Dennis L. Schmitt

Potential Mechanisms for Cancer Resistance in Elephants and Comparative Cellular Response to DNA Damage in Humans

IMPORTANCE Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. OBJECTIVE To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). DESIGN, SETTING, AND PARTICIPANTS A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. EXPOSURES Ionizing radiation and doxorubicin. MAIN OUTCOMES AND MEASURES Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. RESULTS Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis at higher rates than human lymphocytes proportional to TP53 status (ionizing radiation exposure: patients with LFS, 2.71% [95% CI, 1.93%-3.48%] vs human controls, 7.17% [95% CI, 5.91%-8.44%] vs elephants, 14.64% [95% CI, 10.91%-18.37%]; P < .001; doxorubicin exposure: human controls, 8.10% [95% CI, 6.55%-9.66%] vs elephants, 24.77% [95% CI, 23.0%-26.53%]; P < .001). CONCLUSIONS AND RELEVANCE Compared with other mammalian species, elephants appeared to have a lower-than-expected rate of cancer, potentially related to multiple copies of TP53. Compared with human cells, elephant cells demonstrated increased apoptotic response following DNA damage. These findings, if replicated, could represent an evolutionary-based approach for understanding mechanisms related to cancer suppression.

Ultrasonography of the Urogenital Tract in Elephants (Loxodonta africana and Elephas maximus): An Important Tool for Assessing Male Reproductive Function

The success rate of captive elephant breeding programs worldwide is poor. Along with undiagnosed reproductive disorders in females and fatal diseases such as the newly discovered herpesvirus infection, male infertility now is considered a major contributing factor in the failure to maintain self-sustaining captive populations. To address questions related to male reproductive dysfunction, approximately 309 ultrasonographic assessments combined with semen collection were performed in captive (n = 10) and wild (n = 4) African (Loxodonta africana) and captive (n = 61) Asian (Elephas maximus) elephants. Bulls ranged from 4 to 50 years of age and were examined at 9 institutions in North America, 13 in Europe, 2 in Africa, and 7 in Asia. About half of the reproductive assessments were performed in protected contact situations with elephants handled in a restraint device, and half involved assessments of trained Asian bulls managed in free contact. Four wild African and two Asian elephant bulls were evaluated after receiving general anesthesia. Transrectal ultrasound was used to characterize the morphology and functionality of the entire urogenital tract, including the testes and accessory sex organs. Bulls were categorized on the basis of breeding status (breeders vs. non-breeders) and social history (i.e., type of interaction with conspecifics and keepers). Most of the bulls were non-breeders (designated Types I–V). Type I (n = 3 African, 6 Asian) and Type V (n = 1 Asian) were immature and castrate, respectively. On the basis of keeper evaluations, Type II bulls (n = 2, 4) were subordinate to older cows and keepers, whereas Type III bulls (n = 4, 28) were dominated by other bulls. Type IV (n = 1, 8) were older bulls of unknown history that exhibited numerous testicular pathologies resulting in poor semen quality. Non-breeding bulls included those that were exposed to females, but failed to breed, as well as those that had no opportunities to breed. Type VI individuals (n = 4, 14) were proven breeders. The percentage of observable reproductive tract pathology in adult males was remarkably low (14%), even in older bulls. However, apparent infertility of non-organic cause (i.e., not due to specific anatomical abnormalities) in these otherwise healthy bulls was high (32%). Semen quality varied markedly in ejaculates collected from the same bull, as well as from different bulls. In conclusion, although many of these bulls could serve as semen donors for natural mating or artificial insemination, the inconsistent production of good-quality ejaculates raises questions as to the reliability of these individuals to participate in breeding programs. The apparent inhibitory effect of suppressive social interactions on reproductive potential also needs to be investigated. Ultrasound examinations combined with semen collection should be conducted periodically to estimate the reproductive value of each bull and determine whether altered management strategies are needed to enhance captive breeding. Zoo Biol 19:333–345, 2000.

