Low gH/gL (Sub)Species-Specific Antibody Levels Indicate Elephants at Risk of Fatal Elephant Endotheliotropic Herpesvirus Hemorrhagic Disease.

Elephant endotheliotropic herpesviruses (EEHVs), of which eleven (sub)species are currently distinguished, infect either Asian (Elephas maximus) or African elephants (Loxodonta species). While all adult elephants are latently infected with at least one EEHV (sub)species, young elephants, specifically those with low to non-detectable EEHV-specific antibody levels, may develop fatal hemorrhagic disease (EEHV-HD) upon infection. However, animals with high antibody levels against EEHV(1A) gB, an immunodominant antigen recognized by antibodies elicited against multiple (sub)species, may also occasionally succumb to EEHV-HD. To better define which animals are at risk of EEHV-HD, gB and gH/gL ELISAs were developed for each of the Asian elephant EEHV subspecies and assessed using 396 sera from 164 Asian elephants from European zoos. Antibody levels measured against gB of different (sub)species correlated strongly with one another, suggesting high cross-reactivity. Antibody levels against gH/gL of different subspecies were far less correlated and allowed differentiation between these (sub)species. Importantly, while high gB-specific antibody levels were detected in the sera of several EEHV-HD fatalities, all fatalities (n = 23) had low antibody levels against gH/gL of the subspecies causing disease. Overall, our data indicate that (sub)species-specific gH/gL ELISAs can be used to identify animals at risk of EEHV-HD when infected with a particular EEHV (sub)species.

EEHV1A glycoprotein B subunit vaccine elicits humoral and cell-mediated immune responses in mice.

Asian elephants are an endangered species facing many threats, including severe hemorrhagic disease (HD) caused by the elephant endotheliotropic herpesvirus (EEHV). EEHV-HD is the leading cause of death in captive juvenile Asian elephants in North America and Europe, and also affects elephants in their natural range countries. Significant challenges exist for successful treatment of EEHV-HD, which include timely recognition of disease onset and limited availability of highly effective treatment options. To address this problem, our goal is to prevent lethal disease in young elephants by developing a vaccine that elicits robust and durable humoral and cell-mediated immunity against EEHV. EEHV glycoprotein B (gB) is a major target for cellular and humoral immunity in elephants previously exposed to EEHV. Therefore, we generated a vaccine containing recombinant EEHV1A gB together with a liposome formulated TLR-4 and saponin combination adjuvant (SLA-LSQ). CD-1 mice that received one or two vaccinations with the vaccine elicited significant anti-gB antibody and polyfunctional CD4+ and CD8+ T cell responses, while no adverse effects of vaccination were observed. Overall, our findings demonstrate that an adjuvanted gB protein subunit vaccine stimulates robust humoral and cell-mediated immune responses and supports its potential use in elephants.

Young elephants in a large herd maintain high levels of elephant endotheliotropic herpesvirus-specific antibodies and do not succumb to fatal haemorrhagic disease.

Elephant endotheliotropic herpesviruses (EEHVs) have co-existed with elephants for millions of years, yet may cause fatal haemorrhagic disease (EEHV-HD), typically in elephants between 1 and 10 years of age. EEHV is omnipresent in (sub)adult elephants, and young elephants with low EEHV-specific antibody levels are at risk for EEHV-HD, suggesting that fatal disease may occur due to an insufficiently controlled primary infection. To further address this hypothesis, sera of three large elephant cohorts were subjected to a multiple EEHV species ELISA: (I) 96 Asian elephants between 0 and 57 years, including 13 EEHV-HD fatalities, from European zoo herds typically sized five to six elephants, (II) a herd of 64 orphaned elephants aged 0-15 years at the Elephant Transit Home in Sri Lanka and (III) 31 elephants aged 8-63 years, part of a large herd of 93 elephants at Pinnawala Elephant Orphanage, Sri Lanka. All Sri Lankan elephants showed high EEHV-specific antibody levels regardless of their age. While antibody levels of most European zoo elephants were comparable to those of Sri Lankan elephants, the average antibody level of the European juveniles (1-5 years of age) was significantly lower than those of age-matched Sri Lankan individuals. Moreover, the European juveniles showed a gradual decrease between 1 and 4 years of age, to be attributed to waning maternal antibodies. Maintenance of high levels of antibodies in spite of waning maternal antibodies in young Sri Lankan elephants is likely due to the larger herd size that increases the likelihood of contact with EEHV-shedding elephants. Together with the observation that low levels of EEHV-specific antibodies correlate with increased numbers of EEHV-HD fatalities, these results suggest that infection in presence of high maternal antibody levels may protect calves from developing EEHV-HD, while at the same time activating an immune response protective in future encounters with this virus.

