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Development of non-invasive diagnostics for endotheliotropic herpesvirus in free-ranging Asian elephants Smiley Evans, Tierra, Alison Jeffrey, Kirsten Gilardi, and Christine Molter.. UC Davis, 1 Jul 2018. Experiment. doi: 10.18258/11525
Elephant study populations: Healthy captive Asian elephants (n = 9) housed at the Houston Zoo will be used for the pilot study of EEHV detection in different biological sample types. Elephants at the Houston Zoo have been monitored closely in recent years for EEHV and have had a total of three lethal EEHV-HD cases (between 2000 and 2008) caused by EEHV1A, as well as subsequent nonlethal, mildly viremic infection episodes by EEHV1A, EEHV1B, EEHV4B, EEHV5A, and EEHV5B1-5. In addition, a trial will be conducted in the Bago Yomo area of Central Myanmar to determine the efficacy of EEHV detection using non-invasive samples in a field setting, where samples will be collected from one wild elephant herd (n = 23) and one semi-captive elephant herd (n = 20). All sample collections will be performed in accordance with recommended guidelines for research involving animals, and this sampling protocol has been approved by the University of California, Davis Institutional Animal Care and Use Committee, the Houston Zoo Investigative Review Committee and the Myanma Timber Enterprise.
Sample collection and processing: For the study at the Houston Zoo, whole blood, trunk wash, oral swab, fecal and chewed plant samples will be collected twice per week over the course of 5 weeks from each trained elephant. In addition, in the event that an active viremic calf is identified during the course of our study period, samples will be taken daily from that individual during the course of shedding. Sampling will be performed on the same day for each biological sample type, to compare EEHV viral loads detectable using each proposed sampling method. For whole blood collection, 2 ml of blood will be collected from an auricular vein into EDTA anti-coagulated tubes. Samples will be aliquoted into 500 ul aliquots and stored at -80 degrees C. until DNA processing. For trunk-wash collection, samples containing elephant nasal secretions will be collected by means of the standard recommended technique for collection of samples for Mycobacterium tuberculosis testing by the USDA. Briefly, 50 mL of sterile saline (0.9% NaCl) solution will be poured into the nares of each elephant, and the proboscis elevated for 20-30 seconds. The elephant will then be instructed to blow the instilled saline solution into a fresh plastic 1-gallon freezer bag. The resulting effluent will be transferred to 50-mL conical tubes and chilled on ice until processed. Trunk wash samples will be centrifuged at 1,500 x g for 10 minutes, the supernatant discarded, and the cell pellet stored at -80 degrees C. until DNA processing. For oral swab collection, a dacron swab will be rubbed along the palette during feeding for 3-5 seconds, swabs placed in viral transport media (ThermoFisher Scientific) and stored at -80 degrees C. For fecal sample collection, a whole fecal bolus (the last produced within a dung pile) will be collected and dissected as soon as possible after observing defecation. Boluses will be split in half and fecal matter collected from central and edge locations of one half. Four 4.5 gram fecal aliquots will be stored in 15 ml conical tubes under the following conditions: 1) placed in 3 ml viral transport media and stored at -80 degrees C; 2) placed in 3 ml RNAlater (Invitrogen) and stored at ambient temperature for 2 weeks followed by storage at -80 degrees C; 3) frozen immediately until processing for glucocorticoid extraction; and 4) dried and stored at ambient temperature for two weeks until glucocorticoid extraction6,7. For chewed plant oral samples, elephants will be given fibrous browse and allowed to chew and discard pieces naturally. Various browse types will be tried in order to determine which is logistically the best choice for recovering chewed plant pieces. The most chewed portions of the plants will be collected and cut using sterile scalpel blades. Two aliquots of chewed plant pieces will be collected and stored in 15 ml conical tubes under the following conditions: 1) chewed plants placed in 3 ml viral transport media and stored at -80 degrees C; and 2) chewed plants placed in 3 ml RNAlater and stored at ambient temperature for 2 weeks followed by storage at -80 degrees C.8.
