The production cost of anthelmintic resistance in lambs.

The economic impact of anthelmintic resistance was investigated in lambs by comparing productivity parameters in groups of animals treated either with a highly effective anthelmintic, or an anthelmintic to which three species of resistant worms were known to be present. Ten farmlets, each stocked with 30 lambs, were rotationally grazed for 5 months, with monthly treatments of either albendazole, to which resistance existed, or a new combination product containing derquantel and abamectin (DQL-ABA) to which there was no resistance. Stock on five farmlets were treated with each anthelmintic and productivity measures, including liveweights, body condition and faecal soiling were assessed throughout. In addition, fleece weights and information on carcass weight and quality was collected at the end of the trial. Anthelmintic efficacy was measured at the last two treatment dates by faecal egg count reduction test with larval cultures. Albendazole demonstrated efficacies of 48.4% and 40.9% for Trichostrongylus spp. and Teladorsagia circumcincta respectively. By contrast, the DQL-ABA treatments were >99% effective against all genera. The difference in live-weight gain was 9 kg in favour of the DQL-ABA treatments. This translated into a 4.7 kg increase in carcass weight with a 10.4% increase in carcass value. Significant differences in body condition scores, faecal breech soiling and fleece weights were also recorded, all in favour of the DQL-ABA treatments. The time required for 50% of the animals to reach a target live-weight of 38 kg was significantly shorter (by 17 days) in those animals treated with DQL-ABA. The results show that the production cost of subclinical parasitism as a result of using an anthelmintic product which is less than fully effective due to resistance can greatly exceed the cost of routine testing of anthelmintic efficacy and the adoption of new anthelmintic classes. There is a strong case for many farmers to re-evaluate their position on some of these issues in order to optimise financial performance.

Field efficacy and safety of an oral formulation of the novel combination anthelmintic, derquantel-abamectin, in sheep in New Zealand.

AIM: To evaluate the efficacy and safety of the novel anthelmintic combination, derquantel-abamectin, against gastrointestinal nematode populations in sheep, under field-use conditions. METHODS: Controlled faecal egg count reduction tests (FECRT) were conducted in New Zealand in 14 trials, covering a range of geographic locations, farming enterprises, breeds, nematode populations, and anthelmintic-resistance profiles. Enrolled animals were naturally infected with mixed populations of gastrointestinal nematodes. All trials included a group treated with derquantel-abamectin, and a negative control group. Nine trials included additional groups each treated with a single- or dual-active oral reference anthelmintic, selected from albendazole, levamisole, albendazole-levamisole, ivermectin, abamectin and moxidectin. A total of 838 animals were enrolled across all trials, and were randomly allocated to treatment groups within blocks defined by faecal nematode egg counts (FEC) pretreatment. On Day 0 derquantel-abamectin was administered orally at 1 ml/5 kg bodyweight (2 mg/kg derquantel, 0.2 mg/ kg abamectin), and each reference anthelmintic was given at the recommended label dose. Faecal samples were collected on Day 14 (+/- 1 day), to determine the percentage reduction in mean FEC for each anthelmintic tested. Larval differentiation was also performed post-treatment, to estimate efficacy at the genus level. Animals were weighed on or before Day 0, and on Day 14 (+/- 1 day) in 13 trials. RESULTS: The efficacy of derquantel-abamectin against mixed strongyle populations was > or =99.2%, based on the percentage reduction in geometric mean FEC. Nematodirus sp. was present in six trials at a level sufficient for efficacy calculations to be conducted; in all cases, the efficacy of derquantel-abamectin was 100%. In those trials where the efficacy of at least one reference anthelmintic was <95% against strongyles and/or Nematodirus sp., derquantel-abamectin was 100% effective. In five trials, the mean gain in bodyweight was significantly greater in the derquantel- abamectin group than the negative controls. CONCLUSIONS AND CLINICAL RELEVANCE: When administered orally at 1 ml/5 kg bodyweight, derquantel-abamectin is highly effective for the treatment of gastrointestinal nematodes in sheep, including populations of strongyles and Nematodirus sp. with resistance to one or more single- or dual-active anthelmintics. Derquantel-abamectin presents sheep producers with a unique opportunity to introduce a new class of anthelmintic to their nematode control programmes, with the added benefits offered by a combination anthelmintic.