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country.

Elephant endotheliotropic herpesviruses (EEHVs) may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Prevalence of EEHV in different elephant populations is still largely unknown. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight into its spread among elephants, we developed novel ELISAs based on EEHV1A gB and gH/gL. Performance of the ELISAs was assessed using sera from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, indicating that EEHV prevalence has been highly underestimated so far. Reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.

Evidence of high EEHV antibody seroprevalence and spatial variation among captive Asian elephants (Elephas maximus) in Thailand.

BACKGROUND: Elephant endotheliotropic herpesviruses (EEHV) can cause an acute highly fatal hemorrhagic disease in young Asian elephants (Elephas maximus), both ex situ and in situ. Amongst eight EEHV types described so far, type 1 (subtype 1A and 1B) is the predominant disease-associated type. Little is known about routes of infection and pathogenesis of EEHV, and knowledge of disease prevalence, especially in range countries, is limited. METHODS: A large cross-sectional serological survey was conducted in captive elephants (n = 994) throughout Thailand using an EEHV-1A glycoprotein B protein antigen specific antibody ELISA. RESULTS: Antibody seroprevalence was 42.3%, with 420 of 994 elephants testing positive. Associations between seropositivity and potential risk factors for EEHV infection were assessed and included: elephant age, sex, camp cluster size, management type (extensive versus intensive), sampling period (wet vs. dry season) and location of camp (region). Univariable regression analysis identified management system and region as risk factors for the presence of EEHV antibodies in elephants, with region being significant in the final multivariable regression model. Prevalence was highest in the North region of the country (49.4%). CONCLUSIONS: This study produced baseline serological data for captive elephants throughout Thailand, and showed a significant EEHV burden likely to be maintained in the captive population.

The Use of Genomics in Conservation Management of the Endangered Visayan Warty Pig (Sus cebifrons).

The list of threatened and endangered species is growing rapidly, due to various anthropogenic causes. Many endangered species are present in captivity and actively managed in breeding programs in which often little is known about the founder individuals. Recent developments in genetic research techniques have made it possible to sequence and study whole genomes. In this study we used the critically endangered Visayan warty pig (Sus cebifrons) as a case study to test the use of genomic information as a tool in conservation management. Two captive populations of S. cebifrons exist, which originated from two different Philippine islands. We found some evidence for a recent split between the two island populations; however all individuals that were sequenced show a similar demographic history. Evidence for both past and recent inbreeding indicated that the founders were at least to some extent related. Together with this, the low level of nucleotide diversity compared to other Sus species potentially poses a threat to the viability of the captive populations. In conclusion, genomic techniques answered some important questions about this critically endangered mammal and can be a valuable toolset to inform future conservation management in other species as well.

THE INFLUENCE OF ULTRAVIOLET-B RADIATION ON THE GROWTH OF MARABOU STORK (LEPTOPTILOS CRUMENIFERUS) NESTLINGS IN RELATION TO PLASMA CALCIUM, PHOSPHORUS, AND VITAMIN D3 CONCENTRATIONS.

