H.J. Bertschinger

Immunocontraception of African elephants

Concerted efforts to nurture elephant populations have resulted in elephant overpopulation in several areas, which in turn has led to damaging levels of browsing. As an alternative to culling entire family groups in order to control this damage, we have developed an immunocontraceptive vaccine from pig zona pellucida which safely and successfully controls free-roaming African elephants.

Contraceptive potential of the porcine zona pellucida vaccine in the African elephant (loxodonta africana)

Immunocontraception has been successful in controlling free-roaming equids; however, what is the potential for the immunocontraceptive control of the African elephant (Loxodonta africana)? The porcine zona pellucida (pZP) glycoproteins share antigenic domains with the African elephant zona pellucida (elZP) glycoproteins, and anti-zona pellucida serum antibodies have been successfully stimulated. To determine the cross-reactivity of the pZP and elZP, immunocytochemistry was evaluated by light and electron microscopy. Specifically, the binding of polyclonal antibodies against total heat-solubilized-porcine zona pellucida to fixed elephant ovary sections was evaluated. The elZP of primary, secondary and tertiary follicles was recognized by the rabbit-anti-pZP serum, but there was no apparent recognition of the primordial follicles. The ability of anti-pZP antibodies to recognize the elZP demonstrates that there is molecular homology between the pZP and elZP glycoproteins. This homology makes the African elephant a candidate for pZP immunocontraception. Three captive elephants were vaccinated with 400 micrograms pZP with a synthetic trehalose dicorynomycolate (S-TDCM) adjuvant. The elephants received 2 boosters of 600 micrograms pZP at 4 wk and 10 m.o. after the primary vaccination. The vaccinated female elephants developed significant (P < 0.05) titers to pZP over prevaccination levels. These levels persisted for 12 to 14 m.o. after the third vaccination. This preliminary evidence shows that the female elephant can develop significant serum antibody levels to pZP. These levels of antibodies are comparable to those required in horses for successful immunocontraception. Thus, porcine zona pellucida immunocontraception might be used to control elephant populations.

Concentrations of faecal glucocorticoid metabolites in physically injured free-ranging African elephants Loxodonta africana

Abstract Free-ranging African elephants Loxodonta africana use their front feet frequently during the process of foraging and this could be the reason for the high prevalence of physical injuries to these parts of the body. Although the occurrence of severe lameness caused by foot lesions in adult elephants has already been investigated and the clinical and pathological findings have been reported, the effect of foot injuries on glucocorticoid levels as a potential physiological stress response has not been examined. Given the practical difficulties involved in monitoring unpredictable events in free-ranging animals, like the occurrence of foot injuries in elephants, it is not surprising that information regarding the endocrine correlates of physical injury is still limited for elephants. In our study we investigated the effects of foot injuries on concentrations of faecal glucocorticoid metabolites (GCM), body condition score (BCS) and reproductive behaviour in two GPS/radio-collared elephant bulls in the Kruger National Park, South Africa. We monitored the bulls aged 40+ (Bull 1) and 30+ (Bull 2) 2-3 times per week for 13 months starting in June 2007 and frequently collected faecal samples for non-invasive hormone monitoring. Faecal samples were lyophilised, extracted and assayed with an enzyme immunoassay which detects GCM with a 3α-hydroxy-11-oxo-structure. Both bulls acquired foot injuries (right-front), which caused temporary lameness, but the effect of injury on GCM concentration differed between bulls (P < 0.001). In Bull 1 the injury lasted ± 250 days and was associated with an up to four-fold increase in GCM concentrations (P < 0.001) and his BCS reduced from ‘good‘ to ‘very thin‘ by the end of the injury period. In Bull 2 the injury lasted 65 days and was associated with a smaller increase in GCM concentrations (P = 0.03) together with a reduced loss in condition when compared to Bull 1. Following recovery, the condition of both bulls improved progressively and faecal GCM returned to baseline concentrations. Collectively, the data clearly underlined the value of non-invasive hormone measurements as a tool to provide information on the level of stress experienced by elephants. Thus, monitoring GCM levels could help improve the assessment of an elephants state of health.