Santalbic acid from quandong kernels and oil fed to rats affects kidney and liver P450.

Kernels of the plant Santalum acuminatum (quandong) are eaten as Australian 'bush foods'. They are rich in oil and contain relatively large amounts of the acetylenic fatty acid, santalbic acid (trans-11-octadecen-9-ynoic acid), whose chemical structure is unlike that of normal dietary fatty acids. When rats were fed high fat diets in which oil from quandong kernels supplied 50% of dietary energy, the proportion of santalbic acid absorbed was more than 90%. Feeding quandong oil elevated not only total hepatic cytochrome P450 but also the cytochrome P450 4A subgroup of enzymes as shown by a specific immunoblotting technique. A purified methyl santalbate preparation isolated from quandong oil was fed to rats at 9% of dietary energy for 4 days and this also elevated cytochrome P450 4A in both kidney and liver microsomes in comparison with methyl esters from canola oil. Santalbic acid appears to be metabolized differently from the usual dietary fatty acids and the consumption of oil from quandong kernels may cause perturbations in normal fatty acid biochemistry.

The delta 5-3 beta-hydroxy steroid acyl transferase activities in tissues of the male rat and sheep.

Pregnenolone and dehydroepiandrosterone and their sulphate and fatty acid ester derivatives are concentrated in the mammalian brain, as compared to the peripheral organs. Although biological functions have been postulated for the free steroid and sulphate conjugates, the role of steroid fatty acid esters in the central nervous system (CNS) has yet to be established. A comparison of the esterifying capacities of brain and peripheral organs of the male rat and sheep is made here. The male rat brain was found to possess a higher esterifying capacity than all other tissues examined, with 5.32 +/- 2.46 pmol dehydroepiandrosterone fatty acid esters synthesized/mg protein/h and 1.89 +/- 0.42 pmol pregnenolone fatty acid esters synthesized/mg protein/h. Sheep adrenal and sheep liver homogenates were found to have higher esterifying capacities than sheep brain. Within the brain of both species. delta 5-3 beta-hydroxy steroid acyl transferase enzyme(s) were concentrated in the microsomal fraction. The addition of a lecithin:cholesterol acyl transferase inhibitor to incubation assays resulted in a loss in enzyme activity. This effect was more significant in sheep brain microsomes than rat brain microsomes. It is likely that lecithin:cholesterol acyl transferase is more active in the sheep brain than the rat brain. Results from this study indicate that the steroid acyl transferase enzymes that are active in the mammalian brain are substrate specific.

Efficacy of doramectin against naturally acquired adult and inhibited larval infections by some nematode parasites in cattle in New Zealand.

A study was conducted to determine the therapeutic efficacy of 1% doramectin injected subcutaneously at 200 microg/kg into cattle harbouring naturally acquired infections of inhibited Ostertagia ostertagi larvae. Sixteen yearling Friesian bulls, grazed without anthelmintic treatment throughout the autumn-winter, were selected on the basis of similar body weights and serum pepsinogen activities. After removal from pasture on day -23 they were weighed and randomly assigned to two treatment groups on the basis of this weight. On day 0, one group was given saline (1 ml/50 kg) while the second was treated with doramectin (200 microg/kg). Both treatments were given by subcutaneous injection. All stock were slaughtered 14-15 days after treatment. Moderate to high levels of adult O. ostertagi and Trichostrongylus axei and early and late 4th larval stages of O. ostertagi were recovered from saline-treated calves at necropsy. Doramectin was highly effective in eliminating all stages of O. ostertagi (99.9%; p<0.0001) and T. axei (100%; p<0.0001). No evidence of lesions were detected at the injection sites at necropsy. These results confirm that doramectin is an extremely effective broad-spectrum avermectin anthelmintic with efficacy against inhibited as well as maturing larval and adult forms of O. ostertagi.