Field Trial in Myanmar: We will conduct sampling of 20 semi-captive MTE elephants once per week over the course of 4 weeks in both dry and monsoon seasons (December/January 2018-19 and June/July 2019). Elephants selected for this study will be evenly divided between sex and age class. Substantial seasonal variation in mean monthly glucocorticoid concentrations have been documented in semi-captive populations of Myanmar elephants, suggesting that stress levels vary considerably between seasons of the year (peaking during the monsoon season)9and EEHV shedding is hypothesized to follow similar patterns. During each sampling event, non-invasive samples (based on the method optimized from the Houston zoo study*) for EEHV detection and a fecal sample for glucocorticoid detection will be collected as described above. In addition to our semi-captive elephant sampling, non-invasive samples will be collected from the local ranging elephant herd once per week over the course of 4 weeks during the dry season (n=23 elephants). For each sampling event, data on sample quality (i.e. perceived age of sample, environmental conditions that could affect sample quality such as rain and temperature) will be collected.
Management, demographic and behavioral data collection: For all captive and semi-captive elephants included in this study, information on the following will be collected for each elephant at each time point a biological sample is collected: age, sex, exercise schedule, feeding parameters, recent changes in herd hierarchy, recent illness, recent acts of aggression between elephants, recent injuries, enrichment diversity and reproductive history. For Myanmar elephants, data on working hours and type of work and recent elephant transfers will also be collected. The majority of this data is already recorded on a daily basis in detailed log books maintained by the Houston Zoo and the Myanma Timber Enterprise.
Diagnostics: DNA extraction - DNA from whole blood will be extracted using standard protocols available for the DNeasy Blood and Tissue kit (Qiagen Inc., Valencia CA, USA). DNA from cells in trunk washes and oral swabs will be extracted using a modified protocol for the DNeasy Blood and Tissue kit described by Hardman, et al.10. Chewed plant samples will be processed according to protocols described previously by Smiley Evans, et al.11and sample supernatant will then be extracted using the same protocol described for trunk washes. DNA from fecal samples will be extracted using standardized protocols for the QIAamp DNA stool mini kit (Qiagen Inc.; Valencia CA, USA).
Quantitative real-time PCR - 5 ul of each DNA preparation will be screened using three independent qPCR assays targeting the EEHV1 MDBP gene, EEHV3/4 Terminase gene, and the EEHV5 DNA polymerase gene2,3,12. Asian elephant tumor necrosis factor alpha (TNF) will be used as an internal control3. All assays have been developed, validated and standardized in the laboratory of Dr. Paul Ling, Baylor College of Medicine. A sub-set of samples per elephant, positive by qPCR in Myanmar will be sequenced and genotyped to determine potential transmission patterns within herds as part of a larger epidemiologic study3,5.
Glucocorticoid Enzyme Immunoassay -Dry feces will be gently separated from plant material, and hormones extracted from fecal material using a protocol validated for Asian elephants7,9. The corticosterone enzyme immunoassay has been validated for Asian elephants by demonstrating parallelism between serially diluted samples and the respective standard curve, 90% recovery of added standard hormone to pooled samples, and physiological glucocorticoid increases in response to known stressors7,9,13.
1 Long, S. Y., Latimer, E. M. & Hayward, G. S. Review of Elephant Endotheliotropic Herpesviruses and Acute Hemorrhagic Disease. ILAR journal / National Research Council, Institute of Laboratory Animal Resources 56, 283-296, doi:10.1093/ilar/ilv041 (2016).
2 Stanton, J. J. et al. Detection of pathogenic elephant endotheliotropic herpesvirus in routine trunk washes from healthy adult Asian elephants (Elephas maximus) by use of a real-time quantitative polymerase chain reaction assay. Am J Vet Res 71, 925-933, doi:10.2460/ajvr.71.8.925 (2010).
3 Stanton, J. J.et al. Kinetics of viral loads and genotypic analysis of elephant endotheliotropic herpesvirus-1 infection in captive Asian elephants (Elephas maximus). Journal of zoo and wildlife medicine 44, 42-54, doi:10.1638/1042-7260-44.1.42 (2013).
4 Atkins, L.et al. Elephant endotheliotropic herpesvirus 5, a newly recognized elephant herpesvirus associated with clinical and subclinical infections in captive Asian elephants (Elephas maximus). Journal of zoo and wildlife medicine 44, 136-143, doi:10.1638/1042-7260-44.1.136 (2013).
5 Zong, J. C.et al. Comparative genome analysis of four elephant endotheliotropic herpesviruses, EEHV3, EEHV4, EEHV5, and EEHV6, from cases of hemorrhagic disease or viremia. Journal of virology 88, 13547-13569, doi:10.1128/JVI.01675-14 (2014).
6 Lynsdale, C. L.et al. A standardised faecal collection protocol for intestinal helminth egg counts in Asian elephants, Elephas maximus. Int J Parasitol Parasites Wildl 4, 307-315, doi:10.1016/j.ijppaw.2015.06.001 (2015).
7 Watson, R.et al. Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species. Gen Comp Endocr 186, 16-24, doi:10.1016/j.ygcen.2013.02.001 (2013).
8 Evans, T. S.et al.Detection of viruses using discarded plants from wild mountain gorillas and golden monkeys. American journal of primatology, doi:10.1002/ajp.22576 (2016).
9 Mumby, H. S.et al.Stress and body condition are associated with climate and demography in Asian elephants. Conserv Physiol 3 (2015).
10 Hardman, K.et al. Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR. The Veterinary record 170, 205, doi:10.1136/vr.100270 (2012).
11 Smiley-Evans, T.et al. Detection of primate viruses in dropped forage food from free-ranging mountain gorillas and golden monkeys. American journal of primatology, doi:10.1002/ajp.22576 (2015).
12 Stanton, J. J., Nofs, S. A., Peng, R., Hayward, G. S. & Ling, P. D. Development and validation of quantitative real-time polymerase chain reaction assays to detect elephant endotheliotropic herpesviruses-2, 3, 4, 5, and 6. Journal of virological methods 186, 73-77, doi:10.1016/j.jviromet.2012.07.024 (2012).
13 Brown, J. L.et al. Successful artificial insemination of an Asian elephant at the National Zoological Park. Zoo biology 23, 45-63, doi:10.1002/zoo.10116 (2004).
14 Hernes, S. S.et al. Swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs. Eur J Clin Microbiol Infect Dis 30, 159-165, doi:10.1007/s10096-010-1064-2 (2011).
In the event, that non-invasive methods are determined to not be feasible sample types based on the Houston zoo study, we will continue with validating the effectiveness of the collection of oral swab samples as a detection method for EEHV with MTE semi-captive elephants. This method, while still requiring elephant contact, is substantially less invasive than the collection of blood or trunk washes and requires comparatively less training.
Data Analyses and Final Product: To determine the optimal non-invasive sample type, collection and storage method, viral load as well as sensitivity and specificity will be calculated for samples collected from elephants at the Houston Zoo. The EEHV viral load in each sample type will be based on differences in cycle threshold (CT) values for qPCR assays. A comparative analysis of viral load in different sample types will be performed using generalized estimating equation (GEE) models, which allows for repeated observations on individuals and clustering of individuals over time. Equivalency of detection rates in different biological sample types (i.e. presence or absence of viral DNA) will be assessed by McNemar test. Using EEHV detection in blood or trunk wash as the best available “gold standard”, sensitivity and specificity will also be calculated for each alternative non-invasive sample type and protocol. Based on these data, the optimal sample type, collection and storage procedure will be selected as our non-invasive protocol for field trials in Myanmar. Using Myanmar semi-captive and wild herds, additional field conditions will be evaluated for their impact on EEHV detection in non-invasive samples using GEE models, including weather, temperature, and time spent processing samples in the field. While a longitudinal study of potential environmental and management related influences on EEHV shedding and disease severity is beyond the scope of this pilot project, we will begin collecting and analyzing covariate data collected during our two 4-week sampling points to help guide future studies involving the large semi-captive and wild elephant populations in Myanmar. Similar GEE models will be used to evaluate covariate data collected, that may be associated with glucocorticoid levels and EEHV shedding. Our final product for this proposed research project will be a practical, non-invasive protocol for the detection of EEHV in captive and wild elephants that will be published in the Journal of Zoo and Wildlife Medicine and disseminated through the EEHV Asian Working Group for use in future studies in Asian elephant range countries.
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