Controlled-release components of PZP contraceptive vaccine extend duration of infertility

Successful immunocontraception of wildlife relying on repeated access to individuals for boosters has highlighted the need to incorporate primer and booster immunisations into one injection. We have investigated use of controlled-release polymers (lactide–glycolide) in small pellets to provide delayed in vivo delivery of booster porcine zona pellucida (PZP) antigen and adjuvant. This report reviews pellet-making methodology, in vitro testing of controlled-release pellets and in vivo effects of controlled-release PZP vaccine. We assessed 3 different manufacturing approaches for producing reliable, cost-effective pellets: (1) polymer melting and extrusion; (2) solvent evaporation from polymer solution; and (3) punch and die polymer moulding. In vitro testing of release patterns of controlled-release formulations, towards development of a 3-year duration vaccine, provided estimates for in vivo use of pellet preparations. These in vitro studies demonstrated protein release delay up to 22 months using 100% l-lactide or polycaprolactone polymers. For in vivo tests, pellets (1-, 3-, and 12-month release delay) serving as boosters were administered intramuscularly with PZP/adjuvant liquid primer to wild horses (Equus caballus), white-tailed deer (Odocoileus virginanus) and African elephants (Loxodonta africana). Horse field studies assessed fertility via offspring counts and/or faecal-hormone pregnancy testing. Treatment decreased fertility 5.3–9.3-fold in Year 1 and 3.6-fold in Year 2. In preliminary testing in deer, offspring counts revealed treatment-associated fertility reduction of 7.1-fold Year 1 and 3.3-fold Year 2. In elephants, treatment elevated anti-PZP titres 4.5–6.9-fold from pretreatment (no fertility data).

Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Africa.

Blood specimens from wild dogs (n=301) were obtained from De Wildt Cheetah and Wildlife Centre (Pretoria) and five game reserves (4 in the North-West Province and 1 in Limpopo Province), South Africa. Specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. Positive results were obtained in 18 (6%) wild dogs. Sixteen specimens were found positive for Babesia rossi and two dogs were Hepatozoon sp. positive. It appears that these tick-borne pathogens are not widely distributed in wild dog populations.

The use of a GnRH vaccine to suppress mare ovarian activity in a large group of mares under field conditions

The aim of this study was to evaluate the effect of active immunisation against GnRH on ovarian activity and serum progesterone concentrations in a large group of mares (10 control and 55 experimental) under field conditions as a model for wildlife species such as zebra and African elephants. Within the experimental group, mares were subdivided into three age categories: Category 1 (4 years and younger, n = 26), Category 2 (4–10 years old, n = 18), and Category 3 (≥11 years old, n = 11). Experimental mares were vaccinated intramuscularly with 2 mL (400 μg) of the GnRH vaccine Improvac (Pfizer Animal Health, Sandton, South Africa). Control mares received the same amount of saline solution. The vaccinations were repeated 35 days later. The ovaries and reproductive tracts of each mare were examined by means of rectal palpation and ultrasonography on Days 0, 35 and 70. Blood was collected weekly for determination of serum progesterone concentration until Day 175. On Day 35 after primary vaccination all of the control mares and 14.5% of the experimental mares showed evidence of ovarian activity on the basis of clinical examination and serum progesterone concentration. On Day 70, all control mares and none of the experimental mares showed evidence of cyclic activity. No age-related effect within treatment groups was found. The serum progesterone concentration indicated that all experimental mares remained in anoestrus until Day 175. Five of the control mares fell pregnant between Days 35 and 70. The five non-pregnant control mares continued to cycle until the end of the observation period. Having achieved such promising results in this trial we now plan to test the GnRH vaccine in Burchell’s zebra mares and African elephant cows.