Efficacy of doramectin against experimental infections by some nematode parasites in cattle in New Zealand.

A study was undertaken to determine the efficacy of the novel avermectin, doramectin, against experimental larval and adult infections of three species of nematode parasite important to cattle production in New Zealand. Eighteen worm-free dairy bull beef calves were randomly allocated on live weight to three similar treatment groups. Each calf was given 30,000 Ostertagia ostertagi, 20,000 Cooperia spp. and 10,000 Trichostrongylus axei infective larvae as a single dose. One group was treated with doramectin 6 days after infection while the remaining groups received saline or doramectin 27 days after infection. Given as a single subcutaneous injection behind the ear, doramectin at 200 microg/kg removed 99.9-100% of adult and larval stages of O. ostertagi, Cooperia spp. and T. axei when compared to infections established in untreated controls (p<0.001). No adverse reactions were observed following treatment in the doramectin-treated animals. No injection site lesions were found by palpation following treatment or by injection site examination at necropsy.

Development of a radioimmunoassay method for ethynylestradiol in plasma using a monoclonal antibody.

An ethynylestradiol (EE2) radioimmunoassay method (RIA) using a monoclonal antibody (MAB) reagent is described. 76 plasma and 67 buffer blanks spiked with known concentrations of EE2 were used as positive samples. The steroid was extracted using dichloromethane method with an extraction efficiency of 78 +/- 2.6%. The plasma extracts were not purified. Separation of the free from bound tritiated EE2 was by centrifugation with dextran-coated charcoal suspension in phosphate-gelatin buffer. Within 95% confidence limits, the sensitivity of the assay was 10 pg/ml in buffer samples, and 43 pg/ml in unpurified plasma samples. The accuracy and precision of the method were lower for the plasma samples than for the buffer solutions but within accepted limits (i.e., 80-120% for accuracy, and up to 20% for precision). The mean recovery accuracy (R) of the method for measuring 38 pg/ml EE2 in buffer was 101 +/- 0.9% (n = 67), while for 2.39 pg/ml to 18.0 pg/ml EE2 in plasma was 97 +/- 12% (n = 76). The buffer solutions and plasma samples containing 600 pg/ml EE2 had a mean R of 102% (n = 18) and 90% (n = 16), respectively. The average interassay variation (%CV) between buffer samples was 14 +/- 1.2%, and between plasma samples was 19 +/- 8%. Plasma samples with 18 pg/ml and 2.39 pg/ml EE2, together with the blank negative plasma samples showed high interassay variations (CV > 20%). Except for the above samples, however, the precision of the method in all sample groups was within accepted limits. The intra-assay variation of replicate assays of a single plasma sample containing 1.2 ng/ml EE2 and 4 ng/ml levonorgestrel was 13% (n = 7). In the presence of very high concentration of closely related levonorgestrel, the R of the method was 106%. The increase in average variability (%CV) between results for the plasma samples (CV 19 +/- 8%) over that of the spiked buffer solutions (CV 14 +/- 1.2%) suggest some possible interference by co-extracted materials from plasma, for example lipids, which interfered in the ligand-antibody reactions. Further investigation may prove that a pre-assay purification of the sample, e.g., HPLC, would eliminate this interference. Nevertheless, the immunoassay described above completely eliminates the problem of cross-reactivity caused by the natural estrogens in plasma with the conventional polyclonal antibodies. It possesses an acceptable intra- and interassay variation with very high specificity to EE2.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus brevis.

The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced. The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively. Sequence comparisons at the amino acid level to the B. subtilis degS-degU genes showed 74% and 84% similarity, respectively. On a multicopy vector the B. brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B. subtilis rec- strain as well as in a B. subtilis sacU(HY) strain.

Characterization, cloning and sequencing of a thermostable endo-(1,3-1,4) beta-glucanase-encoding gene from an alkalophilic Bacillus brevis.

