Michael W. Fleming

Ascaris suum: Role of ecdysteroids in molting

Three studies were conducted to examine the function of ecdysteroids in the development of parasitic nematodes. Ecdysone and 20-hydroxyecdysone were extracted, separated chromatographically, and measured in the reproductive tracts of adult female Ascaris suum. Perienteric fluid and the body wall did not contain measurable levels of these steroids. Levels of 20-hydroxyecdysone were correlated with the third and fourth molts of larvae grown in vitro from the third stage. In a bioassay, addition of ecdysteroid extracted from the female reproductive tract or synthetic ecdysteroid increased the proportion of third-stage larvae that molted after 4 days in culture. This evidence supports the role of ecdysteroids in molting in A. suum, as well as suggesting a function in gametogenesis and embryogenesis.

Cortisol as an indicator of severity of parasitic infections of Haemonchus contortus in lambs (Ovis aries).

Two experiments were conducted with lambs to examine the effects of experimental infections of Haemonchus contortus on circulating cortisol levels. In both experiments, naive lambs were divided into three groups based on inoculum doses of 0 (control), 2000 (low dose) and 20,000 (high dose) infective larvae. In experiment 1, the lambs were sampled once daily from day 1 post-inoculation (p.i.) until day 39 p.i. Lambs from the high-dose group had significantly higher sustained levels of cortisol throughout the course of the infection than either the low-dose group or control lambs. In experiment 2, blood was sampled at 30-min intervals for 4 hr on day 21 p.i. The high-dose group also had highest cortisol levels. The consequences of chronic stress and the resultant elevated cortisol concentrations potentially could compromise animal health and animal well-being.

Effects of exogenous progesterone and/or prolactin on Haemonchus contortus infections in ovariectomized ewes.

Reproduction can alter the course of ovine nematodiasis; fecal nematode egg concentrations often increase near lambing and throughout lactation, a phenomenon referred to as the periparturient rise. To identify the host mechanisms that might link these disparate events, i.e. lactation and the fecundity of gastrointestinal trichostrongyles, ovariectomized ewes were injected daily with progesterone and/or prolactin, or saline. Progesterone treatment commenced 20 days before inoculation with Haemonchus contortus and prolactin was administered throughout the 30-day infection period. Ovariectomized ewes receiving prolactin during the experimental infection maintained higher daily fecal egg concentrations than the progesterone, progesterone/prolactin, or the control treatment groups. However, ovariectomized ewes that received 20 days of pre-inoculation exposure to progesterone, as well as prolactin during the infection, had greater numbers of nematodes that were larger than the other treatment groups. Thus, the sequential delivery of these hormones that are associated with the reproductive cycle in ewes produced some of the same results that occur during periparturient rise.

Ecdysteroids during embryonation of eggs of Ascaris suum

1. The optimal temperature for in vitro development of fertilized eggs of Ascaris suum was 24 degrees C. 2. Samples (2 X 10(7) eggs) were obtained from in vitro embryonating cultures every 3 days for 4 weeks; lipids were extracted, partially purified, fractionated with HPLC and analyzed for ecdysteroids by radioimmunoassay. 3. Free ecdysone and 20-hydroxyecdysone (20-HE) were at low levels (less than 20 pg) in freshly excised eggs and rose to maximal values on day 6 of embryonation. 4. Conjugated ecdysone and conjugated 20-HE rose to maximal values on day 9. 5. Both free and conjugated ecdysteroids were undetectable from days 15 to 27 of cultivation. 6. These profiles indicate that ecdysteroids might have a selective role in nematode embryonation and/or tanning of the egg shell.

Effects of plumbagin on development of the parasitic nematodes Haemonchus contortus and Ascaris suum.

1. Plumbagin (5-hydroxy,2-methyl-1,4-napthoquinone) inhibited the motility and survival of Haemonchus contortus first-stage larvae (L1) with an ED50 of 1 microgram/ml, but was less effective in preventing the development of H. contortus to infective third-stage larvae in a faecal slurry assay. 2. Of the structural analogs tested, plumbagin was the most potent in preventing development of L1 followed in decreasing order of potency by 1,4-naphthoquinone, 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone. 3. Plumbagin had a biphasic effect on development of the fourth-stage Ascaris suum larvae that caused an increase in growth at low concentrations but was lethal at higher doses. 4. Plumbagin and 1,2-napthoquinone partially inhibited embryonation of A. suum eggs.

Sex behavior and the sexually dimorphic hypothalamic nucleus in male zucker rats

To determine whether or not impaired male sex behavior in obese male Zucker rats is accompanied by any anatomical alterations in a hypothalamic area implicated in the control of sex behavior, 6 lean and 5 obese male Zucker rats were studied behaviorally and anatomically at 14 months of age. Obese males showed markedly decreased male sex behavior relative to lean males, in spite of serum levels of testosterone and testicular weights comparable to those of lean rats. Obese rats had significant decreases in brain weight and volumes of sexually dimorphic nuclei per g of brain, relative to lean rats; volumes per g brain of other structures (paraventricular and suprachiasmatic nuclei) were not different between groups. It is suggested that an incomplete expression of sexually dimorphic features of the preoptic area-anterior hypothalamus, due perhaps to an impaired process of perinatal brain androgenization, may contribute to decreased male sex behavior in adult obese rats.

Ecdysteroids during development in the ovine parasitic nematode, Haemonchus contortus

1. Samples of embryonating eggs, infective larvae, fourth-stage larvae, young adults, and male and female mature adults of Haemonchus contortus were collected for steroids analysis; lipids were extracted, partially purified, fractionated with HPLC, and analyzed for ecdysteroids by radioimmunoassay. 2. Free ecdysone and 20-hydroxyecdysone were detected in all samples; maximal concentrations occurred in the fourth-stage larvae and young adults. 3. Conjugated ecdysone and 20-hydroxyecdysone were detected in all samples but the infected larvae; maximal concentrations occurred in the fourth-stage larvae and young adults. 4. Patterns of ecdysteroid occurrence support regulatory roles in embryogenesis, cuticular deposition, and gonadogenesis. 5. Relative concentrations of ecdysteroids are comparable to those previously reported in eggs and adults of Ascaris suum.

Acute or chronic administration of prolactin alters ovine infections of Haemonchus contortus

Prolactin has been implicated as a modulator in the phenomenon of periparturient egg rise during ovine nematodiasis. In the first experiment, lambs (4 months of age) with patent infections (10,000 infective larvae) of Haemonchus contortus were injected daily (Days 28-39 post-inoculation (PI)) with prolactin (20 or 200 IU) or saline. Fecal egg concentrations and total daily egg production were monitored for each lamb. After 12 days, lambs injected with the lower dose of prolactin had the highest daily egg production. Fewer adult worms were collected from prolactin-treated lambs, and the female worms were larger than those from the saline-injected controls. Worm size and number were not correlated. In the second experiment, lambs were injected daily with ovine prolactin (25 IU) or saline. Five days after initiation of treatments, each lamb was inoculated with 3000 infective larvae of H. contortus. After 42 days of injections (Day 37 PI), prolactin-treated lambs had larger and fewer worms, with higher fecundity, than saline-treated controls. Increases in endogenous circulating prolactin during late pregnancy and lactation in ewes, therefore, might contribute to periparturient egg rise irrespective of the developmental stage of the parasite when the hormone exposure occurs.

Peripheral androgen levels in peripuberal rams infected with Fasciola hepatica

Three-month-old, Polled Dorset rams infected with Fasciola hepatica and uninfected control rams were monitored throughout the peripuberal phase for peripheral concentrations of testosterone and for body weight. No differences between groups occurred in these measurements from early spring to mid-autumn. However, the infected postpuberal rams demonstrated a reduced ability to remove exogenous testosterone from the circulation. Liver damage induced by moderate infections of flukes apparently impairs hepatic catabolism of this steroid but compensatory mechanisms appear to maintain circulating levels comparable to those of uninfected rams.

Experimental Inoculations with Ostertagia ostertagi or Exposure to Artificial Illumination Alter Peripheral Cortisol in Dairy Calves (Bos taurus)

A series of experiments were conducted on dairy calves (Bos taurus) to assess, by way of circulating cortisol, the impact of a parasitic infection as a systemic stressor. The first study was designed to assess the effects of chronic stress on dairy calves resulting from a large bolus inoculation of the nematode parasite, Ostertagia ostertagi. Peripheral cortisol concentrations and adrenal cortical competency to adrenocorticotropic hormone (ACTH) challenge were utilized as indicators of chronic stress for 5 weeks. Calves were cleared of nematodes by anthelminthic treatment after the third week of infection. Calves were challenged with ACTH on weeks 0 and 2, and blood samples were obtained at a 12 x 10-min bleeding schedule. Cortisol concentrations were significantly higher (P < 0.05) in the infected calves than in the uninfected calves. The maximal response level to the ACTH challenge was also higher while the calves were infected. Two additional experiments were conducted to investigate the effects of experimental procedures that became evident during Experiment 1. Firstly, calves that had previously been fitted with jugular cannulae were sampled from 3 hr predawn until 5 hr after dawn under red- or white-light incandescent illumination. Calves under red lights had lower initial cortisol concentrations but increased to the concentrations in calves under white lights, indicating a compounding effect of lighting with the procedures of blood-sample acquisition. Secondly, 12 calves were inoculated with 10,000, 100,000, or 200,000 third-stage, infective larvae of O. ostertagi. Blood samples were obtained similarly to the regimen in Experiment 1. Cortisol concentrations were elevated only in the 200,000-dose group during week 3, correlating with the period immediately after emergence of the young adult parasites from the gastric glands. Continuous emergence of these parasites might induce chronic hyperadrenocorticism and the concomitant negative consequences on metabolic and immunological processes.