In order to prevent metabolic bone disease in growing captive-bred marabou storks (Leptoptilos crumeniferus), three hatchlings were exposed twice a day for 30 min each time to ultraviolet-B (UVB) radiation. During their first 35 days of life, body weights were monitored weekly, and blood was collected to determine total calcium, phosphorus, 25(OH) cholecalciferol, and 1.25(OH)2cholecalciferol plasma levels. Data were compared with those obtained from two marabou stork nestlings that were raised before, without being exposed to UVB. These two birds developed metabolic bone disease, while the UVB-exposed birds developed into healthy adult animals. Plasma chemistry data obtained in this study demonstrate that nestling marabou storks produce vitamin D3under the influence of UVB radiation. The absence of clinical metabolic bone disease in the nestlings that received UVB compared to the nestlings that were raised with the same diet without UVB radiation and that developed MBD demonstrates the importance of UVB radiation for normal development in this species.

A novel antigen capture ELISA for the specific detection of IgG antibodies to elephant endotheliotropic herpes virus.

BACKGROUND: Elephants are classified as critically endangered animals by the International Union for Conservation of Species (IUCN). Elephant endotheliotropic herpesvirus (EEHV) 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. RESULTS: 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. CONCLUSIONS: 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 (whether captive-born or wild-caught) is high.

Effect of calcium and cholecalciferol supplementation on several parameters of calcium status in plasma and urine of captive Asian (Elephas maximus) and African elephants (Loxodonta africana).

The aim of the current study was to assess the effect of oral calcium and cholecalciferol supplementation on several parameters of calcium status in plasma and urine of captive Asian (Elephas maximus; n=10) and African elephants (Loxodonta africana; n=6) and to detect potential species differences. Calcium and cholecalciferol supplementation were investigated in a feeding trial using a crossover design consisting of five periods of 28 days each in summer. From days 28-56 (period 2), elephants were fed the Ca-supplemented diet and from days 84-112, elephants were fed the cholecalciferol-supplemented diet (period 4). The control diet was fed during the other periods and was based on their regular ration, and the study was repeated similarly during winter. Periods 1, 3, and 5 were regarded as washout periods. This study revealed species-specific differences with reference to calcium and cholecalciferol supplementation. Asian elephants showed a significant increase in mean plasma total calcium concentration following calcium supplementation during summer, suggesting summer-associated subclinical hypocalcemia in Western Europe. During winter, no effect was seen after oral calcium supplementation, but a significant increase was seen both in mean plasma, total, and ionized calcium concentrations after cholecalciferol supplementation in Asian elephants. In contrast, evidence of subclinical hypocalcemia could be demonstrated neither in summer nor in winter in African elephants, although 28 days of cholecalciferol supplementation during winter reversed the decrease in plasma 1,25(OH)2-cholecalciferol and was followed by a significant increase in mean plasma total calcium concentration. Preliminary findings indicate that the advisable permanent daily intake for calcium in Asian elephants and cholecalciferol in both elephant species at least during winter might be higher than current guidelines. It is strongly recommended to monitor blood calcium concentrations and, if available, blood parathyroid hormone levels to adjust the nutritional supplementation for each individual elephant.

Delayed postpartum fetotomy in an Asian elephant (Elephas maximus).

A 37-yr-old Asian elephant (Elephas maximus) started parturition after 640 days of pregnancy but no fetal parts entered the birth canal. Despite veterinary intervention, the calf was not delivered. After 13 mo calving resumed and a full-term dead calf advanced into and lodged within the vagina. With standing xylazine tranquilization, the dam received a vagino-vestibulotomy to permit total fetotomy of the calf, which presented with bilateral carpal arthrogryposis. Severe infection of the caudal vaginal vestibulum complicated wound healing, and over the following year two corrective surgeries were performed, which resolved the fistula 3 mo after the second debridement. The elephant not only survived the procedures but also resumed normal estrous cycles, as demonstrated by blood progesterone concentration monitoring.

Retrospective serological investigation of bovine tuberculosis in two gemsbok (Oryx gazelle gazelle) and an onager (Equus hemionus onager).

In 1997 a 26-yr-old gemsbok (Oryx gazelle gazelle) died of bovine tuberculosis in a zoo. Three remaining gemsbok were administered the comparative tuberculin skin test repeatedly over a period of 5 mo. Two animals showed inconclusive results on the second test. All three gemsbok were euthanatized. Mycobacterium bovis was isolated from one of those with an inconclusive skin test result, whereas Mycobacterium fortuitum was detected in the other gemsbok. Eight years later, an onager (Equus hemionus onager) died of bovine tuberculosis. This animal had been kept in the same building as the gemsbok. Three herd mates were culled after administering the comparative tuberculin skin test. They were all nonreactors and produced no evidence of tuberculosis at postmortem examination. Retrospectively, using plasma samples collected from the gemsbok and onagers, three antibody tests, Elephant TB STAT-PAK, multiantigen print immunoassay (MAPIA), and dual-path platform (DPP) VetTB (Chembio Diagnostic Systems Inc., Medford, New York, 11763, USA), were used to assess their diagnostic value for these species. The M. bovis-infected gemsbok tested strongly positive by Elephant TB STAT-PAK at the time of euthanasia and 5 mo earlier when the skin test was negative. This animal was not antibody reactive in MAPIA and DPP VetTB. No M. bovis-specific antibody was detected in the other two gemsboks by any of the immunoassays. Among the onagers, Elephant TB STAT-PAK, MAPIA, and DPP VetTB revealed gradually increasing antibody response in the animal that died of bovine tuberculosis, but not in the three disease-free herd mates euthanatized. Seroconversion in the M. bovis-infected onager was first noticed 5 yr before death when the tuberculin skin test was negative.

Preliminary validation of assays to measure parameters of calcium metabolism in captive Asian and African elephants in western Europe.

Hypocalcemia is a well known cause of dystocia in animals, including elephants in captivity. In order to study calcium metabolism in elephants, it is of utmost importance to use properly validated assays, as these might be prone to specific matrix effects in elephant blood. The aim of the current study was to conduct preliminary work for validation of various parameters involved in calcium metabolism in both blood and urine of captive elephants. Basal values of these parameters were compared between Asian elephants (Elephas maximus) and African elephants (Loxodonta africana). Preliminary testing of total calcium, inorganic phosphorus, and creatinine appeared valid for use in plasma and creatinine in urine in both species. Furthermore, measurements of bone alkaline phosphatase and N-terminal telopeptide of type I collagen appeared valid for use in Asian elephants. Mean heparinized plasma ionized calcium concentration and pH were not significantly affected by 3 cycles of freezing and thawing. Storage at 4 °C, room temperature, and 37 °C for 6, 12, and 24 hr did not alter the heparinized plasma ionized calcium concentration in Asian elephants. The following linear regression equation using pH (range: 6.858-7.887) and ionized calcium concentration in heparinized plasma was utilized: iCa(7.4) (mmol/l) = -2.1075 + 0.3130·pH(actual) + 0.8296·iCa(actual) (mmol/l). Mean basal values for pH and plasma in Asian elephant whole blood were 7.40 ± 0.048 and 7.49 ± 0.077, respectively. The urinary specific gravity and creatinine concentrations in both Asian and African elephants were significantly correlated and both were significantly lower in Asian elephants.

Compressive myelopathy of the cervical spine in Komodo dragons (Varanus komodoensis).

Cervical subluxation and compressive myelopathy appears to be a cause of morbidity and mortality in captive Komodo dragons (Varanus komodoensis). Four cases of cervical subluxation resulting in nerve root compression or spinal cord compression were identified. Three were presumptively induced by trauma, and one had an unknown inciting cause. Two dragons exhibited signs of chronic instability. Cervical vertebrae affected included C1-C4. Clinical signs on presentation included ataxia, ambulatory paraparesis or tetraparesis to tetraplegia, depression to stupor, cervical scoliosis, and anorexia. Antemortem diagnosis of compression was only confirmed with magnetic resonance imaging or computed tomography. Treatment ranged from supportive care to attempted surgical decompression. All dragons died or were euthanatized, at 4 days to 12 mo postpresentation. Studies to define normal vertebral anatomy in the species are necessary to determine whether the pathology is linked to cervical malformation, resulting in ligament laxity, subsequent instability, and subluxation.

Serologic survey for selected viral pathogens in free-ranging endangered European mink (Mustela lutreola) and other mustelids from south-western France.

To investigate the possible role of selected pathogens in the decline of endangered European mink (Mustela lutreola) populations and the potential for these pathogens to affect mink survival, a serologic survey was conducted using serum samples collected from March 1996 to March 2003 in eight departments of south-western France. In total, 481 free-ranging individuals of five mustelid species (including the European mink) were tested. Sympatric mustelids can serve as sentinels to determine the presence of antibodies to viruses in the study area that could potentially infect mink. Antibodies to Canine distemper virus (CDV) were detected in all species; 9% of 127 European mink, 20% of 210 polecats (Mustela putorius), 5% of 112 American mink (Mustela vison), 33% of 21 stone marten (Martes foina) and 5% of 20 pine marten (Martes martes). Antibody prevalence was significantly higher in stone marten and polecats, possibly because their ranges overlap more closely with that of domestic species than that of the other species tested. Antibodies to Canine adenovirus were detected in all species but the pine marten; antibody prevalence estimates ranging from 2% to 10%. Antibodies to canine parainfluenza virus were detected in 1% of European mink, 1% of American mink and 5% of tested polecats but were not detected in Martes species. Antibodies to Rabies virus (RV) were detected in three animals, possibly because of interspecies transmission of bat lyssaviruses as the sampling area is considered to be free of RV, or to a lack of test specificity, as antibody titers were low. The high antibody prevalence to potentially lethal CDV suggests that this pathogen could have significant effects on the free-ranging populations and has implications for the conservation efforts for the endangered European mink.

Vaccination against highly pathogenic avian influenza H5N1 virus in zoos using an adjuvanted inactivated H5N2 vaccine.

Highly pathogenic avian influenza (HPAI) H5N1 virus infections have recently caused unprecedented morbidity and mortality in a wide range of avian species. European Commission directive 2005/744/EC allowed vaccination in zoos under strict conditions, while reducing confinement measures. Vaccination with a commercial H5N2 vaccine with vaccine doses adapted to mean body weight per species was safe, and proved immunogenic throughout the range of species tested, with some variations between and within taxonomic orders. After booster vaccination the overall homologous geometric mean titre (GMT) to the vaccine strain, measured in 334 birds, was 190 (95% CI: 152-236), and 80.5% of vaccinated birds developed a titre of >or=40. Titres to the HPAI H5N1 virus followed a similar trend, but were lower (GMT: 61 (95% CI: 49-76); 61%>or=40). The breadth of the immune response was further demonstrated by measuring antibody titres against prototype strains of four antigenic clades of currently circulating H5N1 viruses. These data indicate that vaccination should be regarded as a beneficial component of the preventive measures (including increased bio-security and monitoring) that can be undertaken in zoos to prevent an outbreak of and decrease environmental contamination by HPAI H5N1 virus, while alleviating confinement measures.

Highly pathogenic avian influenza (H7N7): vaccination of zoo birds and transmission to non-poultry species.

In 2003 an outbreak of highly pathogenic avian influenza virus (H7N7) struck poultry in The Netherlands. A European Commission directive made vaccination of valuable species in zoo collections possible under strict conditions. We determined pre- and post-vaccination antibody titres in 211 birds by haemagglutination inhibition test as a measure of vaccine efficacy. After booster vaccination, 81.5% of vaccinated birds developed a titre of > or =40, while overall geometric mean titre (GMT) was 190 (95% CI: 144-251). Birds of the orders Anseriformes, Galliformes and Phoenicopteriformes showed higher GMT, and larger percentages developed titres > or =40 than those of the other orders. Antibody response decreased with increasing mean body weight in birds > or =1.5 kg body weight. In the vicinity of the outbreak, H7N7 was detected by RT-PCR in wild species (mallards and mute swans) kept in captivity together with infected poultry, illustrating the potential threat of transmission from poultry into other avian species, and the importance of protecting valuable avian species by means of vaccination.