A Bacillus brevis gene coding for an endo-(1,3-1,4)-beta-glucanase was cloned in Escherichia coli and sequenced. The open reading frame contains a sequence of 759 nucleotides encoding a polypeptide of 252 amino acid residues. The amino acid sequence of the beta-glucanase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-beta-glucanases. The optimum temperature and pH for enzyme activity were 65-70 degrees C and 8-10, respectively. When held at 75 degrees C for 1 h, 75% residual activity was measured. The molecular mass was estimated to be about 29 kDa on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and the enzyme was found to be resistant to SDS.

Evidence for multiple anthelmintic resistance in two nematode parasite genera on a Saanen goat dairy.

Multiple resistance was demonstrated within the experimental milking goat herd at the Ruakura Agricultural Centre. The development of this resistance was probably exacerbated by the atypical animal management and parasite control programmes under which the dairy was managed. Resistance by Teladorsagia (Ostertagia) spp. to members of the three broad-spectrum families of anthelmintic is reported for the first time. Worm counts in a controlled slaughter trial demonstrated multiple anthelmintic resistance in a mixed population of Teladorsagia (T. circumcincta and T. trifurcata) and Trichostrongylus spp. The efficacies of oxfendazole, morantel citrate and ivermectin in reducing the Teladorsagia population were 43.8%, 75.6% and 93.1%, respectively. Ivermectin removed more than 99% of the small intestinal Trichostrongylus spp. but oxfendazole and morantel were less effective, removing 37.9% and 87.7%, respectively. Only treatment with ivermectin totally eliminated faecal nematode egg counts 8 days after treatment. The lack of agreement between faecal nematode egg depressions and worm counts in lambs given ivermectin suggests that the drug may have affected the fecundity of the surviving nematode population or the assay lacked the sensitivity required. These data suggest that the sole use of faecal egg depression would be inappropriate for accurate assessment of anthelmintic effectiveness.

The in vitro biosynthesis of epitestosterone and testosterone from C19 steroid precursors in the testis of the lizard Tiliqua rugosa.

The metabolism of androgens in the testis of the lizard Tiliqua rugosa has been studied in vitro by incubating cellular homogenates with radiolabeled C19-steroid substrates. The identification 17 beta-oxidoreductase and 3 beta-hydroxysteroid dehydrogenase/isomerase activities. Aromatase, 5 alpha-reductase, and 17 alpha/beta-epimerase activities were not detected. The 17 alpha-oxidoreductase activity was temperature dependent (maximal at 32 degrees), while the 17 beta-oxidoreductase activity was temperature independent. Time yield and dual-label studies indicated that testosterone biosynthesis mainly involves the 4-ene pathway (via androstenedione), whereas the formation of epitestosterone uses both the 4-ene and 5-ene (via 5-androstene-3 beta, 17 alpha-diol) pathways. The function of alternative pathways in androgen biosynthesis is discussed, as is the role of temperature in the intratesticular regulation of androgen production.

A sensitive direct radioimmunoassay for assessing D-norgestrel levels in human plasma.

A direct radioimmunoassay was developed for assessing the levonorgestrel levels in human plasma without the need for heat, chromatographic or extraction pretreatment. D-norgestrel-3 (0-carboxymethyl)-oximino-BSA conjugate was used to raise antibodies having a low binding affinity towards other endogenous steroids. The rivanol-purified antiserum after dilution was used as the binding protein. The direct assay of levonorgestrel in human plasma was then compared to an extraction procedure. Lower intra-assay variability was shown by the direct assay when compared to the extraction method used at a sensitivity level of 0.15 nmol/L. The performance of the direct assay in quality control tests was more than favourable when compared with the extraction procedure. An examination of the effects of protein concentration on extraction efficiency was carried out together with an assessment of levonorgestrel levels in plasma in eight normal healthy women currently taking oral contraceptives and eight women who were not, at 0-24 h after the ingestion of 150 micrograms of D-NG and 30 micrograms of ethynyl oestradiol.

The comparative efficacy of four broad spectrum anthelmintics against some experimentally induced trichostrongylid infections in sheep and goats.