Manual collection and characterization of semen from Asian elephants (Elephas maximus)

The implications of collecting semen from elephants for use in artificial insemination programs are profound in the context of propagating captive elephants. Using a manual manipulation technique, semen was collected and characterized from five adult Asian elephants (Elephas maximus) and ejaculate fluid was obtained from one castrated elephant bull. The penis was stimulated to protrusion and erection by rectal massage of the pelvic portion of the urethra. During an ejaculatory response, massage was also directed onto the area of the ampulla of the ductus deferens. Sperm rich ejaculates were usually collected as a result of each ejaculatory contraction. Ejaculates were evaluated for spermatozoal concentration and pH (when possible) and sperm rich fractions combined for determination of total volume. Mean total volume of each collection was 27.5+/-4.4 ml. Mean concentration of the first and second ejaculatory responses from a collection was 2.05+/-0.17 x 10(9) and 1.34+/-0.19 x 10(9) sperm/ml, respectively. Measurement of seminal pH revealed no significant differences between the fractions. Mean pH of the first and second ejaculatory responses were 7.05+/-0.07 and 7.04+/-0.13. This method of collecting elephant sperm can be utilized for semen evaluation of bulls of unknown reproductive status in conjunction with other evaluation techniques (i.e. ultrasonographic, endocrinologic). It also has the potential for providing valuable genetic material for genome resource banks and for use with assisted reproductive techniques like artificial insemination.

CLINICAL AND PATHOLOGICAL FINDINGS OF A NEWLY RECOGNIZED DISEASE OF ELEPHANTS CAUSED BY ENDOTHELIOTROPIC HERPESVIRUSES

The unique clinical and pathological findings in nine Asian (Elephas maximus) and two African (Loxodonta africana) elephants from North American Zoos with a highly fatal disease caused by novel endotheliotropic herpesviruses are described. Identification of the viruses by molecular techniques and some epidemiological aspects of the disease were previously reported. Consensus primer polymerase chain reaction (PCR) combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and the second in African elephants. Disease onset was acute, with lethargy, edema of the head and thoracic limbs, oral ulceration and cyanosis of the tongue followed by death of most animals in 1 to 7 days. Pertinent laboratory findings in two of three clinically evaluated animals included lymphocytopenia and thrombocytopenia. Two affected young Asian elephants recovered after a 3 to 4 wk course of therapy with the anti-herpesvirus drug famciclovir. Necropsy findings in the fatal cases included pericardial effusion and extensive petechial hemorrhages in the heart and throughout the peritoneal cavity, hepatomegaly, cyanosis of the tongue, intestinal hemorrhage, and ulceration. Histologically, there were extensive microhemorrhages and edema throughout the myocardium and mild, subacute myocarditis. Similar hemorrhagic lesions with inflammation were evident in the tongue, liver, and large intestine. Lesions in these target organs were accompanied by amphophilic to basophilic intranuclear viral inclusion bodies in capillary endothelial cells. Transmission electron microscopy of the endothelial inclusion bodies revealed 80 to 92 nm diameter viral capsids consistent with herpesvirus morphology. The short course of the herpesvirus infections, with sudden deaths in all but the two surviving elephants, was ascribed to acute cardiac failure attributed to herpesvirus-induced capillary injury with extensive myocardial hemorrhage and edema.

Ultrasonography of the estrous cycle in female African elephants (Loxodonta africana)

The endocrinology of the elephant estrous cycle has been well characterized, but little emphasis has been placed on evaluating corresponding changes in the reproductive tract. Ultrasound was used to document changes in reproductive tract morphology throughout the estrous cycle in four cycling female African elephants. During a 7-month period, frequent ultrasound examinations (n = 190) during the luteal and non-luteal phase were compared with serum progesterone and luteinizing hormone (LH) concentrations during a 7-month period. Ultrasonographic images documented vaginal and cervical edema and changes in mucus consistency during the non-luteal phase. The cross-sectional diameter of the endometrium showed a dramatic increase during the non-luteal phase and followed cyclic changes. A different pattern of follicular development on the ovary was associated with the two LH surges. Follicle growth associated with the first, anovulatory LH surge was characterized by the formation of multiple small follicles, in contrast to the maturation of a single large follicle at the second, ovulatory LH (ovLH) surge. Ovulation and the subsequent formation of a corpus luteum (CL) were observed only after the ovLH surge. Ultrasound data in combination with endocrine assessments suggest that the African elephant is non-ovulatory, although multiple non-ovulatory luteal structures developed during the late nonluteal phase of each cycle. Both ovulatory CL and non-ovulatory luteal structures were present only through one cycle and regressed at the end of the luteal phase in conjunction with the drop in serum progesterone. We conclude that

KINETICS OF VIRAL LOADS AND GENOTYPIC ANALYSIS OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS-1 INFECTION IN CAPTIVE ASIAN ELEPHANTS (ELEPHAS MAXIMUS)

Abstract:  Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elephas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3–8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7–21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants.

