1 Zentrum für Biologische Sicherheit, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
2 Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany
3 Institute for Parasitology and International Animal Health, Freie Universität Berlin, Königsweg 67, 14163 Berlin, Germany
4 Zoologisch-Botanischer Garten Wilhelma, 70342 Stuttgart, Germany
5 Lanada/Lcpa, Bingerville, Côte d'Ivoire
Correspondence
Georg Pauli
PauliG{at}rki.de
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ABSTRACT |
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INTRODUCTION |
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Field surveys of well-studied wild primates, such as chimpanzees, are needed to determine the natural prevalence of STLV-1 and to understand the routes of virus transmission. Blood samples from a large number of wild-caught individuals are required for such surveys. Unfortunately, this would be possible only by anaesthetizing or killing the animals, which is contrary to the conservation of wild-living primate species. A previous study that was performed on necropsy samples from 10 chimpanzees (six of them adults) belonging to the study groups that are described in this investigation indicated a high prevalence of STLV-1 infection in the adult individuals that were analysed (5/6). In addition, STLV-1 genome analyses showed a remarkable variance in STLV-1 strains that were isolated from different chimpanzee groups, within one group or even in strains from a single individual. Two of these new STLV-1 strains had close genetic similarity to strains that were found in two red colobus monkeys (the preferred prey of chimpanzees in this area) from the same region, which suggests interspecies transmission (Leendertz et al., 2004). In order to determine the prevalence of STLV-1, we modified antibody-detection assays to determine STLV-1-specific antibodies in urine samples from all members of three communities that are the subject of long-term, ongoing behavioural research.
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METHODS |
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Chimpanzees younger than 5 years were defined as infants, those of 69 years as juveniles, those of 1015 years as adolescents' and individuals older than 15 years as adults' (Boesch & Boesch-Achermann, 2000).
Validation of urine antibody testing.
Multiple urine samples and blood/serum and/or DNA samples that were extracted from spleen or other lymphatic tissues were available from 11 chimpanzees. Blood/serum samples were tested for STLV-1 antibodies by using an HTLV-1/2 ELISA (Murex Biotech) and/or an HTLV-1/2 Western blot (version 2.4; Genelabs Diagnostics Pte.) (Table 1).
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Antibody detection.
Detection of specific antibodies in urine samples from various primates has been described for simian immunodeficiency virus (SIV) (Santiago et al., 2002, 2003
; Ling et al., 2003
). We used antibody-detection tests that were designed to determine antibodies in human serum or plasma samples. Urine samples were analysed by using a modified version of this protocol for Western blot analysis. After thawing, urine samples were vortexed and then centrifuged at 3000 r.p.m. to remove debris. The following modifications of the test were introduced: 500 µl urine was mixed with 500 µl blotting buffer. Western blot strips were incubated in narrow wells to ensure that they were covered completely by the urinebuffer mixture. Wells were sealed with adhesive tape and incubated overnight on a rocking platform at room temperature; the samples were then aspirated and stored at 20 °C. Strips were washed three times with wash buffer (for 5 min each) and incubated with the enzyme-labelled secondary antibody according to the manufacturer's instructions. Strips were incubated subsequently for 1 h with substrate solution and the reaction was stopped by rinsing with distilled water.
Urine samples that showed reactivity to at least two envelope antigens (rgp46-I and GD21) and at least one core protein (p19 and/or p24) on the Western blots were defined as STLV-1 antibody-positive, those that showed reactivity to either envelope or core structural proteins exclusively were described as indeterminate and strips that showed reactivity against the immunoglobulin control only were described as negative. This algorithm follows the recommendations of the HTLV European Research Network for seroepidemiological HTLV studies (Goubau et al., 1996).
Sensitivity and specificity of the test were determined by using an algorithm for the evaluation of diagnostic tests that was developed by Galen & Gambino (1979) and Abel (1993)
, with a confidence interval of 95 %. The fraction of positive tests per total number of urine samples (rather than number of individuals) was analysed, with confidence limits that were determined given the assumption of binomial sampling. For these calculations, it was assumed that successive samples from one individual were not correlated (Santiago et al., 2003
).
Prevalence estimation.
To investigate the prevalence of STLV-1, we tested 74 urine samples and six blood samples from 80 individuals with unknown STLV-1 antibody status. The blood samples originated from individuals that had died before 2001, the year in which urine sampling was first implemented.
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RESULTS |
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DISCUSSION |
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In order to compare the sensitivity of the HTLV-1/2 Western blot version 2.4 (Genelabs Diagnostics) to another Western blot kit that is used frequently for PTLV diagnostics [the INNO-LIA HTLV I/II (Innogenetics)], we tested 13 urine samples by using both methods. Although bands that were observed with the INNO-LIA Western blot were very faint compared to most bands seen by using HTLV-1/2 Western blot version 2.4, most results were consistent between both kits. Five out of six HTLV-1/2 Western blot version 2.4-positive urine samples tested positive in the INNO-LIA assay and the other was indeterminate; four out of four samples tested negative and three samples that had tested indeterminate in the HTLV-1/2 Western blot version 2.4 were negative in the INNO-LIA Western blot. These results suggest that the INNO-LIA Western blot has a slightly lower sensitivity for the screening of antibodies in urine samples than the HTLV-1/2 Western blot version 2.4. We therefore suggest screening for urine antibodies with the HTLV-1/2 Western blot version 2.4.
Analysis of urine samples by using the particle agglutination test (Serodia HTLV-1; Fujirebio) and ELISA (Murex HTLV-1/2 ELISA) gave no clear results when paired samples of blood and urine were analysed; this may be due to low antibody titres or inhibitors present in urine.
Prevalence
A high STLV-1 antibody prevalence (38/80, 47·5 %) was detected in wild chimpanzees that live in three defined groups in the Taï National Park by using this method. Sixty-seven of 74 urine samples from chimpanzees with unknown STLV-1 antibody status showed distinct positive or negative Western blot results. According to the criteria that are suggested in this paper, urine samples from 34 individuals were STLV-1 antibody-positive and those from 26 individuals were antibody-negative. On the basis of the test algorithm, urine samples from seven of 74 chimpanzees showed an indeterminate Western blot pattern and were considered to be negative for the epidemiological study. Five of these chimpanzees were juveniles; a follow-up of these individuals will be performed in the future.
It was not possible to discriminate between STLV-1, -2 and -3 from the Western blot results, but no reaction of blood or urine samples from Taï chimpanzees to HTLV-2-specific peptides was observed on Western blot strips. Furthermore, an investigation of 10 animals by using PCR with STLV-2 or -3 primers did not give a positive reaction (Leendertz et al., 2004).
The only evidence for a relatively high prevalence of STLV-1 in chimpanzees is from a study by Niphuis et al. (2003), who found that 20 of 37 founder animals (54 %) from a breeding colony of Pan troglodytes verus that originated from Sierra Leone were STLV-1-positive. At present, the route of infection of the founder animals is unclear, as the mother-to-child transmission rate in this breeding colony is extremely low. It is possible that infection of the founder animals might be have been caused iatrogenically by treatment with STLV-contaminated material, as these animals were captured in Sierra Leone as infants (E. J. Verschoor & J. L. Heeney, personal communication).
In our study groups, there was previous evidence for a high STLV-1 prevalence from 10 chimpanzees that had died from natural causes. Five of six adult chimpanzees were positive by STLV-1 serology and PCR (Leendertz et al., 2004). The results that were obtained in this study confirm and extend these findings. To our knowledge, screenings of other chimpanzee subspecies (mainly P. troglodytes troglodytes) revealed a much lower prevalence (Ishikawa et al., 1987
; Georges-Courbot et al., 1996
; Nerrienet et al., 2001
, 2004
). Table 5
gives an overview of published data on STLV-1 prevalence in chimpanzees of different origin. Differences in prevalence may reflect the age at which individuals were caught (Nerrienet et al., 2004
), but differences in chimpanzee subspecies susceptibility, range or number of prey, lower prevalence of STLV-1 in simian prey and even biological properties (such as STLV-1 strain infectivity) should also be considered. Further investigations will be performed on the prevalence of STLV-1-directed antibodies in different subspecies of chimpanzees and in other monkey species that originate from different regions of Africa.
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In young individuals, the possibility of a lower antibody response (and consequently a lower antibody titre in blood and urine) cannot be excluded, which may lead to an underestimation of the frequency of positive infants and juveniles. However, our previous results and additional infant samples that were analysed at a later date showed that tissue/blood samples from six of six chimpanzees that were <10 years old had tested negative, whereas, in contrast, five of six adult chimpanzees had tested positive by using serological and PCR methods (Leendertz et al., 2004).
Significant differences in antibody prevalence in adolescent and adult versus infant and juvenile individuals (71·4 vs 9·7 %) may be due to three important factors in chimpanzee life: in-fighting, sexual intercourse and consumption of other primate species.
By studying the distribution of STLV-positive and -negative female individuals around the time of sexual maturity (10 years; at this age they also start to attract adult males), a clear jump in STLV-1 prevalence can be observed. On sexual maturity, male chimpanzees start to be involved in the male hierarchy and female chimpanzees leave their native group to immigrate into new groups. Both changes in behaviour carry a high potential for conflict and create new sexual partner constellations. In captivity, virus transmission from one chimpanzee to another via fighting or sexual contact is assumed to occur (Georges-Courbot et al., 1996; Niphuis et al., 2003
; Parrish et al., 2004
); these routes also cannot be ruled out as sources of infection for the Taï chimpanzees. Fourteen adult/adolescent chimpanzees were found to be negative for STLV-1 antibodies; among these were high-ranking individuals like Marius, the current top-ranking male of the north group (we tested two independent samples from this male). These results suggest a low rate of STLV-1 transmission via aggression and sexual behaviour in the wild chimpanzees of the Taï National Park, despite frequent fighting and copulation within the group (Boesch & Boesch-Achermann, 2000
).
In previous studies, we described six new STLV-1 isolates (Leendertz et al., 2003, 2004
) in five chimpanzees originating from the same groups that were investigated here. These virus isolates showed surprisingly high heterogeneity, not only in comparison to STLV-1 isolates described previously in other primate species, but also between the different chimpanzee groups, within one group and even between two strains that were isolated from a single individual. We also found STLV-1 in western red colobus monkeys that live in the same area of the Taï National Park. The STLV-1 strains that were detected in these red colobus monkeys were closely related phylogenetically to strains found in two chimpanzees, suggesting that interspecies transmission from prey to hunter via killing and consumption of meat, bones and intestines may be an important route of virus transmission (Leendertz et al., 2004
).
Participation in hunting of different monkey species and thus access to primate meat also becomes more frequent with maturity, awarding the maturing chimpanzee a higher social position within the community. Almost the entire prey is ingested, including bones and skull, which may induce wounds in the oral cavity or digestive tract. Adult male and female chimpanzees consume a mean of 186 and 25 g, respectively, of simian prey per day. Adolescent and younger chimpanzees consume considerably less prey than adults and generally obtain only small pieces that have been discarded by their mothers or other adults. The red colobus represents the most frequent prey by far (80 % of simian prey) of the 10 different simian species that are hunted by chimpanzees (Boesch & Boesch-Achermann, 2000).
The potential risk of infection via copulation and aggression may increase with age (i.e. total number of sexual and aggression contacts). In combination with our previously published molecular data (Leendertz et al., 2004) and observations on transmission routes of STLV-1 in captive chimpanzees (Niphuis et al., 2003
), the data that are presented here on STLV-1 antibody prevalence in different age groups of chimpanzees suggest an important role of horizontal virus transmission from prey to hunter.
Conclusions
Further studies on the molecular epidemiology of STLV-1 infections in monkey species that are hunted by chimpanzees, as well as long-term studies on STLV-1 antibody prevalence in chimpanzees, are necessary to gain deeper insights into the epidemiology of STLV-1 in populations of wild-living primates. Analysis of urine samples for the presence of STLV-1 antibodies provides an important tool for non-invasive screening of such populations.
In contrast to other studies, our data indicate that the risk of acquiring an STLV-1 infection in wild P. troglodytes verus might be different from that of humans for an HTLV-1 infection. As high-ranking individuals like Marius, a vivid hunter that is surrounded by many STLV-1 antibody-positive chimpanzees, are found to be STLV-1-negative, there may be differences in susceptibility to STLV-1 infection at the individual level.
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ACKNOWLEDGEMENTS |
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Received 23 February 2004;
accepted 12 July 2004.
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