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Three recombinant Taenia ovis antigens (To45, To16, To18) each induce protective immunity in lambs or ewes against infection with T. ovis metacestodes. The degree and duration of immunity were assessed in lambs born from vaccinated ewes. Treatment group sizes varied, typically not fewer than 5 animals per group. Ewes were immunised with one T. ovis recombinant protein prior to lambing and the degree and duration of passive immunity in their lambs was assessed by challenge infection up to 18 weeks. Lambs were fully protected up to 6 weeks of age but immunity waned from 6 to 12 weeks and there was no protection when lambs were challenged at 15 weeks. Immunisation of lambs with the homologous recombinant antigen was not effective when vaccinations were given when maternal antibody was high. Lambs were effectively immunised in the presence of passively protective antibody when vaccinated with an antigen that was different to that given to ewes. Vaccination of lambs with a combination of two proteins, To16 and To18, was more effective than giving these single antigens and gave a significant reduction of cyst numbers when lambs were challenged 12 months after immunisation. These results indicate that the use of combinations of T. ovis recombinant antigens could enable complete protection of lambs against infection, if a delivery system becomes available that will maintain antibody at protective levels for 12 months. Alternatively, a third injection given at 6 months may promote the anamnestic response to give long lasting protection.

Brushtail possums exhibit a distinct preovulatory pattern of prolactin (Prl) secretion suggesting that Prl is involved in normal reproductive function. In some mammals, Prl is essential for corpus luteum (CL) function and/or modulation of steroidal effects on hypothalamic–pituitary activity. The aim of this study was to test the effects of biologically active recombinant possum Prl (recPosPrl) on both pituitary gland and CL function in possums. To confirm biological activity, administration of recPosPrl-N2C1 (10 μg) resulted in an 18-fold stimulation (P < 0.05) of progesterone (P4) production by possum granulosa cells in vitro. Based on these findings, minipumps containing either recPosPrl-N2C1 (n = 10) or saline (n = 8) were inserted into lactating female possums. The expression levels of pituitary-derived PRL, LHB, FSHB and GNRHR and CL-derived LHR mRNA were quantified. Following a resumption of reproductive activity, no differences in ovulation incidence or plasma Prl concentrations were observed. Plasma Prl levels were less variable (P < 0.001) in Prl-treated possums, confirming a self-regulatory role for Prl in this species. There was a marked down-regulation (P < 0.001) of FSHB mRNA at the mid-luteal stage in Prl-treated possums, whereas mean PRL, LHB, GNRHR and LHR mRNA expression levels were not different between experimental groups. Plasma P4 concentrations were not different (P = 0.05) in Prl-treated possums, although tended to be higher in the peri-ovulatory and early-luteal phase. We conclude in the brushtail possum that Prl is self-regulated via a short-feedback loop common to all mammals studied and is able to modulate FSHB expression probably at the level of the hypothalamus and/or pituitary gland.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth in sheep, whereas only GDF9 is essential in mice suggesting that the roles of these oocyte-derived growth factors differ among species. At present, however, there is only limited information on the action of BMP15 and GDF9 in other species. Thus, the aim of this experiment was to determine the effect of neutralizing GDF9 and/or BMP15in vivoon ovarian follicular development and ovulation rate in cattle through active immunization using the mature regions of the proteins or peptides from the N-terminal area of mature regions. Immunization with the BMP15 peptide, with or without GDF9 peptide, significantly altered (increased or decreased) ovulation rate. In some animals, there were no functional corpora lutea (CL), whereas in others up to four CL were observed. From morphometric examination of the ovaries, immunization with GDF9 and/or BMP15 reduced the level of ovarian follicular development as assessed by a reduced proportion of the ovarian section occupied by antral follicles. In addition, immunization against GDF9 and/or BMP15 peptides reduced follicular size to

Woodlands sheep have a putative genetic mutation (FecX2(W)) that increases ovulation rate. At present, the identity of FecX2(W) is unknown. The trait does not appear to be due to the previously described mutations in bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), or bone morphogenetic protein receptor type 1B (BMPR1B) that affect ovulation rate in sheep. Potentially, FecX2(W) could be an unidentified genetic mutation in BMP15 or in the closely related GDF9, which interacts with BMP15 to control ovarian function. Alternatively, FecX2(W) may affect ovulation rate by changing the expression patterns in the molecular pathways activated by genes known to regulate ovulation rate. The objectives of these experiments were to sequence the complete coding region of the BMP15 and GDF9 genes, determine the patterns of expression of mRNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development, and characterize the follicular populations in ewes heterozygous for the Woodlands mutation and their wild-type contemporaries. No differences in the coding sequences of BMP15 or GDF9 genes were identified that were associated with enhanced ovulation rate. The expression patterns of GDF9 and BMPR2 mRNAs were not different between genotypes. However, expression of BMP15 mRNA was less in oocytes of FecX2(W) ewes in large preantral and antral follicles. Expression of ALK5 mRNA was significantly higher in the oocytes of FecX2(W) ewes, whereas expression of BMPR1B was decreased in both oocytes and granulosa cells of FecX2(W) ewes. FecX2(W) ewes also had increased numbers of antral follicles

The antral follicle count (AFC) in cattle is consistent throughout the estrous cycle of individual cows, and cows with a lower AFC have lower fertility. We assessed the AFC at random stages of the estrous cycle, examined the correlation between AFC classifications, and determined the relationship between the most rapid and practical laboratory-based AFC classification (AFC of follicles of ≥ 2 mm in diameter) and fertility measures in New Zealand lactating dairy cows. Cows detected in estrus (n = 202) or not (n = 239) during the first 4 weeks of the breeding season were subjected to ultrasonography and classified as having a high, medium, or low AFC at the time of scanning (on-site classification). Images from ultrasound scanning were recorded onto video for accurate follicle counting in an imaging laboratory. A strong association (P < 0.05) between the AFC of follicles with a diameter of 2 mm or greater and fertility was observed. Cows with a high AFC had a shorter (P < 0.05) interval from calving to conception by artificial insemination (AI; 82.4 ± 1.6 vs. 87.3 ± 1.2 days) and greater pregnancy rates (PRs; i.e., PR to the first AI [68.1% vs. 45.3%], 6-week PR [81.9% vs. 67.3%], and overall PR [91.3% vs. 79.7%]) than cows with a low AFC. The AFC was positively associated (P < 0.0001) with age. Progesterone concentrations during diestrus were greater (P < 0.05) in high-AFC cows (7.6 ± 0.3 ng/mL) than in low-AFC cows (6.5 ± 0.3 ng/mL), whether these were pregnant (7.7 ± 0.3 ng/mL) or not (6.3 ± 0.2 ng/mL). A rapid on-site scoring system determined that cows classified as having a high AFC had a shorter (P < 0.05) interval from calving to the first AI (76.5 ± 1.7 vs. 82.3 ± 1.9 days) and were more likely to show estrus (P < 0.01; 56.8% vs. 36.4%) and have a CL at the beginning of the breeding season (P < 0.01; 93.4% vs. 79.6%) than cows with a low on-site AFC. Collectively, we have confirmed an association between AFC2 and fertility, and these results support the hypothesis that cows with a greater number of antral follicles are more fertile than cows with a lesser number of follicles. Although the on-site classification was related to resumption of estrous cycles after calving, associations with other fertility measurements could not be observed, highlighting a need for further refinement of the on-site classification system for rapid phenotyping of the AFC.

The oocyte derived growth differentiation factor (GDF) 9 and bone morphogenetic protein 15 (BMP15; also known as GDF9b) are essential for normal follicular growth. However, little is known about expression of these factors during ovarian development. Therefore, we determined the ontogeny of expression of GDF9 and BMP15 mRNA in the developing ovary of the brushtail possum. Ovaries were collected from pouch young ( n =3–5 per group) around times of key developmental events namely: (1) morphological sexual differentiation (i.e. days 1–5 following birth), (2) after sexual differentiation (i.e. days 10–15), (3) before and during initiation of germ-cell meiosis (i.e. days 22–45), (4) shortly after initiation of follicular growth (i.e. days 78–85), (5) during preantral follicular growth (i.e. days 96–113) and (6) during antral follicular growth (i.e. days 155–190). Ovaries were also collected from three juvenile and four adult animals and gene expression was determined by in situ hybridization. The mRNAs encoding GDF9 and BMP15 were first observed in oocytes of newly-formed primordial follicles (i.e. days 78–85). Expression of both mRNAs was restricted to the oocyte and was present in follicles irrespective of whether they were non-growing primordial follicles or undergoing preantral or antral development. Thus, since the mRNAs encoding GDF9 and BMP15 were not observed until follicular formation, it is unlikely that these proteins have any role in early germ cell development. Nevertheless, the findings that the mRNAs encoding both proteins were observed in oocytes from the primordial stage of follicular formation suggest a possible role for these proteins in the maintenance of primordial follicles as well as a key role during follicular development. These results highlight important species differences in the ontogeny of expression of GDF9 and BMP15 between possums and other species such as the human, sheep or rat.

Ovarian development and function have been extensively studied in eutherian species, with stem cell factor and its receptor, c-kit, having been shown to play key roles at various stages of these processes. In contrast, relatively little is known regarding ovarian development in marsupials. The aims of this study were, first, to establish the timing of key events during germ cell maturation and follicular development and, second, to determine the timing and cellular localization of gene expression for c-kit in the ovaries of a marsupial, the brushtail possum (Trichosurus vulpecula). For this study, ovaries were collected from possums ranging in age from Day 1 after birth to adult. Using stereology, the number of germ cells was found to increase rapidly during the first 60-100 days of life. This was followed by a sharp decline in number, wherein almost 90% of germ cells had disappeared by Day 180. From histological examinations, the time of initiation of meiosis, follicular formation, and follicular growth were determined to occur on Days 35, 50, and 60, respectively. Using in situ hybridization, c-kit gene expression was localized to germ cells and somatic cells during the first 15 days of life; however, after Day 30 and into adult life, c-kit expression was exclusive to germ cells. Results from this study suggest that the pattern of ovarian development is similar in marsupials to eutherians, and that c-kit may play a key role in germ cell development at various stages throughout life.

Previous trials of an experimental Taenia ovis vaccine using the recombinant antigen GST--45W(B/X) established that it was possible to achieve90% protection against a single artificial challenge of T. ovis eggs. This trial was undertaken to assess vaccine efficacy against artificial challenge and natural infection acquired by lambs grazing contaminated pasture. Two hundred Romney lambs were vaccinated at 6 and 12 weeks of age. One hundred control lambs were not vaccinated but were allowed to run with the vaccinated mob. At 15 weeks of age, 10 controls and 18 vaccinated lambs were artificially challenged with 2000 T. ovis eggs. The remaining control and vaccinated lambs were allowed to graze contaminated pasture for 3 weeks and were then moved to clean pasture for 5 months. The artificially challenged lambs plus 24 of the field-infected lambs were slaughtered and the carcasses dissected to obtain cyst counts. The remaining field-infected lambs were slaughtered at a commercial processing plant and the carcasses examined by conventional meat inspection. The results showed that the vaccine provided a high level of protection against artificial challenge (92%) and natural infection (98%) when assessed by carcass dissection. The data from commercial meat inspection showed that vaccination provided 89% efficacy against downgrading or condemnation compared to non-vaccinated control lambs. The average difference in carcass values between vaccinated and non-vaccinated groups was 4.36 dollars, representing a 35% loss in value due to T.ovis infection in non-vaccinated lambs.

In recent years the possibility of environmental oestrogens affecting the reproduction of vertebrates has become an issue of both public and scientific interest. Although the significance of such chemicals remains controversial there is clear evidence that, in some contexts, environmental oestrogens can influence the fertility of vertebrates. Highly endangered species represent a situation in which even modest reductions in the fertility of key individuals may have implications for the survival of the entire species. This paper reports the screening of both natural and supplementary foods of the kakapo (Strigops habroptilus), a critically endangered New Zealand nocturnal parrot, for oestrogenic activity using a recombinant yeast based bioassay. Low levels of oestrogenic activity were detected in one of the ‘chick-raising’ foods, but no oestrogenic activity was detected in the adult supplementary foods. The oestrogenicity of a range of phytochemicals possibly associated with the kakapo natural diet was also examined. Two such phytochemicals, podocarpic acid and its reduced derivative podocarpinol, showed weak oestro-genic activity (approximately 10 –6 and 10 –4 of the activity of 17-b -oestradiol, respectively).

Gonadotropin-releasing hormone receptors (GnRH-Rs) expressed in the pituitary of eutherian species of mammal are unique in lacking the cytoplasmic C-terminal tail characteristic of GnRH-Rs of nonmammalian vertebrates and other G protein-coupled receptors. To further investigate evolutionary relationships among vertebrate GnRH-Rs, a full-coding region cDNA of the pituitary GnRH-R was cloned from a metatherian marsupial mammal, the Australian brushtail possum (Trichosurus vulpecula). We have determined the pharmacological characteristics and internalization kinetics of this GnRH-R from an early evolved, metatherian species of mammal and compared it with the corresponding receptors in eutherian species of mammal and nonmammalian vertebrates. The predicted GnRH-R protein from the possum pituitary has high homology with the other mammalian GnRH-Rs (80% identity) and, in common with other mammals, lacks an intracellular C-terminal tail. The ligand selectivity of the possum GnRH-R transfected into COS-1 cells, assessed using inositol phosphate assays and radioreceptor binding assays, was similar to that of the other mammalian GnRH-Rs, and distinct from those of the nonmammalian GnRH-Rs. The pharmacological characteristics of the possum GnRH-R were similar to those of other mammalian GnRH-Rs, for a selection of agonists (including naturally occurring GnRH ligands and superagonists) and antagonists. Receptor-mediated internalization of GnRH agonist by the possum GnRH-R was slightly more rapid than that of the human GnRH-R, while the internalization kinetics of the chicken GnRH-R, in which a cytoplasmic C-terminal tail is present, was considerably more rapid. In terms of the evolution of the GnRH-R in vertebrates, the possum (a metatherian mammal) GnRH-R has a striking resemblance, in both structure and pharmacological characteristics, to GnRH-Rs in eutherian mammals, which are quite distinct from the nonmammalian vertebrate GnRH-Rs, and are unique among G protein-coupled receptors in lacking an intracellular C-terminal tail. The distinct structure of the pituitary GnRH-R in mammalian vertebrates is likely to have important functional consequences in the reproductive physiology of mammals.

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.

Follicle-stimulating hormone (FSH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands by using the following purification techniques: fractional ammonium sulfate precipitation, triazinyl-dye affinity chromatography, hydrophobic interaction chromatography, and gel filtration. A yield of 18 micrograms of FSH per gram of pituitary, with a recovery of 12%, was obtained from 1400 glands (20.3 g wet weight). The purified FSH activity per gram of protein was 1320 times more potent than the initial pituitary homogenate. Contamination with possum luteinizing hormone (LH) was < 0.02%. The amino acid composition of possum FSH was similar to that of ovine FSH. Amino-terminal sequencing for 11 cycles indicated that the alpha subunit has the same sequence as ovine FSH except for residue 7, where the possum FSH alpha subunit contains isoleucine compared to the ovine subunit which contains threonine. The beta subunit has two substitutions in the first 11 residues and does not contain the N terminal serine that is found in ovine FSH. Amino acid sequencing did not detect any contaminating proteins. Possum FSH bound possum and bovine testicular receptors with similar affinities. It was also able to stimulate in vitro cAMP production by Chinese hamster ovary cells which express recombinant FSH receptors. In the receptor assays and the bioassay possum FSH has about 21% of the potency of ovine FSH (USDA-oFSH-19-SIAFP-RP2). An RIA was developed for possum FSH using 125I-possum FSH and an antiserum raised against human FSH. The RIA has a sensitivity of 0.3 ng/ml, a 50% displacement of 2.7 ng/ml, and a cross reactivity of 0.05% against possum LH. Plasma FSH levels in male possums (10.4 +/- 2.4 ng/ml, n = 4) were higher (P < 0.05) than levels in females (1.0 +/- 0.1 ng/ml, n = 4). Five days after gonadectomizing these possums the plasma FSH levels increased (P < 0.05) to 27.1 +/- 0.2 ng/ml in the males and to 6.6 +/- 2.0 ng/ml in the females. In summary, we have purified and partially characterized possum FSH. We have also set up an RIA for the hormone and shown that males have higher levels than females and that plasma FSH increases after gonadectomy.

Immunoblotting and in vitro oncosphere-killing were used to identify a putative protective molecule in Echinococcus granulosus mature oncospheres. A range of sera from sheep that had been shown to be protected against E. granulosus, and from those that were not, were tested. The sera used were obtained from sheep hyperimmunized with E. granulosus oncospheres, or immunized with oncosphere non-denatured extract, with immature oncosphere extract or with denatured extracts of oncospheres. Results indicated the involvement of native antigens of 23, 25, 30, 34 and 40 kDa in the protective response to E. granulosus infection. The rapid appearance of antibodies to the 23, 25 kDa antigens, their association with early onset of protection and in vitro oncosphere lysis by affinity-purified antibodies obtained from these fractions, indicated that these antigens contained protective epitopes. Final confirmation was provided by immunization of sheep with fractions prepared by preparative SDS/PAGE, and challenge infection. Only the fraction containing the 23 and 25 kDa molecules was able to stimulate protection. Antisera against this pair of molecules should provide a useful probe for screening an E. granulosus oncosphere cDNA library to identify clones expressing protective molecules.

Oncosphere antigens of Taenia ovis were solubilised in sodium dodecyl sulphate and separated by electrophoresis in polyacrylamide gels (SDS-PAGE). Antigen-containing gel fractions cut from the region covering 18–12 kDa were shown to be highly immunogenic in sheep challenge experiments. Specific antisera against 2 candidate antigens at 18 and 16 kDa were used to screen a cDNA library prepared from T. ovis oncosphere mRNA. Recombinant proteins selected with antibody to the 16 and 18 kDa native antigens were expressed as GST fusion proteins. Vaccination trials using either of the 2 fusion proteins To16.17-GST and To18-GST, revealed that each was capable of inducing high levels of immunity in sheep against challenge infection with T. ovis eggs. Antibodies induced by vaccination with the recombinant antigens reacted specifically with their respective 18 or 16 kDa native oncosphere antigens. There was no apparent homology between the T. ovis cDNA coding for To18 and To16.17, or with another host-protective antigen, To45W, described previously. These additional host-protective antigens should prove a valuable adjunct to To45W and permit the development of effective vaccination strategies.

Sheep were fully protected against challenge infection following immunization with a homogenate of T. ovis oncospheres. Ultracentrifugation of sonicated oncospheres either alone or in the presence of a range of detergents did not reduce the immunogenicity of the extracts. Solubilization of oncosphere extracts in non-ionic detergents or sodium dodecyl sulphate (SDS) enabled analysis of host-protective antigens by isoelectric focusing (IEF) and electrophoresis in polyacrylamide gels (SDS-PAGE), respectively. Immunoblotting analysis of oncosphere antigens with immune sheep sera identified predominantly two groups of antigens with relative mobilities of 31–34 kDa and 47–52 kDa with a common isoelectric point of 5.8. The immunogenicity of these antigens was confirmed in vaccination trials using appropriate fractions cut from SDS-PAGE gels and agarose IEF gels. Affinity-purified antibodies prepared against the candidate antigens were used to select the corresponding recombinant DNA-derived polypeptides, one of which was subsequently found to be host-protective.

Echinococcus granulosus worm excretory/secretory antigens (WES) were used in ELISA for diagnosis of E. granulosus infection in dogs and compared with protoscolex somatic antigens (PSM). Sera from 224 dogs were tested. There was no correlation between ELISA absorbance values and E. granulosus worm burdens using either antigen. There was a significant linear relationship between absorbance values of sera tested in the ELISA using WES (W-ELISA) and the ELISA using PSM (P-ELISA). However, there was a small but significant difference between the absorbance values of the sera tested against the two antigens. Western blot analysis of WES using sera from E. granulosus-infected and uninfected dogs revealed antigenic components of relative molecular mass (Mr) larger than 94,000, Mr 94,000-68,000 and Mr 43,000-39,000 in worms, and these were specific for E. granulosus and not identified in PSM; these antigenic differences may be responsible for differences in reactivity in ELISA. The sensitivities of W-ELISA and P-ELISA were 80.8% and 75.6%, respectively. The specificities of W-ELISA and P-ELISA were 93.7% and 97.9%, respectively. The reduced specificity in W-ELISA was mainly attributable to increased background reactivity of sera from Taenia hydatigena-infected dogs. Despite the reduction in specificity, both ELISAs are valuable epidemiological tools to determine the prevalence of antibody to E. granulosus in dog populations and to monitor the success of hydatid control campaigns.

Fourteen ewes were orally dosed with 2000 E. granulosus eggs at 2 weeks of age, were mated at 19 months, and produced lambs when the cysts were 2 years old. One week after parturition, all 14 ewes had ‘Arc 5’ antibodies in their serum, as did 11 14 of their lambs. Fourteen uninfected ewes were immunized three times before parturition with E. granulosus eggs injected intramuscularly. Cysts grew at the first, or first and second site, but not the third, indicating that the ewes were immune prior to parturition. Most sera from these ewes and their lambs, and from 14 control ewes and their lambs, produced precipitin arcs with hydatid cyst fluid, but no ‘Arc 5’. All lambs were challenged with 500 eggs 2 weeks after birth. At necropsy, cyst numbers within groups ranged from 3 to > 200, but there was no significant difference between the three groups of lambs. The immunized ewes did not pass a protection to their lambs that was effective when the lambs were challenged. ‘Arc 5’ antibodies were induced by prolonged infection with cysts, and were not seen in the sera of ewes immunized with eggs, although the eggs developed into cysts at the injection sites. ‘Arc 5’ antibodies did not protect lambs against infection, and were not correlated with protection in ewes. Subcutaneous injection of oncospheres into four ewes from each group at the time of lamb challenge showed that the immunized ewes were immune to this method of challenge, but the infected and control ewes were not. Ewes and lambs of the immunized group, and to a lesser extent the 2-year-infected group, had in vitro oncosphere-killing capacity in their serum collected 1 week after parturition, while those from the control group did not. Killing capacity in the sera of two hyperimmunized lambs was predominantly in the IgG2 subclass. It would appear that the IgG1 subclass, which is the major immunoglobulin in ewe colostrum, either does not carry protective antibodies, or is not able to participate in killing oncospheres in vivo in young lambs.

An important goal in the intensive conservation management of New Zealand’s critically endangered nocturnal parrot, kakapo (Strigops habroptilus), is to increase the frequency of breeding attempts. Kakapo breeding does not occur annually but rather correlates with 3–5-year cycles in ‘mast’ seeding/fruiting of kakapo food plants, most notably podocarps such as rimu (Dacrydium cupressinum). Here we advance a hypothetical mechanism for the linking of kakapo breeding with such ‘mast’ seeding/fruiting. The essence of the hypothesis is that exposure to low levels of dietary phytochemicals may, in combination with hepatic gene ‘memory’, sensitise egg yolk protein genes, expressed in female kakapo livers, to oestrogens derived from developing ovarian follicles. Only in those years when the egg yolk protein genes have been sufficiently ‘pre-sensitised’ by dietary chemicals do kakapo ovarian follicles develop to ovulation and egg-laying occurs. While speculative, this hypothesis is both physiologically and evolutionarily plausible and suggests both future research directions and relatively simple interventions that may afford conservation workers some influence over kakapo breeding frequency.

Sheep (Ovis aries) are a highly diverse species, with more than 900 different breeds that vary significantly in their physiological characteristics, including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates than their wild-type counterparts. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile owing to arrested follicular development. In bovine and ovine ovaries, GDF9 was expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 was expressed exclusively in oocytes from the primary stage: no data for the ontogeny of BMP15 expression are currently available for cattle. In vitro, ovine growth differentiation factor 9 (oGDF9) has no effect on 3H-thymidine incorporation by either bovine or ovine granulosa cells, whereas ovine bone morphogenetic protein 15 (oBMP15) has modest (1.2- to 1.6-fold; P < 0.05) stimulatory effects. Ovine GDF9 or oBMP15 alone inhibited progesterone production by bovine granulosa cells, whereas in ovine cells only oGDF9 was inhibitory. The effects of oGDF9 and oBMP15 together were often cooperative and not always the same as those observed for each factor alone. Active immunisation of ewes with BMP15 and/or GDF9 peptides affected ovarian follicular development and ovulation rate. Depending on the GDF9 and/or BMP15 vaccine formulation, ovulation rate was either increased or suppressed. A primary and single booster immunisation of ewes with a BMP15 peptide in a water-based adjuvant has led to 19–40% increases in lambs born per ewe lambing. Collectively, the evidence suggests that oocyte signalling molecules have profound effects on reproduction in mammals, including rodents, humans and ruminants. Moreover, in vivo manipulation of these oocyte signalling molecules provides new opportunities for the management of the fertility of ruminants.

Reverse transcription-PCR has been used to obtain a cDNA sequence from the follicle-stimulating hormone (FSH) beta-subunit gene of the Australian brushtail possum (Trichosurus vulpecula). Comparisons of the possum FSHbeta-mRNA coding region nucleotide sequence with that of six eutherian mammal homologues reveals a mean percent identity of 77.3% and 76.8% at the nucleotide and predicted amino acid-sequence levels respectively. Furthermore, the predicted amino acid sequence of the possum FSHbeta mature protein shows evolutionary conservation of twelve cysteine residues and two potential N-linked glycosylation sites. The protein lacks the CAGY motif present in most reported glycoprotein beta-subunit sequences. The translation termination codon and consensus polyadenylation sequence overlap, a feature observed in other mammalian FSHbeta genes. Northern hybridization of total RNA from adult female possum pituitary revealed three hybridizing transcripts of approximately 2.8, 1.2 and 0.5 kb which may arise from utilizing alternative polyadenylation signals. In situ hybridization localized the FSHbeta transcripts to a sub-population of anterior pituitary cells interpreted as being gonadotropes. In summary the results indicate considerable evolutionary conservation of the structure of the FSH beta-subunit gene between the marsupial and eutherian mammalian lineages.

Extract Taenia ovis is a parasite which infects dogs (the definitive host) and sheep and goats (intermediate hosts). It has been the subject of a control programme in New Zealand based largely on the 6-weekly dosing of rural dogs with praziquantel. Since the patent for praziquantel has expired, various generic formulations have become available, including the formulation tested here. It is debatable whether such products should be tested for bioequivalence and biological activity against the target parasite or for bioequivalence only. In this instance, although the drug had been registered on the basis of blood levels of praziquantel achieved, it was felt that a biological test would be beneficial if the generic drug, mixed with other anthelmintics, was to be used on a wide scale to control Taenia ovis in New Zealand.

Harris A. , Heath D. D. , Lawrence S. B. and Shaw R. J. 1987. Ultrastructure of changes at the surface during the early development phases of Taenia ovis cysticerci in vitro . International Journal for Parasitology 17 : 903–910. Transmission and scanning electron microscopy and conventional, interference, and u.v. light microscopy were used to examine the epithelial membrane and underlying cytoplasm during the early development of Taenia ovis cysticerci in vitro . For 2 h after activation of the oncosphere, blebs of plasma membrane containing cytoplasm and secretory granules were released. Two h later the surface area of the plasma membrane had increased by the elaboration of microvilli. Microvilli increased in length and number for 48 h. Synthesis of granules was observed in two of the four lobes of the penetration glands for at least 4 h in vitro . Granules were present in the surface epithelium 4 h after activation. At 24 h granules remaining in the epithelium were electron lucid. In the presence of heat inactivated serum from sheep immunised against T. ovis oncospheres, an electron dense material was precipitated on and between microvilli within 24–48 h of culture. Material in this position reacted positively with fluorescein-labelled antisheep IgG. When metacestodes became motile after 4–5 days, microvilli were sloughed and replaced by a form of microtriche.

Extract Madam;– The Cysticercus ovis survey of McNab and Robertson,(1)conducted in New Zealand from 1967-70, concluded that meat inspection results seriously underestimated the incidence of C.ovis, and that the true incidence could be 5-10 times that based simply on routine inspection. This survey found that figures were 50% higher on average than the figures produced by the specific works on that day, indicating that an interested observer looking for only T.oviscysts, could find more cysts than the meat inspector. From 190 carcases that had no observable cysts at meat inspection, 50(26%) were found to contain cysts when finely sliced. The estimate of true incidence was therefore based on the combination of these two factors. No figures were available for the prevalence of cysts in sheep and lambs, and neither were the figures available to relate the number of cysts observed at meat inspection to the number of cysts actually present in the carcase. An estimate, based on figures supplied by Gemme...

A single oral treatment with mebendazole for the control of Taenia crassiceps larval infections in rats. International Journal for Parasitology 9 : 73–76. Rats infected with Taenia crassiceps larvae were treated with mebendazole. At a sublethal dose level of 50 mg/kg, a single large oral treatment proved to be markedly more effective in killing cysts than the same amount of drug divided into 10 daily smaller doses. Levamisole promoted a vigorous host cellular response to the intraperitoneal cysts, but when incorporated with mebendazole, it did not enhance the action of the latter drug.

Cysticercosis caused by larval tapeworms is a major public health problem and a cause of substantial economic losses in the farm-animal industries. Taenia ovis in sheep is a particularly important example. Immunity to reinfection with the larvae has a central role in regulating natural transmission of the parasites, and vaccination with antigens from the early larval oncosphere stage can induce complete protection against infection. As it is impractical to obtain enough oncospheres for a commercial vaccine against these tapeworms, an alternative approach is to use recombinant DNA methods to generate a cheap and plentiful supply of antigens. We report here the expression in Escherichia coli of complementary DNA encoding T. ovis antigens as fusion proteins with the Schistosoma japonicum glutathione S-transferase. Vaccination of sheep with these fusion proteins gave significant, although not complete, immunity against challenge infection with T. ovis eggs. Commercial development of a vaccine is being pursued.

When cultured in vitro in the presence of serum from a number of sheep infected with Echinococcus granulosus cysts, varying proportions of oncospheres died within 24 h. Of the survivors, some died during reorganization into cysts; others were able to develop normally but showed evidence of precipitates in the outer layers of the cyst. The lethal effects were removed by heating the serum to 56°C for 30 min and could be restored by the addition of freshly-collected normal sheep serum. In the presence of serum from sheep immunized against E. granulosus, most oncospheres were dead within 24 h, and few or none of the survivors were able to reorganize into cysts.

Extract Sir:–The widespread practice of feeding macerated raw carcasses and offal of cull sheep to fitch (Mustela putorius Juro) prompted some concern as to whether fitch could be a definitive host of Echinococcus granulosus, Taenia hydatigena or Taenia ovis. Because mustelids do not normally feed on sheep carcasses, the lack of records of sheep parasites in mustelids does not necessarily mean that the association could not occur.

The epithelium of artificially hatched and activated oncospheres of E. granulosus was studied ultrastructurally over the first 8 days of metacestode development in vitro. Within 4 h of activation, the epithelium was transformed from a thin cytoplasmic layer into a much wider layer packed with penetration gland granules and containing mitochondria and Golgi apparatus. Microvilli were extended from the outer plasma membrane and the basal lamina on the inner epithelial surface virtually disappeared. Microvilli increased in number and length over the first 24 h of development while granules in both the epithelium and penetration gland decreased in number. The granules appear to be involved in microvilli formation. After 3 days of development, the first lamination resolved ultrastructurally as shortened microvilli and some microtriches extending from the epithelium surrounded by an electron-dense microfibrillate material containing sloughed microvilli. By 6 days post-activation, no microvilli remained and only double-walled truncated microtriches extended from the epithelium. The microfibrillate material had become more electron-dense and was closer to the epithelium than at day 1. Within 8 days of metacestode development, a second lamination had developed. Both microfibrillate and particulate material of a greater electron density than the first lamination was added to the microthrix side of the first lamination.

stimulation of SOD activity by an as yet unknown mechanism. Because we found the difference in reticulocytosis between infected (2.7%) and uninfected mice (0.4%) to be small, and because the SOD activity in the infected mice showed no correlation with the reticulocyte level, the observed differences in SOD activity between the red blood cells from the two groups of mice should not arise from the reticulocytes alone. No variation in SOD level was observed in the young and mature erythrocytes in humans (Michelson, 1977. In Frontiers in physicochemical biology, B. Pullman (ed.). Academic Press, New York, p. 351). Finally, enzyme activity and protein-stained patterns of extracted SOD from red cells of uninfected and infected mice and SOD from isolated parasites appeared identical. The parasite may be able to use this enzyme for combating toxicity of oxygen or oxidant stress during the conversion of ingested hemoglobin (containing heme (Fe II)) to yield hemozoin pigment (containing hemin (Fe III)). Supertimulation of SOD activity by an as yet unnown mechanism. Becaus we found the diference in reticulocyt sis between inf cted .7%) and uninfected mice (0.4%) to be small, d because the SOD activity n he fect d oxide ca arise from the reduction of oxygen bound to hemoglobin during the oxidation of Fe (II) of heme to Fe (III) of hemin (Rotilio et al., 1976. In Superoxide and superoxide dismu ase, J. M. McCord and I. Fridovich (eds.). Academic Press, New York, pp. 239-244). Disulfiram can potentiate oxygen toxicity in rats by interacting with SOD (Forman et al., 1976, J. Pharmacol. Exp. Therapeut. 212: 452-455). Perhaps the antimalarial action of a bioconverted product of disulfiram, diethyldithiocarbamate, on P. falciparum is related to its inhibition of the parasite's SOD activity (Scheibel et al., 1979, Proc. Natl. Acad. Sci. USA 76: 5303-5307). We found that diethyldithiocarbamate at concentration as low as 0.2 mM inhibited the SOD extracted from the isolated P. berghei and also that from infected red blood cells from 20 mice. We thank UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (Project no. 780001) for financial support.

The excretory and secretory antigens from the evaginated scoleces of Taenia ovis were collected for 3 days in vitro, and used in an ELISA test to detect antibodies to T. ovis in the serum of dogs. When tested on sequentially collected sera, diagnostic ELISA values could be detected in many dogs 4 wk after infection, and remained for an average of a further 4 wk after worms were removed from dogs with an anthelmintic. Using an ELISA discriminant value that eliminated all false positives from 70 uninfected laboratory dog sera and from 57 uninfected farm dog sera, 54/62 true positives were found in sera from dogs infected with various numbers of T. ovis for various intervals. Sera from dogs infected with T. hydatigena gave 11/15 false positive reactions, whereas sera from 15 dogs infected with Echinococcus granulosus or 7 dogs infected with T. pisiformis were all negative. For T. ovis the test had a high repeatability, was not greatly influenced by the number of worms carried by the dog and higher titres were correlated with long-standing infections. Approximately 1,000 scoleces could be recovered from each experimentally infected sheep. Using the ELISA test with undiluted antigen and serum diluted 1:40, approximately 10 sera could be tested in duplicate with the excretions and secretions from each T. ovis scolex.

A high level of resistance to oral infection with Echinococcus granulosus eggs was stimulated in lambs by two or more subcutaneous injections of oncospheres given 14 days apart. The degree of resistance was significantly higher than that resulting from a single injection. Resistance was apparently stimulated by the activated oncosphere or a stage of cyst development prior to 14 days of age. Studies on oncospheres cultured in vitro in sera collected from animals during immunization or after oral challenge showed that most cysts were killed before 7 days of culture had elapsed. This confirmed the observation that resistance was stimulated by an early stage of cyst development. The in vitro test also showed that two injections of oncospheres resulted in a marked increase in the lethal effects of serum. These lethal effects decreased with time, providing circumstantial evidence that the high degree of resistance stimu- lated by two or more injections may only be transient.