifically, our study revealed that the Central, Northeast, East, West and South regions were lower in prevalence compared to the North. This result confirmed a higher incidence of EEHV in northern regions [28] based on sample tissue submissions and reported elephant deaths. Specifically, between 2006 and 2017, 32 clinical cases of EEHV-HD in Thailand were confirmed by PCR techniques, and of those, a third (n = 11) were found in the North. Overall, two thirds of EEHV antibody seropositive elephants were found in North, South and West regions of Thailand. By contrast, only two cases (2/32) occurred in the Central region, an area with only a few facilities close together, with limited exchange of animals from the outside. Understanding spatial differences in seroprevalence is complicated, however, by uncontrolled/unregistered elephant movements and transfers, particularly among facilities within those regions, and so needs further study.
The type of elephant management system was a significant risk factor to positive EEHV antibody seroprevalence in the univariable model, with 47% antibody seroprevalence in extensive systems compared to 37% in more intensive systems. The North and West regions include areas along the border with Myanmar, and contain more than half of the captive elephant population in Thailand. Although there is clinical evidence of EEHV-HD in captive elephants in Myanmar, there has been no molecular confirmation to date (Charernpan P., personal communication, National Elephant Health Service, DLD Thailand, 2017). However, given the close contact and/or transport of captive elephants between the Thai-Myanmar borders, transmission of the viral disease to elephants in the North and West of Thailand from Myanmar is possible, similar to what has been documented for foot and mouth disease viral transmission across these regions [29, 30]. Captive elephants in Myanmar are maintained in more natural habitats (extensive care system), particularly at night. Most are allowed to forage in nearby forests while on long chains, so there is potential for more interaction between wild and captive elephants in that country, whereas in Thailand, captive and wild elephants are found to cohabitate mainly in western regions.
Captive elephants in the South also had a relatively high antibody seroprevalence. In general, these were working elephants from the North and West that are taken to rest at their owner’s home in the South during the low tourist season. Conversely, elephants in the Central, East and Northeast regions live in more urban areas, closer to humans, where land and especially forest, is limited, and generally they are not exchanged between camps. The camps in intensive management systems also are less likely to transport elephants or recruit elephants from outside those regions than those in extensive systems. That may limit the degree of exposure to the virus, and agrees with our finding that most elephants living in isolated areas were seronegative. It is likely the seropositive elephants that experienced recent infection or reactivation might be related to camps with frequent or with rare viral reactivations.
Elephants sampled in this study were involved in tourism or logging, which requires tame elephants; hence, the higher numbers of adults than other age categories, and females being more prevalent than males. Trending towards significance (OR = 1.29) was a sex effect, with more males being seropositive, although the relevance of this is unknown. By contrast, elephant age, camp cluster size, and sample collection period were not significant risk factors for EEHV antibody seroprevalence. Our finding that 42.3% of captive elephants in Thailand were seropositive for EEHV antibodies suggest a high rate of viral exposure in this population. Extrapolating to the total captive population in the country (n = 4016 elephants), upwards of 1600 may have been exposed to the virus. The EEHV antibody seroprevalence survey showed that “region” was a significant risk factor associated with the disease incidence, particularly in the North, which is likely to be related to management or perhaps genetic relationships. Since the first diagnosed case of EEHV HD in 1999, this disease has resulted in elephant deaths, particularly calves, around the world, although it is more sporadic than epidemic in captive populations. Long et al. [3] suggested that disease severity is related to primary infection, and that around 20% of young elephants are susceptible.
Finally, a potential limitation of the EEHV-1A gB protein antigen ELISA may be that it has insufficient sensitivity to detect low antibody titers, which could lead to an underrepresentation of seropositive animals. Van den Doel et al. [23] suggested that one or both OD ratios should be ≥ 3 to indicate true seropositivity, while a cut off OD ratio ≥ 4 for both would be stricter. Hence, in addition to the analysis presented in Table 4, we examined the distributions of positive and negative test results in camps using cut off OD ratios of ≥ 3 respectively ≥ 4 for both dilutions (Additional file 1: Figure S1) and found they were similar with a peak around OD ratio 2. Both distributions showed a small elevation around OD ratio 5 (dilution 1:100) or OD ratio 6 (dilution 1:200), which might indicate distribution in a population with recent reactivation of virus or recent infection, whereas the elevation around OD ratio 2 might be the mode of an uninfected population. Obviously a more strict definition of positive animals e.g. both OD ratios ≥ 4 could lead to classifications of animals with low antibody titers as EEHV-negative. Comparison of the risk factor analysis for individual elephant data, criterion for qualification as a positive animal “one or both OD ratios ≥3” (Table 5) with risk factor analysis using the more stringent qualification criterion OD ratio ≥ 4 showed the same results (Additional file 3: Table S2). Until we are able to grow virus in culture to establish ELISA sensitivities, there is the risk of misinterpreting OD ratio results, either positive or negative, depending on what cutoff value is used.