Serological evidence of herpesvirus infection in gibbons

Background Herpesviruses are not only infectious agents of worldwide distribution in humans, but have also been demonstrated in various non-human primates as well. Seventy-eight gibbons were subjected to serological tests by ELISA for herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Results The prevalence of IgG antibodies against HSV-1, HSV-2, EBV and CMV was 28.2%, 28.2%, 14.1% and 17.9%, respectively. Conclusions Antigenic cross-reactivity is expected to exist between the human herpesviruses and gibbon herpesviruses. Gibbons have antibodies to human herpesviruses that may reflect zoonotic infection with human herpesviruses or infection with indigenous gibbon herpesviruses. Therefore, it is difficult to draw concrete conclusions from serological studies alone. Identification should be based on further isolation and molecular characterization of viruses from seropositive animals.


Background
Gibbons (Hylobates spp.) have become valuable animals for zoological, medical and psychological research. Their small size (the smallest of the anthropoids), ease of handling and maintenance in captivity and their close phylogenetic relationship to humans represent only a few of their desirable characteristics as laboratory animals. Gibbons are found throughout the tropical rainforest of South and Southeast Asia, including Thailand, Malaysia and Indonesia. Illegal pet trade is the main cause of the decreasing gibbon population in Thailand. Since gibbons were categorized as a conserved species in Thailand, hundreds of appropriated and abandoned animals have been handed over to the authorities of the Royal Forest Department (RFD). An infectious disease screening process is necessary to interrupt the spread of diseases, including herpesvirus infection. Little is known regarding natural or experimental herpesvirus infections in this interesting arboreal primate and reports of outbreaks or natural disease are still limited. Hence, screening is required to prevent the spread of infectious diseases, including herpesvirus.
Herpesviruses have been isolated from a wide variety of mammalian and non-mammalian species. The eight human herpesviruses, herpes simplex virus type 1 and 2 (HSV-1 and -2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and human herpesviruses-6, -7 and -8 (HHV-6, -7 and -8), represent a significant public health problem worldwide. These viruses have been further classified as members of the alphaherpesvirus (HSV-1, HSV-2 and VZV), the beta-herpesvirus (CMV, HHV-6 and HHV-7) and the gamma-herpesvirus subgroups (EBV and HHV-8). The virus-host relationship is characterized by the benign nature of HSV infection in its usual host, man, and by the fatal disease it causes in accidental hosts, such as the owl monkey or gibbon. Fatal infections caused by HSV have been documented in gibbons, patas and colobus monkeys [1,2]. A natural epizootic model of Herpesvirus hominis is splenectomized gibbons, which clinically mimic the disease as it occurs in man [3]. EBV causes 85% of infectious mononucleosis cases, whereas CMV is responsible for most of the remaining cases. Both are mainly transmitted via exchange of saliva. Cells infected with human herpesviruses such as CMV, HSV-1, HSV-2 and EBV express specific virus-encoded receptors capable of binding the F C domain of IgG [4]. Three major approaches can be employed for herpesvirus diagnosis. The first is isolation of virus from clinical specimens using mammalian cell culture systems and subsequent identification by biological, biochemical and immunological procedures. The second is by identification of anti-herpesvirus antibodies in sera of infected individuals. A third method, PCR, can also be used to amplify and sequence herpesvirus DNA. Although the PCR technique is widely used to detect herpesviruses, we did not utilize it for this study due to the high costs. Since clinical specimens are often unavailable, the first approach is not always possible; the second approach is therefore more routinely used.
In this study, our objective was to determine whether there is evidence of herpesvirus infection in gibbons. Since test kits for gibbon herpesviruses are not currently available, the serological tests were performed using human HSV-1, HSV-2, EBV and CMV strains by detecting IgG antibodies to these viruses. However, due to possible cross-reaction, further studies would need to be performed to differentiate between actual human herpesviruses and indigenous gibbon herpesviruses.

Discussion
Despite the rather limited data on herpesvirus infection in wild animals, our results showed a high prevalence of HSV-1 and HSV-2 infection in gibbons, comparable with previous serological studies on the incidence variation of herpesvirus infections in different species of apes. Of 24 gibbon serum samples tested, 8 (33.3%) were positive and reacted more strongly with the HSV-1 antigen than with any of the other herpesvirus antigens [5].
Both western blot assay and virus neutralization tests were done with sera from 15 gibbons. Antibodies against HSV-1 and HSV-2 were detected in four (26.6%) healthy gibbons [6]. In addition, neutralizing antibodies against HSV were found in 16 of the 84 (19.0%) animals in the colony [3]. Cerebral infarction and myocardial fibrosis were reported in a white-handed gibbon (Hylobates lar), which was serologically positive for HSV-1 and EBV [7]. However, the serological tests can not positively determine if this was the cause of illness. Most of the gibbons are healthy, with herpesvirus infection in those animals apparently being in the latent phase.
Isolation of viruses related to HSV from primates is scarce. However, a number of primate species, including apes, have been surveyed for antibodies to many different simian and related human viruses [5]. These studies have reported that the incidence of herpesvirus antibody in gorillas, orangutans, and gibbons is very low, whereas chimpanzees showed a much higher prevalence. In our study, we found that the prevalence for HSV-1 and HSV-2 was equal (28.2%). It has been reported that both viruses share a common antigen, which can cause serologic crossreaction [8].
Sera obtained from 50 non-human primates and 45 lower mammals were tested for complement fixation (CF) antibodies to partially purified antigens prepared from viruscontaining cells of Burkitt's lymphoma cultures. All five non-human primate species tested (chimpanzee, baboon, cynomolgous, rhesus macaque and African green monkey) displayed a high incidence of CF antibodies to EBV, whereas none of the sera from nine species of laboratory or domestic animals reacted with this antigen [9]. The prevalence of herpesvirus antibody among non-human primate is shown in Table 3.
Since it was not possible to use species-specific antibodies in the ELISA test, the negative results obtained for gibbons might be attributable to non-reactivity of their sera with anti-human IgG. There has been evidence of cross-species reactivity as both human and non-human (Old World) primates exhibit close antigenic relationships between their immunoglobulins. In previous studies, both fluorescent-labeled goat anti-human globulin and anti-monkey globulin reacted well with chimpanzee [10] and non-human primate globulins [11]. For this reason, goat anti-human immunoglobulin G (IgG) conjugated with alkaline phosphatase may be used for both human and non-hu-man (Old World) primates based on the close antigenic relationship between their immunoglobulins [12]. Crossreactivity has also been reported between human and monkey herpesviruses [13]. Hence, it is important to determine whether the antibodies detected in the gibbons in this study are actually directed against human herpesviruses (HSV-1, HSV-2, EBV and CMV) or other related viruses. To resolve this problem, ELISA assays using human herpesvirus and monkey herpesvirus infected cell antigens could be used. The non-human primate herpesviruses are much more similar to each other than to human herpesviruses. Therefore, the sera will react more strongly with
The animals we screened were at high risk of contracting herpesviruses from humans. For example, hand rearing of gibbon infants by zoo personnel would provide ample opportunity for human herpesvirus transmission to gibbons. Moreover, the exclusive HSV serotypes within species suggests that these viral infections may have originated from a common source within a species, with subsequent spread from animal to animal, rather than from isolated human-to-gibbon transmissions. Accordingly, HSV-2 infections have been observed to spread from chimpanzee to chimpanzee [5].
Based on the high level of cross-reactivity expected to exist between the human herpesviruses likely to be contracted by animals in captivity and any indigenous gibbon viruses, it is difficult to draw any concrete conclusions from serological studies alone. The most convincing approach toward identification of an indigenous gibbon herpesvirus would be virus isolation from seropositive animals and subsequent molecular characterization.

Conclusions
It can be stated that infections with herpesvirus-like viruses are relatively common in gibbons. The viruses found in these gibbons are either human herpesviruses or indigenous gibbon herpesviruses that are antigenically similar to human herpesviruses. Further studies on molecular characterization of gibbon herpesviruses should be performed in order to arrive at concrete conclusions.

Materials and methods
Seventy-eight healthy gibbons kept at the Krabok Koo Wildlife Breeding Center, Royal Forest Department, Cha-Cheng-Sao province, Thailand were included in the study. All of the animals examined, although born in the wild, were raised by humans and have since been kept in captivity at the National Wildlife Research Center. Demographic data for the gibbons are shown in Table 1. Blood samples were obtained by femoral venipuncture during a brief period of anesthesia by Ketamine hydrochloride, according to routine health care procedures. The sera were separated by centrifugation and stored at -70°C until further analysis.
As no screening test is available for gibbon herpesviruses, screening for serum antibodies against HSV-1, HSV-2, EBV and CMV was performed by ELISA using the HUMAN-ELI-SA-Antibodies-Test (Human, Wiesbaden, Germany). The procedure applied followed the manufactured recom-