A controlled slaughter trial was undertaken to compare the efficacies of oxfendazole (5 mg/kg), morantel citrate (10 mg/kg), levamisole (8 mg/kg) and ivermectin (0.2 mg/kg) against experimentally induced infections of adult Haemonchus contortus, Ostertagia spp., Trichostrongylus colubriformis and Cooperiacurticei in sheep and goats. Ivermectin and oxfendazole achieved similar levels of efficacy in both hosts against all four worm genera as did levamisole and mortantel against H. contortus and C. curticei. Against Ostertagia spp. and T. colubriformis, however, the latter two drugs were less effective in goats than sheep. Neither the numbers of Ostertagia spp. removed from goats by levamisole (81% reduction) nor the numbers of T. colubriformis removed from goats by morantel (56% reduction) were statistically significant (P>0.05).

Identification of testosterone sulfate in the plasma of the male lizard Tiliqua rugosa.

Plasma (solvolyzed and unsolvolyzed) from the male lizard Tiliqua rugosa was analyzed using gas chromatography, high-performance liquid chromatography (HPLC), and mass spectroscopy. HPLC with electrochemical detection was also used to characterize ketosteroid conjugates. Testosterone was identified in the solvolyzed plasma extract, and a compound corresponding to testosterone sulfate was detected in unsolvolyzed extracts. Concentrations were approximately 500 nmol/liter in intact male plasma, less than 100 nmol/liter in castrates, and undetectable in female plasma. Steroid glucuronides appeared to be absent from plasma. Epitestosterone conjugates were not detected, although the free steroid is found in high concentrations in T. rugosa. The presence of testosterone sulfate in the blood of T. rugosa may indicate a possible role for sulfoconjugates in controlling the availability of biologically active androgens.

Annual cycles of plasma and testicular androgens in the lizard Tiliqua (Trachydosaurus) rugosa.

Androgen production during the annual reproductive cycle was investigated in the male scincid lizard Tiliqua (Trachydosaurus) rugosa. Concentrations of testosterone and epitestosterone were measured in plasma and testis (incubated and nonincubated) using radioimmunoassay. Morphological and histological techniques were used to determine the anatomical changes in the testis. Mating behavior was observed during spring, and sperm were most numerous in the testis at this time. Histological evidence and changes in testicular weight indicated that spermatogenesis begins in autumn and culminates in spring. Testicular regression occurred soon after mating. Plasma concentrations of both androgens were maximal during spring and minimal during summer. The maximal concentrations were approximately 500 and 150 nmol/liter (epitestosterone and testosterone, respectively). The minimal plasma concentrations were 250 and 15 nmol/liter, respectively. Plasma and testicular androgen cycles followed a pattern similar to that of the spermatogenetic cycle, suggesting the possible involvement of one or both of these androgens in the control of spermatogenesis and mating behavior in these lizards.

Effect of temperature on the seasonal production of testicular androgens, in vitro, by the lizard Tiliqua rugosa.

The effect of temperature on the seasonal production of testicular androgens, in vitro, was examined in the scincid lizard Tiliqua (Trachydosaurus) rugosa. Testicular tissue was incubated, in vitro, at various temperatures (18, 25, 32 and 37 degrees C). Endogenous androgens, testosterone and epitestosterone, were measured by radioimmunoassay. Epitestosterone production was maximal at 37 degrees C and minimal at 18 degrees C. There was no consistent effect of incubation temperature on testosterone production. Incubation temperature had no effect on the seasonal pattern of androgen production. The results suggest that temperature may play a part in the regulation of androgen biosynthesis in T. rugosa.

Changes in blood progesterone concentration during pregnancy in the lizard Tiliqua (Trachydosaurus) rugosa.

Progesterone concentrations during pregnancy were measured in the plasma of the viviparous lizard (scincid) Tiliqua (Trachydosaurus) rugosa, using a radioimmunoassay specific for progesterone. The length of gestation in this Australian lizard was 140-170 days. The average number of young was 2.8 and the mean neonatal weight 58 g (maternal body weight 500-700 g). The mean concentration of progesterone was greatest (7.2 nmol/l) during the second trimester of pregnancy; mean values during the first and third trimesters, and in non-pregnant females, were 1.8, 0.9 and 0.2 nmol/l respectively.