Skewed birth sex ratio and premature mortality in elephants

Sex allocation theories predict equal offspring number of both sexes unless differential investment is required or some competition exists. Left undisturbed, elephants reproduce well and in approximately even numbers in the wild. We report an excess of males are born and substantial juvenile mortality occurs, perinatally, in captivity. Studbook data on captive births (CB, n=487) and premature deaths (PD, <5 years of age; n=164) in Asian and African elephants in Europe and North America were compared with data on Myanmar timber (Asian) elephants (CB, n=3070; PD, n=738). Growth in CB was found in three of the captive populations. A significant excess of male births occurred in European Asian elephants (ratio: 0.61, P=0.044) and in births following artificial insemination (0.83, P=0.003), and a numerical inclination in North American African elephants (0.6). While juvenile mortality in European African and Myanmar populations was 21-23%, it was almost double (40-45%) in all other captive populations. In zoo populations, 68-91% of PD were within 1 month of birth with stillbirth and infanticide being major causes. In Myanmar, 62% of juvenile deaths were at >6 months with maternal insufficient milk production, natural hazards and accidents being the main causes. European Asian and Myanmar elephants PD was biased towards males (0.71, P=0.024 and 0.56, P<0.001, respectively). The skewed birth sex ratio and high juvenile mortality hinder efforts to help captive populations become self-sustaining. Efforts should be invested to identify the mechanism behind these trends and seek solutions for them.

Obstetrics in elephants

Obstetrics, one of the oldest fields in veterinary medicine, is well described and practiced in domestic and exotic animals. However, when providing care during elephant birth or dystocia, veterinary intervention options differ greatly from any domestic species, and are far more limited due to the dimensions and specific anatomy of the elephant reproductive tract. In addition, aging of captive elephant populations and advanced age of primiparous females make active birth management increasingly important. Intrauterine infection, uterine inertia and urogenital tract pathologies are emerging as major causes for dystocia, often leading to foetal and dam death. This paper reviews the current knowledge on elephant birth and the factors associated with dystocia. It then summarises recommendations for birth and dystocia management. As Caesarean section, the most common ultima ratio in domestic animal obstetrics, is lethal and therefore not an option in the elephant, non-invasive medical treatment, induction of the Fergusson reflex or the conscious decision to leave a retained foetus until it is expelled voluntarily, are key elements in elephant obstetrics. Surgical strategies such as episiotomy and foetotomy are sometimes inevitable in order to try to save the life of the dam, however, these interventions result in chronic post-surgical complications or even fatal outcome. Limited reliable data on serum calcium concentrations, and pharmacokinetics and effect of exogenous oestrogen, oxytocin, and prostaglandins during birth provide the scope of future research, necessary to advance scientific knowledge on obstetrics in elephants.

USE OF FAMCICLOVIR FOR THE TREATMENT OF ENDOTHELIOTROPHIC HERPESVIRUS INFECTIONS IN ASIAN ELEPHANTS (ELEPHAS MAXIMUS)

Abstract Two juvenile Asian elephants (Elephas maximus) presented with an acute onset of facial edema and lethargy. Examination of the oral cavity of each animal revealed cyanosis of the tip and distal margins of the tongue suggestive of endothelial inclusion body disease (EIBD) of elephants. Whole-blood samples were obtained, and polymerase chain reaction tests confirmed the presence of elephant herpesvirus. The animals were administered famciclovir (Famvir, SmithKline Beecham Pharmaceuticals, Philadelphia, Pennsylvania 19101, USA), a potent human anti-herpesvirus drug, in the course of their disease, and recovery followed a treatment regime of 3–4 wk. These are the first known cases of elephants surviving EIBD.

Foetal age determination and development in elephants

Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown–rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85–90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation.