Daily prolactin pulse inhibits the corpus luteum during lactational quiescence in the marsupial, Macropus eugenii.

The corpus luteum (CL) of the tammar wallaby is inhibited by prolactin during lactation and seasonal quiescence. In seasonal quiescence a daily transient pulse of prolactin (PRL) of less than 2h duration is sufficient to maintain inhibition. We investigated whether the same inhibition applies in lactation and, if so, how. Our results show that inhibition of the CL during lactation is maintained by a transient pulse of prolactin once a day. They also show that the minimum time without a PRL pulse for the CL to escape inhibition is more than 48 h and less than 72 h. Nevertheless, some animals had a longer refractory period than 72 h, which was reflected in a longer interval to the progesterone peak and birth. These results support the previous conclusion that PRL exercises its effect on a rate-limiting step in progesterone synthesis and secretion rate from the CL, which precedes any increase in its mass. Therefore, we conclude that the role of PRL is to act as a luteostatic agent, an effect that is in marked contrast to its luteotrophic effect in many eutherian species, including rodents.

A review of complementary mechanisms which protect the developing marsupial pouch young.

Marsupials are born without a functioning adaptive immune system, into a non-sterile environment where they continue to develop. This review examines the extent of exposure of pouch young to microorganisms and describes the protective mechanisms that are complementary to adaptive immunity in the developing young. Complementary protective mechanisms include the role of the innate immune system and maternal protection strategies, such as immune compounds in milk, prenatal transfer of immunoglobulins, antimicrobial compounds secreted in the pouch, and chemical or mechanical cleaning of the pouch and pouch young.

Physical mapping of innate immune genes, mucins and lysozymes, and other non-mucin proteins in the tammar wallaby (Macropus eugenii).

Sequencing of the tammar wallaby (Macropus eugenii) genome has the potential to be an extremely valuable resource for investigating evolutionary and developmental aspects of the mammalian immune system. However, the tammar wallaby genome has only been sequenced to a 2-fold depth and consists of small contigs, leaving many sequence gaps, many putative orthologs unpredicted and the location of genes within the genome unknown. In the case of low sequenced genomes, physical maps of genes on chromosomes can help identify specific genes if they map to conserved regions. Genes corresponding to adaptive immunity have been mapped in the tammar wallaby; however, genes corresponding to the innate immune system have not been investigated. We predict 2 types of genes important to the innate immune system, mucins and lysozymes, in the tammar wallaby and compare the predicted peptide sequences and locations of the genes with the South American opossum (Monodelphis domestica) and human. We use fluorescence in situ hybridization to physically map the genes to tammar wallaby chromosomes, demonstrating the importance of identifying and mapping genes when genomes have low sequence coverage. As mucins and lysozymes play protective roles in young animals, we also propose that their immunological role in developing marsupials warrants further investigation.

Single-dose pharmacokinetics of oxytetracycline and penicillin G in tammar wallabies (Macropus eugenii).

The pharmacokinetics of oxytetracycline and penicillin G was investigated in tammar wallabies (Macropus eugenii). Groups of eight healthy tammar wallabies were administered i.v. oxytetracycline hydrochloride (40 mg/kg), i.m. long-acting-oxytetracycline (20 mg/kg), i.v. sodium penicillin G (30 mg/kg), or i.m. procaine/benzathine penicillin G (30 mg/kg). Plasma concentrations of oxytetracycline were determined using high-performance liquid chromatography. Pharmacokinetic parameters were comparable to those reported for eutherians of equivalent size and suggest that the practice of adjusting allometrically scaled doses to account for the lower metabolic rate of marsupials may not be valid. Long-acting oxytetracycline and penicillin G both demonstrated depot effects. However, the plasma concentrations achieved question the therapeutic efficacy of the long-acting preparations.

Comparative pathology of pulmonary hydatid cysts in macropods and sheep.

The development and appearance of hydatid cysts of Echinococcus granulosus in experimentally infected tammar wallabies (Macropus eugenii) and sheep during the period 9-17 months post-infection (mpi) were studied. Cysts of unknown age were also examined from mature, naturally infected sheep. The cysts grew more rapidly and became fertile within a shorter period in wallabies compared with sheep. Cysts from the wallabies were larger in absolute size and were larger relative to the size of the lungs. Microscopical examination revealed that wallaby hydatid cysts developed in small bronchioles. Hydatid cysts in the wallabies had a thicker germinal membrane, with more nuclei and a thicker laminated layer (LL), than hydatid cysts of similar age found in sheep. In contrast, the adventitial layer was thicker in the ovine cysts, comprising a hyalinized layer of degenerate collagen and necrotic cellular debris surrounded by a layer of granulation tissue that was largely absent from lesions in the wallabies. Multilocular cysts were present in sheep, but not in wallabies. The greater thickness of the germinal membrane in wallaby cysts suggests greater parasite activity, which may explain the more rapid growth rate in this host, whereas the thicker adventitial layer in sheep cysts may be restrictive to growth while simultaneously protecting the hydatid from the host immune response. These differences in the parasite-host relationship between macropods and sheep may reflect the relatively recent introduction of the parasite into Australia.

Efficacy of the EG95 hydatid vaccine in a macropodid host, the tammar wallaby.

In Australia, macropodids are common intermediate hosts for the cestode Echinococcus granulosus, and sylvatic transmission is maintained via wild dogs. The parasite causes mortality in a number of macropodid species and the sylvatic cycle provides a source of infection to domestic livestock and humans. We determined the efficacy of the hydatid vaccine, EG95 in the tammar wallaby, Macropus eugenii, challenging either 1 or 9 months post-vaccination. EG95 provides similar protection to that seen in sheep (96-100%). Control tammars were significantly more likely to become infected (odds ratio 29.44; CI 4.13, 209.97; P=0.001) and to develop more cysts (count ratio 26.69; CI 5.83, 122.19; P<0.001). The vaccination may be beneficial if administered pre-release in captive breeding programmes for endangered macropodids. Further work to develop oral delivery methods may enable vaccine administration of wild animals and thereby a reduction in sylvatic transmission.

Progesterone concentration in the marsupial Sminthopsis macroura: relationship with the conceptus, uterine glandular regeneration and body weight.

Close examination of hormonal profiles and uterine morphology in the marsupial reproductive cycle highlights significant differences between pregnant and non-pregnant cycles. In the polyovular dasyurid marsupial Sminthopsis macroura, we identified changes associated with gestation by comparing ovarian and plasma progesterone concentrations, uterine weights, uterine epithelial mitoses, body weights and gestation lengths between pregnant and non-pregnant luteal phases. The plasma progesterone profile of S. macroura was biphasic, peaking during unilaminar blastocyst expansion and on the day of implantation. Periods of rapid embryonic development were associated with increasing plasma progesterone concentrations and animal body weight. For the first time in a polyovular marsupial, we identified 1) a correlation between ovarian progesterone concentration and conceptus number during the luteal phase just prior to implantation (total ovarian progesterone), indicating a conceptus influence on progesterone concentration; 2) a pulse of uterine epithelial mitotic activity at the time of implantation and 3) increased mitotic activity in pregnant animals during unilaminar blastocyst formation compared with non-pregnant animals. Gestation length was reduced by up to 15%, due to the loss of, or reduction in, the four-cell arrest and more rapid definitive blastocyst expansion. This is the first time a conceptus influence on gestation length has been identified in a dasyurid. This study provides further evidence for the modification of the luteal phase by pregnancy in S. macroura.

Biological control of vertebrate pests using virally vectored immunocontraception.

Species-specific viruses are being genetically engineered to produce contraceptive biological controls for pest animals such as mice, rabbits and foxes. The virus vaccines are intended to trigger an autoimmune response in the target animals that interferes with their fertility in a process termed virally vectored immunocontraception. Laboratory experiments have shown that high levels of infertility can be induced in mice infected with recombinant murine cytomegalovirus and ectromelia virus expressing reproductive antigens as well as in rabbits using myxoma virus vectors. The strategies used to produce and deliver species-specific immunocontraceptive vaccines to free-living wildlife are presented in this review. Discussion includes coverage of the likely safety of the proposed vaccines as well as the implications of the approach for fertility control in other species.

Assessment of the immunocontraceptive effect of a zona pellucida 3 peptide antigen in wild mice.

Immunizing laboratory mice against a short peptide to mouse zona pellucida protein 3 (mZP3; amino acids 328-342) reduces fertility in some strains. This antigen was therefore tested to see if it is suitable for use in an immunocontraceptive vaccine to control wild mice. Mouse zona pellucida protein 3 peptide conjugated to a carrier protein (keyhole limpet hemocyanin) was considerably more immunogenic and effective in reducing fertility in wild mice when compared with inbred BALB/c mice. Fertility of the immunized wild mice was reduced by over 50% compared with controls, whereas BALB/c mice showed no reduction. Variation in the responses between individual animals to mZP3 peptide was observed and infertility correlated to the presence of cross-reacting antibodies to native zona pellucida in wild, but not BALB/c, mice.

Ecological basis for fertility control in the house mouse (Mus domesticus) using immunocontraceptive vaccines.

Laboratory studies confirm the potential for fertility control in the house mouse Mus domesticus using mouse cytomegalovirus (MCMV) as a vector for an immunocontraceptive vaccine. This article presents an overview of key results from research in Australia on enclosed and field populations of mice and the associated epidemiology of MCMV. The virus is geographically widespread in Australia. It also persists in low population densities of mice, although if population densities are low for at least a year, transmission of the virus is sporadic until a population threshold of approximately 40 mice ha(-1) is reached. The serological prevalence of MCMV was high early in the breeding season of four field populations. Enclosure studies confirm that MCMV has minimal impact on the survival and breeding performance of mice and that it can be transmitted to most adults within 10-12 weeks. Other enclosure studies indicate that about two-thirds of females would need to be sterilized to provide effective control of the rate of growth of mouse populations. If this level is not maintained for 20-25 weeks after the commencement of breeding, the mouse population can compensate through increased recruitment per breeding female. The findings from this series of descriptive and manipulative population studies of mice support the contention that MCMV would be a good carrier for an immunocontraceptive vaccine required to sustain female sterility levels at or above 65%.

Experimental infection of European red foxes (Vulpes vulpes) with canine herpesvirus.

We report on the pathogenicity of canine herpesvirus (CHV) for European red foxes. In the first experiment, we inoculated 10 adult foxes intravenously with a canine isolate of CHV. All foxes became infected and shed CHV in saliva and genital secretions for up to 14 days post-inoculation (p.i.) as evaluated by PCR and/or by virus isolation. All foxes developed clinical signs such as fever, lethargy and evidence of respiratory tract disease. Two foxes died on day 6 p.i., one on day 7 p.i., and one fox was euthanased on day 6 p.i. Tissues taken from the four dead foxes were positive for CHV by PCR. The remaining six foxes recovered after approximately 14 days p.i. Virus particles with morphology typical of herpesviruses were found by electron microscopy in the liver of an infected animal. All surviving foxes developed serum anti-CHV antibodies. In a second experiment, six foxes were dosed perorally with CHV and paired with six untreated controls. Neither the perorally dosed nor the in-contact control foxes developed clinical signs of disease. Infectious CHV was not isolated from any of the dosed or the in-contact foxes but all perorally-infected foxes and one of the in-contact foxes tested PCR-positive for CHV on several occasions p.i. All perorally-infected foxes, but none of the in-contact foxes, seroconverted. In summary, intravenous CHV inoculation caused a clinical disease in adult foxes much more severe than observed in experimentally-infected adult dogs. No clinical disease or virus spread was observed after peroral dosing although viral infection occurred as evidenced by seroconversion.

Hormonal profiles in the tammar wallaby, Macropus eugenii, following FSH/LH superovulation.

This study investigated the effect of superovulation with exogenous porcine FSH/LH on the normal hormonal milieu of the tammar wallaby (Macropus eugenii). During seasonal and lactational quiescence, groups of 6 females were treated with either multiple doses of porcine follicle-stimulating hormone (FSH) (8 x 6 mg i.m., 12 h apart) followed by a single subcutaneous injection of 4 mg porcine luteinizing hormone (LH) on Day 5 or saline. Blood samples were collected throughout each 10-day experimental period and each female was examined twice daily for signs of a recent copulation. On Day 9, females were killed and their reproductive tracts removed for examination and flushed for eggs. During both seasonal and lactational quiescence, treatment with porcine FSH/LH induced circulating concentrations of progesterone, porcine FSH and porcine LH that were within the normal range of the natural tammar oestrous cycle. However, higher plasma oestradiol concentrations (30-50 microg mL(-1)) than would be expected in a natural tammar preovulatory rise and the presence of 'highly stimulated' ovaries in several of the treated animals suggests that some degree of over-stimulation was occurring. During both seasonal sampling periods, behavioural oestrus was detected in treated tammars in the absence of a withdrawal of progesterone. This data suggests that plasma progesterone is not the critical factor inducing behavioural oestrus.

Infertility in mice induced by a recombinant ectromelia virus expressing mouse zona pellucida glycoprotein 3.

Population control has become a major problem in many wildlife species. Fertility control through immunocontraception has been proposed as a method for reducing population size. We have tested the concept that immunocontraception can be achieved with a recombinant ectromelia virus expressing an ovary-specific antigen, the mouse zona pellucida 3 (ZP3) glycoprotein. Female mice infected with the recombinant virus produced autoimmune antibodies against ZP3 and were infertile for 5-9 mo after infection. For almost half the infertile mice, immunity to ZP3 was associated with a disruption of ovarian follicular development and the depletion of mature follicles without observable oophoritis. Mice returned to fertility as the anti-ZP3 antibody level in the serum decreased. Reinfection of the mice with the recombinant virus boosted the anti-ZP3 response and restored infertility.

Examination of the immunocontraceptive potential of recombinant rabbit fertilin subunits in rabbit.

Recombinant fertilin subunits produced in a bacterial expression systems were used to test fertilin as an immunocontraceptive antigen in the European rabbit (Oryctolagus cuniculus). Wild female rabbits (n = 40) were immunized with either recombinant rabbit fertilin alpha or beta subunits by the s.c. or intra-Peyer's patch route. High titers of serum anti-fertilin polyclonal IgG antibodies were achieved in all rabbits after repeated boosts, with fertilin-specific IgG but not IgA antibodies detected in vaginal lavages of all animals. The serum IgG antibodies recognized polypeptides in detergent extracts of rabbit sperm with relative molecular masses of 48, 53, and 85 kDa on reducing SDS-PAGE gels and were shown to bind to the head region of methanol-fixed and live caudal rabbit sperm. Preincubation of rabbit sperm with these anti-fertilin IgG antibodies at concentrations of 400 micrograms/ml blocked sperm binding to zona-intact oocytes and inhibited fertilization in vitro by 60-80%. However, despite the levels of circulating and vaginal IgG antibodies achieved, only 4 immunized does failed to become pregnant out of 33 that ovulated. The remaining animals either showed no effect on fertility (n = 29) relative to control animals or failed to ovulate (n = 7). All control animals ovulated and were either fully fertile (n = 15) or were mated to infertile males (n = 4). In addition, proven-fertile male domestic rabbits (n = 3) were immunized s.c. and boosted three times with fertilin beta. Only one animal subsequently showed impaired fertility. These results show that in the rabbit, high levels of circulating sperm-reactive anti-fertilin antibodies and the presence of vaginal IgG does not ensure infertility.

Interstitial orchitis with impaired steroidogenesis and spermatogenesis in the testes of rabbits infected with an attenuated strain of myxoma virus.

The pathogenesis of infertility associated with acute viral orchitis was investigated in a rabbit model. Infection of postpubertal male European rabbits with an attenuated strain of myxoma virus caused systemic disease with viral replication to high titres in the testes. Infected rabbits developed an interstitial orchitis and epididymitis. This was associated with degeneration of the seminiferous epithelium, decreased serum testosterone concentrations and increased serum LH concentrations. Virus was localized within the interstitial cells. Clearance of infectious virus from the testis occurred between day 20 and day 30 after infection, although viral DNA could still be detected by polymerase chain reaction at 120 days. Viral clearance was associated with a return to normal serum testosterone and LH concentrations. Anti-sperm antibodies were present in the serum as early as 5 days after infection and declined during the recovery phase. Rabbits were infertile at 60 days but returned to normal fertility 60-90 days after infection.

A bait-delivered immunocontraceptive vaccine for the European red fox (Vulpes vulpes) by the year 2002?

An orally-delivered immunocontraceptive vaccine is being developed for the control of fox populations. A number of genes (PH-20, LDH-C4, ZP3) encoding gamete proteins have been cloned, produced in recombinant expression systems and used in fertility trials to test the efficacy of these antigens. As the immunocontraceptive vaccine will be delivered in a bait, there is a requirement for a greater understanding of the immune responses of the reproductive mucosa in canids, and the assessment of the best vaccine delivery system that will evoke a mucosal antibody response. Several vaccine delivery systems including microencapsulated antigens, and both vaccinia virus and bacterial vectors are being investigated. Oral administration of Salmonella typhimurium recombinants expressing different fox sperm antigens stimulates both systemic IgG responses to the antigen and a mucosal immune response within the female reproductive tract in the fox, indicating that salmonella may have potential with respect to the oral delivery of antigen. The enhancement of mucosal immune responses to orally-delivered vaccines is also being examined, research focussing on the possible use of fox-specific cytokines or the beta-subunit of cholera toxin in forming part of the vaccine construct.

Selection of antigens for use in a virus-vectored immunocontraceptive vaccine: PH-20 as a case study.

The European wild rabbit (Oryctolagus cuniculus) has become the major agricultural and environmental pest species in Australia. Current methods of rabbit control are lethal procedures which are increasingly questioned for their overall efficiency, applicability, specificity, cost and humaneness. New initiatives are required. One such initiative is virus-vectored immunocontraception. In this approach, the lagomorph-specific myxoma virus will be genetically engineered to include genes encoding components of rabbit gametes which can induce an immune response that causes infertility. Central to such a strategy is the ability to identify antigens capable of inducing an immunocontraceptive response. A strategy for identifying such antigens has been described previously. A case study of one sperm antigen, PH-20, is reported here. The issues involved in developing this antigen to the stage where it could be considered as a candidate for insertion into a recombinant myxoma virus with the ultimate goal of testing for immunocontraceptive efficacy are discussed. Techniques for inserting genes into myxoma virus have been described previously. The knowledge gained from research with this particular antigen are broadly applicable to other antigens used for both immunocontraceptive vaccines in general and, specifically, for virus-vectored immunocontraception.

Regulation of reproductive tract immunoglobulins by oestradiol-17beta in the European Red Fox.

The effect of the ovarian hormone, oestradiol-17beta, on reproductive tract immunity in the female fox was investigated. Reproductive tract antibody responses were induced by either Peyer's patch immunization with a recombinant fox sperm protein, or by oral immunization with live, attenuated Salmonella typhimurium. The effect of exogenous oestradiol-17beta or the stage of the oestrous cycle on reproductive tract immunity was assessed. The secretion of specific vaginal IgA, but not vaginal IgG, antibodies was reduced by exogenous treatment with oestradiol-17beta, while both specific vaginal IgA and vaginal IgG levels declined during the period of natural oestrus. It is concluded that oestradiol-17beta, and probably other reproductive hormones, are involved in the regulation of antibody-secretion in the fox reproductive tract, and that reproductive status is an important factor to consider in the design and application of vaccines which aim to induce immunity within the female reproductive tract.

Circulating levels of prolactin and progesterone in a wild population of red kangaroos (Macropus rufus) Marsupialia: Macropodidae.

Circulating progesterone and prolactin levels were measured in shot and live-caught wild red kangaroos using radioimmunoassays validated for the red kangaroo. The objective of the study was to correlate hormone profiles with reproductive status and determine if red kangaroos follow the general pattern elucidated for other macropodids. During Phase 2a lactation ( < 70 days) plasma progesterone concentrations were < 189 pg/ml (n = 41). This value increased to > 600 pg/ml (n = 32) during the transition to Phase 3 lactation (181 to 235 days) when the quiescent corpus luteum and embryo were reactivated. Progesterone concentrations then decreased to < 300 pg/ml (n = 29) during dual lactation when females were suckling a neonate and a young at foot. Concentrations of prolactin during Phase 2a were < 6 ng/ml (n = 17). Coincident with the period of reactivation of the diapausing blastocyst (181 to 235 days), plasma prolactin concentrations increased to 15 ng/ml (n = 32), then decreased and remained low through the subsequent stage of dual lactation. These results indicate that progesterone and prolactin profiles in wild red kangaroos follow patterns found previously in other macropodid species, the tammar and Bennett's wallabies.

Hormones of oestrus and ovulation and their manipulation in marsupials.

Oestrus and ovulation occur spontaneously in the majority of marsupials, with behavioural oestrus usually occurring 1-2 days before ovulation. The hormone changes that occur at this time have been described in the most detail for the monovular tammar wallaby Macropus eugenii. The respective roles of the Graafian follicle, corpus luteum and the pituitary in the events leading up to oestrus and ovulation in this species are also reviewed. Recently, various protocols have been developed for superovulation of marsupials, including Australian species, such as the brush-tailed possum, fat-tailed dunnart, brush-tailed bettong and tammar wallaby, and the American laboratory opossum, Monodelphis domestica. These protocols provide an opportunity for studying the regulation of ovarian activity and for the collection of larger quantities of material for the study of gamete maturation, in vitro fertilization and embryonic development.