BACKGROUND
the family herpesviridae comprises a diverse group of viruses, which are divided into three subfamilies   <cit> .
most commonly, herpesvirus infection presents as a benign, self-limiting disease with a mild viraemia.
herpesvirus rarely causes widespread disseminated disease in the natural host, although cross-species transmission to non-reservoir hosts can also result in lethal infections  <cit> .
in general neonates and immune-compromised hosts are at higher risk of developing a fatal herpesvirus infection  <cit> .
the elephant endotheliotropic herpesvirus  is a poorly characterized virus of elephants that was first observed in healthy wild african elephants  by mccully et al.
<cit>  in  <dig> and subsequently in asian elephants  with no apparent disease  <cit> .
in  <dig> the death of a 3-year old female asian circus elephant was associated with herpesvirus by ossent et al.
<cit> .
pathologic changes in the capillary endothelial cells with herpetic inclusion bodies and weak cross-neutralization in serum neutralization tests with bovine herpesviruses suggested that the etiologic agent was a hitherto unidentified herpesvirus  <cit> , which was identified in  <dig>  <cit> .
the disease has a sudden onset characterized by lethargy, oral ulcerations, edema in the skin of the head and proboscis, cyanosis of the tongue, decreased white blood cell and platelet count and massive internal hemorrhages, which lead to death within 12–72 h after onset of the symptoms.
phylogenetic analysis showed that eehv is a distinct branch within the β-herpesviridae group, assigned as the new genus proboscivirus  <cit> .
to date, as many as seven different genotypes of eehv have been identified within this genus  <cit> .
however, xtensive evaluation of several subtypes indicated that eehvs have a large genomic inversion of a 40-kb core segment that is distinct from the roseoloviruses and all other β-herpesviruses.
furthermore, they encode α-herpesvirus-like genes that are absent in β-herpesviruses and contain  <dig> novel open reading frames not found in any other herpesvirus  <cit> .
these findings suggest that this particular virus is so different from other herpesviruses that it may be considered as a new herpesvirus subfamily  <cit> .
eehv poses a threat to the conservation mission of zoos.
several studies have shown that dnaemia can be detected in asian elephants before the onset of clinical signs and in elephants that do not display clinical signs using pcr techniques .
to date, this is the only option to monitor a complete herd, and only allows identification of animals with an active infection.
therefore, the detection of antibodies against eehv likely offers a better strategy to determine which animals are carriers of eehv.
ideally, antibody detection against the whole virus is preferable, but since the virus cannot be cultured in vitro, the best alternative is to use an immunodominant protein, such as glycoprotein b , as an antigen.
the viral envelope protein glycoprotein b of herpesviruses is involved in host cell entry and represents an important target of neutralizing antibodies in the host  <cit> .
in this study a serologic test based on the eehv-gb antigen is described.
this assay was used to assess seroprevalence against eehv-gb in serum and heparin plasma samples from two large cohorts of european and north-american captive asian elephants.
these data are useful to link seroprevalence with eehv virus detection by pcr methods and, at the end, may give better insight into eehv dynamics within elephant populations.
methods
cloning, expression and large scale production of glycoprotein b
the viral sequence of eehv-1a glycoprotein b   <cit>  comprising the start codon up to the transmembrane domain was synthesized  and cloned, using xhoi/psti restriction sites, into the multiple cloning site in frame with a c-terminal myc/6his tag, but in an alternative open reading frame  relative to the shine dalgarno ribosomal binding site, of the bacterial expression vector ptrchis2a .
induction of recombinant glycoprotein b expression was performed in e.coli rosetta2plyss codon plus strain .
this strain supplies trnas for  <dig> rare codons  in order to improve the expression of heterologous proteins.
a single colony transformed with the ptrchis2a-gbmyc/his plasmid was inoculated into fresh lb medium containing 100 μg/ml ampicillin and 50 μg/ml chloramphenicol and grown overnight at 37 °c in a shaker incubator.
the next day  <dig> l fresh lb medium without antibiotics was inoculated  <dig> in  <dig> with the pre-culture and grown in a shaker incubator at 37 °c to an od600 ~  <dig> .
the recombinant protein expression was induced by adding iptg to an end concentration of 1 mm.
the temperature was lowered to 25 °c for optimal recombinant protein expression.
cells were harvested 4 h after induction and pelleted at 6000xg for 10 min in a beckman highspeed centrifuge using the jla <dig>  rotor.
the supernatant was decanted and the cells were resuspended in native lysisbuffer , 5 % v/v glycerol and 10 mm of imidazole and protease inhibitors).
lysozyme was added to an end concentration of 1 mm and the cells were treated 30 min at 4 °c at continuous agitation.
the cells were cracked by three freeze/thaw cycles and sonification with a microtip;  <dig> x 30s at 60 % on ice .
the lysate was cleared by ultracentrifugation for 2 h at  <dig>  rpm in a beckman optima l-90 k ultracentrifuge using the sw32ti rotor.
a similar procedure was conducted with another ptrchis2a vector encoding a  <dig> kd irrelevant 6his-tagged protein that was used as a negative control.
purification of glycoprotein b
the cleared lysate was loaded onto ni-nta resin  which was preconditioned with water and 1x native lysisbuffer.
binding was performed overnight at +4 °c under continuous agitation.
next day, the resin was settled vertically and washed with  <dig> bed volumes of lysisbuffer containing 20 mm of imidazole.
thereafter, the bound recombinant protein was eluted with  <dig> bed volumes of elution buffer , 5 % v/v glycerol and 300 mm of imidazole).
subsequently, salts were removed by dialysis in a slide-a-lyzer cassette  for 48 h at +4 °c.
the dialysis buffer  was refreshed three times.
after dialysis the recombinant protein content was analyzed for protein concentration  and western blot.
a second round of purification was performed by fast protein liquid chromatography  with a 1 ml volume histrap-hptm column  with a pressure flow of  <dig>  ml/min.
the his-tagged proteins were eluted from the column with  <dig> column volumes  of a linear gradient ranging from 50 mm imidazole up to 500 mm imidazole in  <dig>  ml aliquots.
these were analyzed by western blot and the eluates containing the his-tagged proteins were subsequently pooled and dialyzed as described above.
rabbit anti-eehv glycoprotein b serum
five peptide sequences  derived from the amino acid sequence of glycoprotein b of eehv1a  were synthesized and coupled to lph  carrier protein.
ten rabbits  were immunized six times intramuscularly with 10 μg of the respective peptides.
final bleeding was at day  <dig> after the first vaccination.
the polyclonal rabbit sera were produced by biogenes gmbh  according to european law on animal welfare.
all rabbit sera were highly reactive against the respective peptide as tested in a peptide elisa assay.
immunodetection of recombinant glycoprotein b
recombinant glycoprotein b antigen was analyzed by separation of small samples from the purification process on a 10 % sds-page gel  and separated products were subsequently transferred to pvdf membrane .
gels were run in duplicate; one was coomassie blue stained for total protein and the duplicate was analyzed with western blot using an anti-6his tag mouse monoclonal antibody .
as a secondary antibody goat anti-mouse irdye <dig>  was used.
rabbit serum reactivity with recombinant glycoprotein b was detected with western blot by incubating the membranes with rabbit serum  followed by detection with goat-anti-rabbit irdye <dig> .
similar strategy was done for elephant serum , but primary antibody was detected with rabbit-anti asian elephant igg .
sera of rabbits as well as elephants were pre-incubated in buffer containing e.coli lysate  for 1 h at room temperature in order to reduce possible e.coli reactivity.
the signal of the conjugate was detected with the licor odyssey scanner .
glycoprotein b specific capture elisa
to avoid non-specific reaction against e.coli contaminants which co-purified with the 6his-tagged eehv gb antigen during ni-nta purification an optimal concentration of 100 ng/well of mouse-anti 6his antibody  as a capture antibody was coated in high binding 96-well microtiterplate .
the microtiter plates were blocked with 100 μl/well of pbs containing 2 % w/v bsa in pbs for one hour at 37 °c and washed.
all washing steps were performed three times with 200 μl of wash buffer .
to determine the optimal antigen concentration for use in the elisa, the gb antigen was diluted two-fold starting at 2400 ng per well.
a pool of rabbit sera was used as anti-gb positive serum.
the purified eehv-gb antigen was diluted in pbs containing  <dig>  % triton-x to a concentration of 750 ng/well and bound to the capture antibody for 1 h at 37 °c.
after washing, primary incubation was performed with 100 μl/well diluted  elephant serum in elisa buffer containing pbs,  <dig>  % bsa,  <dig>  % non-fat dried powder milk and 3 % w/v nacl and incubated for one hour at 37 °c.
after washing, elephant specific igg’s bound to glycoprotein b were detected by incubation with 100 μl/well of rabbit anti-elephant igg  for one hour at 37 °c followed by an incubation step with swine anti-rabbit igg hrpo conjugate .
after washing 100 μl/well 1xtmb substrate  was added, incubated for 10 min at room temperature.
the reaction was stopped by adding 100 μl  <dig>  m h2so <dig> per well.
the absorbance  was measured at 450 nm with 620 nm reference filter  on a 96-well plate spectrophotometer .
study cohort in europe/usa
the elephants in this study were kept in zoos as part of the european endangered species program  and the species survival program .
within these programs elephants are trained to allow voluntarily blood collection for several reasons: monitoring health parameters, monitoring reproduction parameters and providing  data for new diagnostic technologies for diseases that may threaten their health.
the use of banked elephant blood samples for a retrospective research study falls within the law.
all participating zoos reviewed and approved the research proposal for this study and allowed the use of their banked elephant blood samples.
all elephants in the study were asian elephants; table  <dig>  and  <dig>  summarize the age, sex, and clinical status of elephants which will be discussed in detail in this study.
all participating zoos reviewed and approved the research described in this study.
initially,  <dig> serum or heparin plasma samples of  <dig> asian elephants located in  <dig> european zoos were tested for validation of the capture elisa test.
the cut-off value was calculated as a mean od of  <dig> samples of  <dig> animals which scored consistently on the base line level of the test.table  <dig> age, sex, and clinical status of elephants in the study cohort of european zoos
zoos are numbered, elephants are indicated with a letter and subsequent numbers describe maternal offspring
nd, not detectable
tw, trunk wash
c, conjunctiva
u, urine
o, oral lesion
wb, whole blood
v, vulva
three european zoos  provided asian elephant serum or plasma samples for a longitudinal seroprevalence study ranging from  <dig> up to 600 days for multiple animals in their herds.
moreover, the samples from zoo  <dig> and  <dig>  were also analyzed for eehv presence by qpcr.
in the usa cohort  <dig> asian elephant samples were tested from  <dig> animals in  <dig> zoos in the period  <dig> until  <dig> with a six month interval.
all samples were tested in 1: <dig> and 1: <dig> dilutions and the signal to background od was calculated for both dilutions.
a sample was considered positive when both dilutions produced ods  <dig> times of the background od and undetectable when both dilutions produced less than  <dig> times the background od.
a sample was considered as borderline when one or both dilutions measured an od between 2– <dig> times the background od values.
an animal was considered to be seropositive when all analyzed samples scored positive with an occasional borderline score.
an animal was considered borderline seropositive when the majority of its samples scored borderline.
an animal was considered undetectable when all of its samples were undetectable.
RESULTS
expression and purification of eehv glycoprotein b
positioning of the eehv-gb open reading frame  in an alternative position downstream of the shine dalgarno ribosomal binding site  severely reduced the expression levels of the recombinant eehv-gb, but retained the recombinant protein in the soluble fraction and in its native conformation.
purification of the recombinant eehv-gb was performed using nickel columns .
full length eehv-gb has an expected molecular size of  <dig> kd, and a band at this expected size was observed .
since the portion of recombinant gb protein in the e.coli lysate was low many native proteins of e. coli rosetta <dig> that displayed affinity to nickel in the immobilized metal affinity chromatography  were co-purified during ni-nta purification.
full length as well as many other molecular weight sizes, were detected by anti-6his-tag antibody detection on western blot.
western blot analysis with the eehv-gb peptide specific rabbit  serum showed more bands with different molecular weights compared to the pattern obtained with anti-his antibody .
several specific smaller bands were also detected.
an elephant serum, which did contain eehv-gb specific antibodies, as detected with the described sandwich elisa, did not recognize as many of the eehv-gb fragments on western blot .
since the double purified  lost a portion of the full length eehv-gb  the single ni-nta purified antigen was further used to develop an elisa test.fig.
<dig> sds-page total protein analysis  and western blot analysis with an anti-his monoclonal antibody  demonstrate the purification of full length eehv-gbhis  and several smaller fragments with a his-tag produced from the eehv-gbhis open reading frame .
during purification a small portion of the recombinant protein is lost in the flow through of the ninta column  and during the washing steps .
the empty vector does not yield any his-tagged proteins .
lane m is the molecular weight marker
fig.
<dig> western blot analysis of an irrelevant  <dig> kd his-tagged protein and , single ninta purified eehv-gbhis  and double purified ninta/histrap fplc  with three different antisera.
a western blot with mouse monoclonal anti-his demonstrates the  <dig> kd his-tagged protein in lane  <dig> and the highly fragmented eehv-gbhis in lane  <dig> and  <dig>  double purification results in loss of the full length eehv-gbhis .
bthe rabbit peptide  eehv-gb specific antiserum recognizes non-his tagged bands.
c serum of elephant 4a detects identical bands as the rabbit peptide specific serum in the eehv-gbhis recombinant protein, except for the ~ <dig> kd band which might be an e.coli background band
validation of an eehv glycoprotein b specific capture elisa
to reduce aspecific e.coli protein reactivity  the elisa assay was setup as a capture elisa using a mouse-anti 6his monoclonal antibody.
pooled sera of rabbits immunized with eehv-specific peptides were used as a positive control.
figure  <dig> shows that 100 ng of the capture antibody reduces aspecific reactivity to an acceptable background level of an a450/ <dig> of ~ <dig> .
subsequently, the capture elisa was further validated with an antigen checkerboard titration to test its specificity with various irrelevant rabbit sera.
the results in fig.
<dig> demonstrate a dose-related response only when eehv-gb specific sera were used.
irrelevant rabbit antisera did not react with the antigen.
to detect antibody reactivity in the linear range an optimum of 750 ng antigen/well was used in the elisa, which was used to evaluate the elephant sera cohorts.
once the capture elisa was developed with the eehv-gb specific rabbit sera, a large panel of elephant sera was tested to screen for sera with specific reactivity.
in total  <dig> serum or plasma samples of asian elephants, representing  <dig> animals of  <dig> zoos were tested.
twenty-three animals from  <dig> zoos tested consistently positive ,  <dig> animals of  <dig> zoos tested intermittently positive/borderline/undetectable  and  <dig> animals tested consistently undetectable .
the test was repeated twice in separate experiments and the results were reproducible.
we found three sera with extremely high reactivity in the capture elisa  and several non-reactive sera .
these elephant sera were used to test again the antigen dose and specificity .
the sera of elephant 2b <dig>  4a and 5a showed a dose-dependent reactivity whereas the serum from elephant 2c did not .
serial dilutions of serum of animal 5a  and animal 2c  on eehv-gb antigen or an irrelevant 6his-tagged protein were used in the assay to test the specificity.
only the positive serum of animal 5a reacted in a dose-dependent manner with eehv-gb antigen and not with the irrelevant antigen .fig.
<dig>
e. coli protein contamination in the purified antigen  induces background signal in an indirect elisa .
this background is reduced in a sandwich elisa using a mouse monoclonal anti-his antibody as a capture antibody for the target antigen .
pooled eehv-gb peptide specific rabbit serum was used in a 1: <dig> dilution.
panel a no capture antibody, panel b 100 ng capture antibody, panel c 200 ng capture antibody, panel d 300 ng capture antibody
fig.
<dig> specificity of the eehv-gb sandwich elisa was determined by antigen titration with the eehv-gb peptide specific rabbit serum, one serum of an eehv <dig> pcr positive elephant and two irrelevant rabbit sera.
all sera were diluted 1: <dig> in elisa buffer
fig.
<dig> characterization of the sandwich elisa with elephant sera.
elephant sera react in a dose-dependent manner as shown by antigen titration  and are specific for the eehv-gb antigen only
the majority of eehv pcr positive elephants display eehv specific antibodies
for  <dig> animals  eehv pcr data was available, either from published data  <cit>  or personal communications.
we compared these results with our elisa results.
nineteen out of  <dig> pcr positive animals  displayed antibodies, of which  <dig> had significant antibody titers and three had borderline titers.
however, in six out of  <dig> animals no eehv specific antibodies could be detected  while they had been tested positive with the eehv specific pcr.
eight elephants displayed eehv-gb specific antibodies, but never tested positive in a routine pcr .
four animals were eehv <dig> pcr positive of which three displayed antibodies against eehv-gb.table  <dig> age, sex, and clinical eehv status of elephants in the study cohort of usa zoos
zoos are numbered, elephants are indicated with a letter and subsequent numbers describe maternal offspring
nd, not detectable
tw, trunk wash
c, conjunctiva
u, urine
o, oral lesion
wb, whole blood
v, vulva
seroprevalence of a european study cohort
three european zoos were approached to collaborate in our study; their animals are listed in table  <dig>  and their asian elephant sera collections were analyzed over a substantial period of time .
during the study period the animals of zoo  <dig> and  <dig> were screened for eehv virus presence in whole blood, trunk wash and conjunctiva swabs by either conventional or real time pcr.
figure  <dig> displays the serostatus of these three herds; the observed patterns showed a rise-and-fall in signal to background ratios .
a similar rise-and-fall pattern was observed for animals, which scored below the detection level.
additional clinical evaluations of the animals in this study can be found in .fig.
<dig> serostatus of three european zoos measured as the signal to background ratio over a period of time.
zoo  <dig>  was studied from october  <dig> until april  <dig>  zoo  <dig>  was studied from may  <dig> until may  <dig> and zoo  <dig>  from january  <dig> until september  <dig>  the arrow indicates a positive eehv <dig> pcr of the trunk wash  or whole blood .
elephants with non-detectable levels of eehv antibodies are indicated in black, elephants with an alternating serostatus in orange and elephants with continuous anti-eehv antibodies in red
only two animals had a positive pcr in the study-period in either trunk wash  or whole blood .
as seen in fig.
6a animal 1a was eehv <dig> pcr positive in trunk wash on day  <dig> and, shortly afterwards, had elevated antibody titers up to 8 weeks after the pcr positive trunk wash. as shown in fig.
6b calf 2b <dig> developed a dnaemia and clinical signs of eehv disease on day  <dig> of the study period with typical eehv clinical signs.
however, this calf was already seropositive on day  <dig> of the study period.
he survived this episode with supportive treatment and famciclovir administration.
eighteen days prior to disease onset his half-sister 2e <dig> showed a rise in antibody titers.
unfortunately no samples of the other herd members were available in this period of the study.
discussion
to date, an accurate and dependable test to evaluate eehv seroprevalence in asian elephants has not been developed.
in part, this is due to the inability to culture the virus in vitro and therefore whole virus is not available as the ideal antigen source for an elisa.
production of recombinant proteins from one of the eehv glycoproteins, like gb, has proven to be a major challenge.
here we describe a novel elisa that specifically detects eehv-gb antibodies circulating in asian elephants.
we were able to link seroprevalence to pcr detection of the eehv in approximately 80 % animals.
this novel assay will provide more insight into the epidemiology of this hitherto poorly understood virus.
the antigen expression, production and purification methods for fragmented eehv-gb  driven by a bacterial shine dalgarno promoter in an alternative open reading frame, yielded antigens for a sandwich elisa specific for eehv-gb.
incomplete eehv-gb amino acid sequences were produced from the plasmid presumably due to programmed ribosomal frameshifting  and alternative translation initiation sites.
prf and alternative translation initiation are well-described phenomena  and can occur in prokaryotes, as well as in eukaryotes.
prf occurs when the ribosome encounters a so-called hungry codon  in its a-site followed by a slippery sequence in the p-site of the ribosomal complex.
in our expression system, it is likely that the shine dalgarno sequence rbs initiates the overexpression of a short nonsense peptide of  <dig> amino acids thereby releasing the ribosomes quickly in order to re-initiate translation with a high likelihood to undergo prf since this small orf contains multiple hungry codons.
as a side product, fragments of eehv-gb specific sequences, either with or without a his-tag were made and confirmed by western blot analysis.
this suggested that the non-his-tagged fragments were co-purified during the purification process.
eehv-gb peptide specific rabbit sera were able to detect the antigen fragments, whereas positive elephant sera recognized fewer of these fragmented antigen products.
it is likely that the majority of the anti-eehv-gb antibodies in elephants recognize conformational epitopes, which are linearized in western blot.
this is supported by the fact that the produced antigen was highly reactive in the sandwich elisa test with the positive rabbit sera as well as many elephant sera and not with irrelevant sera.
as a result, it is likely that this elisa would fail to detect antibodies directed to those conformational epitopes of gb which do not form properly in the bacterial expression machinery.
in this study no seronegative elephants were found.
the background of the described assay was set up with sera of elephants, which were consistently found on the non-detectable range of the assay, but still ~ <dig>  od higher than naïve rabbit serum.
this test was developed with a capture antibody, which did not completely reduce the background to an od of  <dig>  therefore, it is highly likely that the cut-off value was leveled unrealistically high.
in the study cohort we found several seropositive elephants, which were routinely diagnosed with other subtypes than eehv1a by pcr.
the obtained data suggest that this test may be able to detect antibodies against gb from eehv subtypes other than eehv1a; at least eehv1b and eehv <dig>  divergence on the amino acid level of the glycoprotein b of the subtypes eehv ranges from 11 %  up to 21 %   <cit> .
however, we cannot exclude that these animals did had an eehv1a co-infection as well.
it is interesting to speculate that genetic background plays a role in susceptibility to develop severe disease.
however, better designed study cohorts, which include serology as well as pcr detection of the virus are required in order to address the question whether the host genetic background has a major role in fatal eehv disease.
CONCLUSIONS
we have shown that it is possible to express the gb of eehv in a bacterial system by using alternative translation upstream of the shine dalgarno sequence.
further, our results and those of recent reports  <cit>  demonstrate that eehv prevalence in captive born as well as wild-born elephants  is much higher than initially anticipated.
these studies underline that elephants may be the natural host of eehv that, in general, does not cause major disease.
the methodology and data presented in this article give a novel insight into the eehv <dig> seroprevalence in asian elephants.
as expected, a large portion of elephants showed antibodies against eehv, but not all.
additionally, the serostatus of elephants within a herd may vary from non-detectable up to high titers.
additional file
additional file 1:
supplementary information.
abbreviations
bcabicinchoninic acid
bsabovine serum albumin
cvcolumn volume
eehvelephant endotheliotropic herpes virus
eependangered species program
elisaenzyme linked immunosorbent assay
emelephas maximus
fplcfast protein liquid chromatography
gbglycoprotein b
hprohorse radish peroxidase
imacimmobilized metal affinity chromatography
iptgisopropyl β-d-thiogalactoside
laloxodonta africana
lbluria bertani broth
lphlimulus polyphemus hemocyanin
ni-ntanickel nitrilotriacetic acid
nmnanometer
odoptical density
orfopen reading frame
pbsphosphate buffered saline
pcrpolymerase chain reaction
prfprogrammed ribosomal frameshifting
pvdfpolyvinylidene difluoride
qpcrquantative polymerase chain reaction
rbsribosomal binding site
sds-pagesodium dodecyl sulphate polyacrylamide gel electrophoresis
sspspecies survival program
tmb <dig> ’, <dig> ’-tetramethylbenzidine
bcabicinchoninic acid
bsabovine serum albumin
cvcolumn volume
eehvelephant endotheliotropic herpes virus
elisaenzyme linked immunosorbent assay
emelephas maximus
fplcfast protein liquid chromatography
gbglycoprotein b
hprohorseradish peroxidase
imacimmobilized metal affinity chromatography
iptgisopropyl b-d-thiogalactoside
laloxodonta africana
lbluria bertani broth
lphlimulus polyphemus hemocyanin
ni-ntanickel nitrilotriacetic acid
nmnanometer
odoptical density
orfopen reading frame
pbsphosphate buffered saline
pcrpolymerase chain reactionprf, programmed ribosomal frameshifting
pvdfpolyvinylidene difluoride
qpcrquantative polymerase chain reaction
rbsribosomal binding site
sds-pagesodium dodecyl sulphate polyacrylamide gel electrophoresis
tmb <dig> ’, <dig> ’-tetramethylbenzidine
competing interests
we declare no relationships or support which could be perceived as a conflict of interest.
authors’ contributions
pd provided conceptual design and technical execution on all aspects of the study.
she wrote the manuscript.
vrp and svrf provided excellent technical assistance in the antigen production procedure.
ws is involved in the european eehv program and study director.
he provided the european asian elephant serum collection and supported pd/bm/ao in the clinical evaluation of the study results.
el contributed to this study by providing the usa asian elephant samples.
lh and pl contributed to this study by kindly allowing pd to use their lab facilities in the usa for performing the sample analysis of the usa cohort.
on account of cites  regulation asian elephant serum samples need to be analyzed in situ.
sc contributed to this study by providing european asian elephant samples.
nm and ad contributed to this study by providing the european asian elephant samples and were involved in the clinical evaluation of the study results.
they provided pcr results of elephants in this study, which were analyzed in their lab facilities.
ao is the principle investigator of the study.
bm supervised all aspects of this study and revised all the draft versions of the manuscript.
all authors read and approved the final manuscript.
the authors thank the following contributors to this study; the keepers, curators, and healthcare staff of the involved zoos; ms. a. brothers ; mrs. e. van der graaf ; dr. r. bodewes for the discussion on the manuscript and dr. r. de vries for the assistance with the image lay-outs.
we highly appreciate the up-to-date international elephant database website maintained by dan koehl.
financial support
zoological society of london

 the family herpesviridae comprises a diverse group of viruses, which are divided into three subfamilies. most commonly, herpesvirus infection presents as a benign, self-limiting disease with a mild viraemia. herpesvirus rarely causes widespread disseminated disease in the natural host, although cross-species transmission to non-reservoir hosts can also result in lethal infections. in general neonates and immune-compromised hosts are at higher risk of developing a fatal herpesvirus infection. the elephant endotheliotropic herpesvirus is a poorly characterized virus of elephants that was first observed in healthy wild african elephants by mccully et al. in and subsequently in asian elephants with no apparent disease. in the death of a 3-year old female asian circus elephant was associated with herpesvirus by ossent et al.. pathologic changes in the capillary endothelial cells with herpetic inclusion bodies and weak cross-neutralization in serum neutralization tests with bovine herpesviruses suggested that the etiologic agent was a hitherto unidentified herpesvirus, which was identified in. the disease has a sudden onset characterized by lethargy, oral ulcerations, edema in the skin of the head and proboscis, cyanosis of the tongue, decreased white blood cell and platelet count and massive internal hemorrhages, which lead to death within 12–72 h after onset of the symptoms. phylogenetic analysis showed that eehv is a distinct branch within the β-herpesviridae group, assigned as the new genus proboscivirus. to date, as many as seven different genotypes of eehv have been identified within this genus. however, xtensive evaluation of several subtypes indicated that eehvs have a large genomic inversion of a 40-kb core segment that is distinct from the roseoloviruses and all other β-herpesviruses. furthermore, they encode α-herpesvirus-like genes that are absent in β-herpesviruses and contain novel open reading frames not found in any other herpesvirus. these findings suggest that this particular virus is so different from other herpesviruses that it may be considered as a new herpesvirus subfamily. eehv poses a threat to the conservation mission of zoos. several studies have shown that dnaemia can be detected in asian elephants before the onset of clinical signs and in elephants that do not display clinical signs using pcr techniques. to date, this is the only option to monitor a complete herd, and only allows identification of animals with an active infection. therefore, the detection of antibodies against eehv likely offers a better strategy to determine which animals are carriers of eehv. ideally, antibody detection against the whole virus is preferable, but since the virus cannot be cultured in vitro, the best alternative is to use an immunodominant protein, such as glycoprotein b, as an antigen. the viral envelope protein glycoprotein b of herpesviruses is involved in host cell entry and represents an important target of neutralizing antibodies in the host. in this study a serologic test based on the eehv-gb antigen is described. this assay was used to assess seroprevalence against eehv-gb in serum and heparin plasma samples from two large cohorts of european and north-american captive asian elephants. these data are useful to link seroprevalence with eehv virus detection by pcr methods and, at the end, may give better insight into eehv dynamics within elephant populations. methods cloning, expression and large scale production of glycoprotein b the viral sequence of eehv-1a glycoprotein b comprising the start codon up to the transmembrane domain was synthesized and cloned, using xhoi/psti restriction sites, into the multiple cloning site in frame with a c-terminal myc/6his tag, but in an alternative open reading frame relative to the shine dalgarno ribosomal binding site, of the bacterial expression vector ptrchis2a. induction of recombinant glycoprotein b expression was performed in e.coli rosetta2plyss codon plus strain. this strain supplies trnas for rare codons in order to improve the expression of heterologous proteins. a single colony transformed with the ptrchis2a-gbmyc/his plasmid was inoculated into fresh lb medium containing 100 μg/ml ampicillin and 50 μg/ml chloramphenicol and grown overnight at 37 °c in a shaker incubator. the next day l fresh lb medium without antibiotics was inoculated in with the pre-culture and grown in a shaker incubator at 37 °c to an od600 ~ . the recombinant protein expression was induced by adding iptg to an end concentration of 1 mm. the temperature was lowered to 25 °c for optimal recombinant protein expression. cells were harvested 4 h after induction and pelleted at 6000xg for 10 min in a beckman highspeed centrifuge using the jla rotor. the supernatant was decanted and the cells were resuspended in native lysisbuffer, 5 % v/v glycerol and 10 mm of imidazole and protease inhibitors). lysozyme was added to an end concentration of 1 mm and the cells were treated 30 min at 4 °c at continuous agitation. the cells were cracked by three freeze/thaw cycles and sonification with a microtip; x 30s at 60 % on ice. the lysate was cleared by ultracentrifugation for 2 h at  rpm in a beckman optima l-90 k ultracentrifuge using the sw32ti rotor. a similar procedure was conducted with another ptrchis2a vector encoding a kd irrelevant 6his-tagged protein that was used as a negative control. purification of glycoprotein b the cleared lysate was loaded onto ni-nta resin which was preconditioned with water and 1x native lysisbuffer. binding was performed overnight at +4 °c under continuous agitation. next day, the resin was settled vertically and washed with bed volumes of lysisbuffer containing 20 mm of imidazole. thereafter, the bound recombinant protein was eluted with bed volumes of elution buffer, 5 % v/v glycerol and 300 mm of imidazole). subsequently, salts were removed by dialysis in a slide-a-lyzer cassette for 48 h at +4 °c. the dialysis buffer was refreshed three times. after dialysis the recombinant protein content was analyzed for protein concentration and western blot. a second round of purification was performed by fast protein liquid chromatography with a 1 ml volume histrap-hptm column with a pressure flow of  ml/min. the his-tagged proteins were eluted from the column with column volumes of a linear gradient ranging from 50 mm imidazole up to 500 mm imidazole in  ml aliquots. these were analyzed by western blot and the eluates containing the his-tagged proteins were subsequently pooled and dialyzed as described above. rabbit anti-eehv glycoprotein b serum five peptide sequences derived from the amino acid sequence of glycoprotein b of eehv1a were synthesized and coupled to lph carrier protein. ten rabbits were immunized six times intramuscularly with 10 μg of the respective peptides. final bleeding was at day after the first vaccination. the polyclonal rabbit sera were produced by biogenes gmbh according to european law on animal welfare. all rabbit sera were highly reactive against the respective peptide as tested in a peptide elisa assay. immunodetection of recombinant glycoprotein b recombinant glycoprotein b antigen was analyzed by separation of small samples from the purification process on a 10 % sds-page gel and separated products were subsequently transferred to pvdf membrane. gels were run in duplicate; one was coomassie blue stained for total protein and the duplicate was analyzed with western blot using an anti-6his tag mouse monoclonal antibody. as a secondary antibody goat anti-mouse irdye was used. rabbit serum reactivity with recombinant glycoprotein b was detected with western blot by incubating the membranes with rabbit serum followed by detection with goat-anti-rabbit irdye. similar strategy was done for elephant serum, but primary antibody was detected with rabbit-anti asian elephant igg. sera of rabbits as well as elephants were pre-incubated in buffer containing e.coli lysate for 1 h at room temperature in order to reduce possible e.coli reactivity. the signal of the conjugate was detected with the licor odyssey scanner. glycoprotein b specific capture elisa to avoid non-specific reaction against e.coli contaminants which co-purified with the 6his-tagged eehv gb antigen during ni-nta purification an optimal concentration of 100 ng/well of mouse-anti 6his antibody as a capture antibody was coated in high binding 96-well microtiterplate. the microtiter plates were blocked with 100 μl/well of pbs containing 2 % w/v bsa in pbs for one hour at 37 °c and washed. all washing steps were performed three times with 200 μl of wash buffer. to determine the optimal antigen concentration for use in the elisa, the gb antigen was diluted two-fold starting at 2400 ng per well. a pool of rabbit sera was used as anti-gb positive serum. the purified eehv-gb antigen was diluted in pbs containing  % triton-x to a concentration of 750 ng/well and bound to the capture antibody for 1 h at 37 °c. after washing, primary incubation was performed with 100 μl/well diluted elephant serum in elisa buffer containing pbs,  % bsa,  % non-fat dried powder milk and 3 % w/v nacl and incubated for one hour at 37 °c. after washing, elephant specific igg’s bound to glycoprotein b were detected by incubation with 100 μl/well of rabbit anti-elephant igg for one hour at 37 °c followed by an incubation step with swine anti-rabbit igg hrpo conjugate. after washing 100 μl/well 1xtmb substrate was added, incubated for 10 min at room temperature. the reaction was stopped by adding 100 μl  m h2so per well. the absorbance was measured at 450 nm with 620 nm reference filter on a 96-well plate spectrophotometer. study cohort in europe/usa the elephants in this study were kept in zoos as part of the european endangered species program and the species survival program. within these programs elephants are trained to allow voluntarily blood collection for several reasons: monitoring health parameters, monitoring reproduction parameters and providing data for new diagnostic technologies for diseases that may threaten their health. the use of banked elephant blood samples for a retrospective research study falls within the law. all participating zoos reviewed and approved the research proposal for this study and allowed the use of their banked elephant blood samples. all elephants in the study were asian elephants; table  and summarize the age, sex, and clinical status of elephants which will be discussed in detail in this study. all participating zoos reviewed and approved the research described in this study. initially, serum or heparin plasma samples of asian elephants located in european zoos were tested for validation of the capture elisa test. the cut-off value was calculated as a mean od of samples of animals which scored consistently on the base line level of the test.table age, sex, and clinical status of elephants in the study cohort of european zoos zoos are numbered, elephants are indicated with a letter and subsequent numbers describe maternal offspring nd, not detectable tw, trunk wash c, conjunctiva u, urine o, oral lesion wb, whole blood v, vulva three european zoos provided asian elephant serum or plasma samples for a longitudinal seroprevalence study ranging from up to 600 days for multiple animals in their herds. moreover, the samples from zoo and were also analyzed for eehv presence by qpcr. in the usa cohort asian elephant samples were tested from animals in zoos in the period until with a six month interval. all samples were tested in 1: and 1: dilutions and the signal to background od was calculated for both dilutions. a sample was considered positive when both dilutions produced ods times of the background od and undetectable when both dilutions produced less than times the background od. a sample was considered as borderline when one or both dilutions measured an od between 2– times the background od values. an animal was considered to be seropositive when all analyzed samples scored positive with an occasional borderline score. an animal was considered borderline seropositive when the majority of its samples scored borderline. an animal was considered undetectable when all of its samples were undetectable.  expression and purification of eehv glycoprotein b positioning of the eehv-gb open reading frame in an alternative position downstream of the shine dalgarno ribosomal binding site severely reduced the expression levels of the recombinant eehv-gb, but retained the recombinant protein in the soluble fraction and in its native conformation. purification of the recombinant eehv-gb was performed using nickel columns. full length eehv-gb has an expected molecular size of kd, and a band at this expected size was observed. since the portion of recombinant gb protein in the e.coli lysate was low many native proteins of e. coli rosetta that displayed affinity to nickel in the immobilized metal affinity chromatography were co-purified during ni-nta purification. full length as well as many other molecular weight sizes, were detected by anti-6his-tag antibody detection on western blot. western blot analysis with the eehv-gb peptide specific rabbit serum showed more bands with different molecular weights compared to the pattern obtained with anti-his antibody. several specific smaller bands were also detected. an elephant serum, which did contain eehv-gb specific antibodies, as detected with the described sandwich elisa, did not recognize as many of the eehv-gb fragments on western blot. since the double purified lost a portion of the full length eehv-gb the single ni-nta purified antigen was further used to develop an elisa test.fig. sds-page total protein analysis and western blot analysis with an anti-his monoclonal antibody demonstrate the purification of full length eehv-gbhis and several smaller fragments with a his-tag produced from the eehv-gbhis open reading frame. during purification a small portion of the recombinant protein is lost in the flow through of the ninta column and during the washing steps. the empty vector does not yield any his-tagged proteins. lane m is the molecular weight marker fig. western blot analysis of an irrelevant kd his-tagged protein and, single ninta purified eehv-gbhis and double purified ninta/histrap fplc with three different antisera. a western blot with mouse monoclonal anti-his demonstrates the kd his-tagged protein in lane and the highly fragmented eehv-gbhis in lane and double purification results in loss of the full length eehv-gbhis. bthe rabbit peptide eehv-gb specific antiserum recognizes non-his tagged bands. c serum of elephant 4a detects identical bands as the rabbit peptide specific serum in the eehv-gbhis recombinant protein, except for the ~ kd band which might be an e.coli background band validation of an eehv glycoprotein b specific capture elisa to reduce aspecific e.coli protein reactivity the elisa assay was setup as a capture elisa using a mouse-anti 6his monoclonal antibody. pooled sera of rabbits immunized with eehv-specific peptides were used as a positive control. figure  shows that 100 ng of the capture antibody reduces aspecific reactivity to an acceptable background level of an a450/ of ~. subsequently, the capture elisa was further validated with an antigen checkerboard titration to test its specificity with various irrelevant rabbit sera. the results in fig.  demonstrate a dose-related response only when eehv-gb specific sera were used. irrelevant rabbit antisera did not react with the antigen. to detect antibody reactivity in the linear range an optimum of 750 ng antigen/well was used in the elisa, which was used to evaluate the elephant sera cohorts. once the capture elisa was developed with the eehv-gb specific rabbit sera, a large panel of elephant sera was tested to screen for sera with specific reactivity. in total serum or plasma samples of asian elephants, representing animals of zoos were tested. twenty-three animals from zoos tested consistently positive, animals of zoos tested intermittently positive/borderline/undetectable and animals tested consistently undetectable. the test was repeated twice in separate experiments and the results were reproducible. we found three sera with extremely high reactivity in the capture elisa and several non-reactive sera. these elephant sera were used to test again the antigen dose and specificity. the sera of elephant 2b 4a and 5a showed a dose-dependent reactivity whereas the serum from elephant 2c did not. serial dilutions of serum of animal 5a and animal 2c on eehv-gb antigen or an irrelevant 6his-tagged protein were used in the assay to test the specificity. only the positive serum of animal 5a reacted in a dose-dependent manner with eehv-gb antigen and not with the irrelevant antigen.fig. e. coli protein contamination in the purified antigen induces background signal in an indirect elisa. this background is reduced in a sandwich elisa using a mouse monoclonal anti-his antibody as a capture antibody for the target antigen. pooled eehv-gb peptide specific rabbit serum was used in a 1: dilution. panel a no capture antibody, panel b 100 ng capture antibody, panel c 200 ng capture antibody, panel d 300 ng capture antibody fig. specificity of the eehv-gb sandwich elisa was determined by antigen titration with the eehv-gb peptide specific rabbit serum, one serum of an eehv pcr positive elephant and two irrelevant rabbit sera. all sera were diluted 1: in elisa buffer fig. characterization of the sandwich elisa with elephant sera. elephant sera react in a dose-dependent manner as shown by antigen titration and are specific for the eehv-gb antigen only the majority of eehv pcr positive elephants display eehv specific antibodies for animals eehv pcr data was available, either from published data or personal communications. we compared these results with our elisa results. nineteen out of pcr positive animals displayed antibodies, of which had significant antibody titers and three had borderline titers. however, in six out of animals no eehv specific antibodies could be detected while they had been tested positive with the eehv specific pcr. eight elephants displayed eehv-gb specific antibodies, but never tested positive in a routine pcr. four animals were eehv pcr positive of which three displayed antibodies against eehv-gb.table age, sex, and clinical eehv status of elephants in the study cohort of usa zoos zoos are numbered, elephants are indicated with a letter and subsequent numbers describe maternal offspring nd, not detectable tw, trunk wash c, conjunctiva u, urine o, oral lesion wb, whole blood v, vulva seroprevalence of a european study cohort three european zoos were approached to collaborate in our study; their animals are listed in table  and their asian elephant sera collections were analyzed over a substantial period of time. during the study period the animals of zoo and were screened for eehv virus presence in whole blood, trunk wash and conjunctiva swabs by either conventional or real time pcr. figure  displays the serostatus of these three herds; the observed patterns showed a rise-and-fall in signal to background ratios. a similar rise-and-fall pattern was observed for animals, which scored below the detection level. additional clinical evaluations of the animals in this study can be found in.fig. serostatus of three european zoos measured as the signal to background ratio over a period of time. zoo was studied from october until april zoo was studied from may until may and zoo from january until september the arrow indicates a positive eehv pcr of the trunk wash or whole blood. elephants with non-detectable levels of eehv antibodies are indicated in black, elephants with an alternating serostatus in orange and elephants with continuous anti-eehv antibodies in red only two animals had a positive pcr in the study-period in either trunk wash or whole blood. as seen in fig. 6a animal 1a was eehv pcr positive in trunk wash on day and, shortly afterwards, had elevated antibody titers up to 8 weeks after the pcr positive trunk wash. as shown in fig. 6b calf 2b developed a dnaemia and clinical signs of eehv disease on day of the study period with typical eehv clinical signs. however, this calf was already seropositive on day of the study period. he survived this episode with supportive treatment and famciclovir administration. eighteen days prior to disease onset his half-sister 2e showed a rise in antibody titers. unfortunately no samples of the other herd members were available in this period of the study. discussion to date, an accurate and dependable test to evaluate eehv seroprevalence in asian elephants has not been developed. in part, this is due to the inability to culture the virus in vitro and therefore whole virus is not available as the ideal antigen source for an elisa. production of recombinant proteins from one of the eehv glycoproteins, like gb, has proven to be a major challenge. here we describe a novel elisa that specifically detects eehv-gb antibodies circulating in asian elephants. we were able to link seroprevalence to pcr detection of the eehv in approximately 80 % animals. this novel assay will provide more insight into the epidemiology of this hitherto poorly understood virus. the antigen expression, production and purification methods for fragmented eehv-gb driven by a bacterial shine dalgarno promoter in an alternative open reading frame, yielded antigens for a sandwich elisa specific for eehv-gb. incomplete eehv-gb amino acid sequences were produced from the plasmid presumably due to programmed ribosomal frameshifting and alternative translation initiation sites. prf and alternative translation initiation are well-described phenomena and can occur in prokaryotes, as well as in eukaryotes. prf occurs when the ribosome encounters a so-called hungry codon in its a-site followed by a slippery sequence in the p-site of the ribosomal complex. in our expression system, it is likely that the shine dalgarno sequence rbs initiates the overexpression of a short nonsense peptide of amino acids thereby releasing the ribosomes quickly in order to re-initiate translation with a high likelihood to undergo prf since this small orf contains multiple hungry codons. as a side product, fragments of eehv-gb specific sequences, either with or without a his-tag were made and confirmed by western blot analysis. this suggested that the non-his-tagged fragments were co-purified during the purification process. eehv-gb peptide specific rabbit sera were able to detect the antigen fragments, whereas positive elephant sera recognized fewer of these fragmented antigen products. it is likely that the majority of the anti-eehv-gb antibodies in elephants recognize conformational epitopes, which are linearized in western blot. this is supported by the fact that the produced antigen was highly reactive in the sandwich elisa test with the positive rabbit sera as well as many elephant sera and not with irrelevant sera. as a result, it is likely that this elisa would fail to detect antibodies directed to those conformational epitopes of gb which do not form properly in the bacterial expression machinery. in this study no seronegative elephants were found. the background of the described assay was set up with sera of elephants, which were consistently found on the non-detectable range of the assay, but still ~ od higher than naïve rabbit serum. this test was developed with a capture antibody, which did not completely reduce the background to an od of therefore, it is highly likely that the cut-off value was leveled unrealistically high. in the study cohort we found several seropositive elephants, which were routinely diagnosed with other subtypes than eehv1a by pcr. the obtained data suggest that this test may be able to detect antibodies against gb from eehv subtypes other than eehv1a; at least eehv1b and eehv divergence on the amino acid level of the glycoprotein b of the subtypes eehv ranges from 11 % up to 21 %. however, we cannot exclude that these animals did had an eehv1a co-infection as well. it is interesting to speculate that genetic background plays a role in susceptibility to develop severe disease. however, better designed study cohorts, which include serology as well as pcr detection of the virus are required in order to address the question whether the host genetic background has a major role in fatal eehv disease.  we have shown that it is possible to express the gb of eehv in a bacterial system by using alternative translation upstream of the shine dalgarno sequence. further, our results and those of recent reports demonstrate that eehv prevalence in captive born as well as wild-born elephants is much higher than initially anticipated. these studies underline that elephants may be the natural host of eehv that, in general, does not cause major disease. the methodology and data presented in this article give a novel insight into the eehv seroprevalence in asian elephants. as expected, a large portion of elephants showed antibodies against eehv, but not all. additionally, the serostatus of elephants within a herd may vary from non-detectable up to high titers. additional file additional file 1: supplementary information. abbreviations bcabicinchoninic acid bsabovine serum albumin cvcolumn volume eehvelephant endotheliotropic herpes virus eependangered species program elisaenzyme linked immunosorbent assay emelephas maximus fplcfast protein liquid chromatography gbglycoprotein b hprohorse radish peroxidase imacimmobilized metal affinity chromatography iptgisopropyl β-d-thiogalactoside laloxodonta africana lbluria bertani broth lphlimulus polyphemus hemocyanin ni-ntanickel nitrilotriacetic acid nmnanometer odoptical density orfopen reading frame pbsphosphate buffered saline pcrpolymerase chain reaction prfprogrammed ribosomal frameshifting pvdfpolyvinylidene difluoride qpcrquantative polymerase chain reaction rbsribosomal binding site sds-pagesodium dodecyl sulphate polyacrylamide gel electrophoresis sspspecies survival program tmb ’, ’-tetramethylbenzidine bcabicinchoninic acid bsabovine serum albumin cvcolumn volume eehvelephant endotheliotropic herpes virus elisaenzyme linked immunosorbent assay emelephas maximus fplcfast protein liquid chromatography gbglycoprotein b hprohorseradish peroxidase imacimmobilized metal affinity chromatography iptgisopropyl b-d-thiogalactoside laloxodonta africana lbluria bertani broth lphlimulus polyphemus hemocyanin ni-ntanickel nitrilotriacetic acid nmnanometer odoptical density orfopen reading frame pbsphosphate buffered saline pcrpolymerase chain reactionprf, programmed ribosomal frameshifting pvdfpolyvinylidene difluoride qpcrquantative polymerase chain reaction rbsribosomal binding site sds-pagesodium dodecyl sulphate polyacrylamide gel electrophoresis tmb ’, ’-tetramethylbenzidine competing interests we declare no relationships or support which could be perceived as a conflict of interest. authors’ contributions pd provided conceptual design and technical execution on all aspects of the study. she wrote the manuscript. vrp and svrf provided excellent technical assistance in the antigen production procedure. ws is involved in the european eehv program and study director. he provided the european asian elephant serum collection and supported pd/bm/ao in the clinical evaluation of the study results. el contributed to this study by providing the usa asian elephant samples. lh and pl contributed to this study by kindly allowing pd to use their lab facilities in the usa for performing the sample analysis of the usa cohort. on account of cites regulation asian elephant serum samples need to be analyzed in situ. sc contributed to this study by providing european asian elephant samples. nm and ad contributed to this study by providing the european asian elephant samples and were involved in the clinical evaluation of the study results. they provided pcr results of elephants in this study, which were analyzed in their lab facilities. ao is the principle investigator of the study. bm supervised all aspects of this study and revised all the draft versions of the manuscript. all authors read and approved the final manuscript. the authors thank the following contributors to this study; the keepers, curators, and healthcare staff of the involved zoos; ms. a. brothers; mrs. e. van der graaf; dr. r. bodewes for the discussion on the manuscript and dr. r. de vries for the assistance with the image lay-outs. we highly appreciate the up-to-date international elephant database website maintained by dan koehl. financial support zoological society of london
to date, identification of latent carriers has been challenging, since there are no serological assays capable of detecting seropositive elephants. eehv infection is detected by pcr in samples from both clinically ill and asymptomatic elephants with an active infection, whereas latent carriers can be distinguished exclusively via serological assays. this study shows that the developed elisa is suitable to detect antibodies specific to eehv. monitoring three asian elephant herds from european zoos revealed that the serostatus of elephants within a herd varied from non-detectable to high titers. results confirm that eehv prevalence among asian elephants is high. elephant endotheliotropic herpesvirus poses a large threat to breeding programs of captive asian elephants by causing fatal haemorrhagic disease. it allows study of eehv seroprevalence in asian elephants. the antibody titers showed typical herpes-like rise-and-fall patterns in time which occur in all seropositive animals in the herd more or less simultaneously. here we describe a novel elisa that specifically detects eehv antibodies circulating in asian elephant plasma/serum.
 elephants are classified as critically endangered animals by the international union for conservation of species. elephant endotheliotropic herpesvirus poses a large threat to breeding programs of captive asian elephants by causing fatal haemorrhagic disease. eehv infection is detected by pcr in samples from both clinically ill and asymptomatic elephants with an active infection, whereas latent carriers can be distinguished exclusively via serological assays. to date, identification of latent carriers has been challenging, since there are no serological assays capable of detecting seropositive elephants.  here we describe a novel elisa that specifically detects eehv antibodies circulating in asian elephant plasma/serum. approximately 80 % of pcr positive elephants display eehv-specific antibodies. monitoring three asian elephant herds from european zoos revealed that the serostatus of elephants within a herd varied from non-detectable to high titers. the antibody titers showed typical herpes-like rise-and-fall patterns in time which occur in all seropositive animals in the herd more or less simultaneously.  this study shows that the developed elisa is suitable to detect antibodies specific to eehv. it allows study of eehv seroprevalence in asian elephants. results confirm that eehv prevalence among asian elephants is high. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords eehvasian elephantglycoprotein belisaseroprevalenceissue-copyright-statement© the author 2015 