Endocrine correlates of musth and the impact of ecological and social factors in free-ranging African elephants (Loxodonta africana)

Sexual activity in mature male African elephants is predominantly associated with the occurrence of musth, a state or condition which refers to a set of physical, physiological and behavioral characteristics, including an elevation in androgen levels. Although musth appears to be energetically costly, the degree to which it is associated with changes in adrenal endocrine function (e.g., glucocorticoid output) is still unclear. To investigate the possible effect of musth on adrenocortical function, and the impact of socioecological changes on androgen and glucocorticoid levels, six adult African elephant bulls were followed for 13months in the Kruger National Park, South Africa, and observations and fecal sample collection for endocrine monitoring was carried out about twice weekly. Our data showed that the occurrence of musth was associated with reduced glucocorticoid output, suggesting that musth does not represent a physiological stress mediated by the hypothalamic-pituitary-adrenal axis. This confirms previous findings in captive-housed animals, providing evidence for a suppressive effect of the musth condition on adrenocortical activity. Furthermore, a seasonal effect on androgen and glucocorticoid levels was found, which appears to vary depending on the reproductive status of the animal. The results also indicate a relationship between the presence or absence of social partners and changes in testicular and adrenal endocrine activity. Finally, the data confirm previous findings in captive-housed elephants, that an elevation in androgen concentrations usually occurs before the onset of physical musth signs, and therefore support the idea that the change in androgen levels represents the initial stimulus for the musth condition.

The use of deslorelin implants for the long-term contraception of lionesses and tigers

Contraception is an essential tool for controlling reproduction in captive and free-ranging lions. This paper describes the treatment and contraception of 23 captive and 40 free-ranging lionesses (Panthera leo) and four captive tigers (Panthera tigris) in South Africa using 3 × 4.7 mg, 2 × 4.7 mg, 9.4 mg or 4.7 + 9.4 mg deslorelin implants. Thirty-one lionesses were treated more than once at 11- to 60-month intervals. In Brazil, two lionesses were treated with 9.4-mg implants and faecal progesterone and oestradiol concentrations were monitored for 920 days. All combinations of deslorelin showed the length of contraception to be around 30 months with one 3 × 4.7 mg treatment lasting 40 months in one captive lioness. The mean time taken to reconception was 30.1 months for the 3 × 4.7 mg combination. The faecal analyses of the lionesses in Brazil reflected quiescent ovarian activity for periods of 17 and 30 months, respectively, when small oestradiol peaks but no progesterone peaks started to appear. This confirmed the field observations in South Africa. No side effects occurred although several of the lionesses were treated repeatedly for up to 8 years. Deslorelin (Suprelorin) is a safe and effective means of controlling reproduction in captive or free-ranging populations of lions. Where contraception is to be maintained, the implementation of implants at 24-month intervals is recommended.

Reversibility of the effects of GnRH‐vaccination used to suppress reproductive function in mares

REASONS FOR PERFORMING STUDY Active immunisation against gonadotrophin-releasing hormone (GnRH) provides a reversible method for control of oestrous behaviour and fertility in mares. Previous reports failed to demonstrate the interval to resumption of cyclic ovarian activity after GnRH-vaccination. HYPOTHESIS Administration of the GnRH-vaccine Improvac in a large group of mares of various ages will result in effective, reliably reversible suppression of ovarian activity within a 2 year period. METHODS The mares, subdivided into 3 age categories, were vaccinated twice (with a 35 day interval) using 400 µg Improvac and monitored via blood samples until Day 720 after initial vaccination for serum progesterone concentration determination by radioimmune assay and anti-GnRH antibody titre by enzyme immunoassay. Samples were collected until individuals resumed cyclic ovarian activity. RESULTS All mares showed suppression of cyclic ovarian activity by clinical examination and serum progesterone concentration (SPC) ≤ 1 nmol/l by Day 70 and 92.2% resumed cyclic activity by SPC at Day 720 with a mean interval = 417.8 days (s.d. ± 23.9; range 232-488 days, median 344 days). A significant age effect (P = 0.028) on the interval, but not on GnRH-antibody titre response, was observed between the youngest (≤ 4 years) and oldest (≥ 11 years) categories. CONCLUSIONS Immunising adult mares of all ages with Improvac resulted in a reversible suppression of cyclic ovarian activity in most mares. An age effect, with the youngest mares showing a longer interval to reversibility, was observed.

Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus)

The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs.