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.

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.

Maintenance of Dispersed Reproductive Cells from Male and Female Ascaris suum.

In vitro cultivation of tissues and cells provides an experimental methodology to define and manipulate physiological mechanisms that are not possible with in vivo techniques. Tissues from the germinative-growth zones of adult Ascaris suum gonads were excised and minced, and then enzymatically dispersed and transferred to an artificial, perienteric fluid-fetal calf-serum-medium complex. Cells were maintained in a viable state for 8 days, with medium replacement every 48 hours. During this period, morphological changes in the gonadal cells included decreased size, dedifferentiation, and degeneration. Two indices of metabolism, evolution of (1)CO from radiolabelled glucose and reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium), decreased by approximately 50% and 60%, respectively. The in vitro procedures developed provide the first opportunity to examine specific cellular functions of nematode reproductive tissues over an extended period of time.

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.

Consequences of dose-dependent immunosuppression by progesterone on parasitic worm burdens in lambs.

Eighteen female lambs with prior exposure to Haemonchus contortus infections were ovariectomized and assigned to 1 of 3 replacement regimens: 0, 25, or 250 mg of progesterone/d delivered IM. After 3 weeks of hormonal treatment, all lambs were inoculated with 100,000 infective larvae of H contortus. After 8 weeks of hormonal treatment, a blastogenic assay was performed on blood lymphocyte populations, and the abomasum from each lamb was obtained for larval and adult worm recoveries of H contortus. Lambs of the 25 mg of progesterone group had significantly (P < 0.05) reduced blastogenic response to concanavalin A and greater adult and larval populations, compared with controls. Lambs of the 250 mg of progesterone group had worm burdens and lymphocyte blastogenesis values intermediate between those of the other treatment groups.

Selection for a strain of Haemonchus contortus that exhibits periparturient egg rise in sheep.

Periparturient egg rise (PPR) is a principal means for some nematode parasites to survive over winter and to provide transmission of infective larvae from ewes to lambs during the spring. Routine laboratory propagation techniques probably have selected for those characteristics that would not promote PPR in conventional laboratory strains of Haemonchus contortus. An established isolate of H. contortus (BPL) was the source of the initial inoculum to select for a strain that readily exhibits characteristics of PPR. The selection process included inoculation of helminth-free pregnant ewes during mid-gestation, collection of nematode eggs during lactation, and storage of infective larvae at 4 C for 4 mo. After 10 generations, the 2 strains, BPL and PPR from lambs and pregnant ewes were compared for reproductive, morphological, and population differences in lambs and pregnant ewes. After lambing, ewes inoculated with the PPR strain had significantly higher fecal egg concentrations. Lambs inoculated with the PPR strain had higher egg concentrations, higher total daily egg production, fewer adult worms, larger female worms, and higher fecundity. Repeated selection in the appropriate host, after prolonged storage of the inoculum, produced a PPR strain of H. contortus morphologically and reproductively distinct from the initial source isolate.

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.

Catecholamines during development of the parasitic nematode, Haemonchus contortus.

1. Catecholamines from infective larvae of Haemonchus contortus were extracted, separated on HPLC, and measured on an electrochemical detector. DOMA 18 ng/100,000 larvae: NE 6 ng: E 1 ng: DA 1 ng 2. Levels of norepinephrine decreased below detection level rapidly after gassing with CO2. 3. Levels of 3,4-dihydroxymandelic acid (DOMA), a metabolite of norepinephrine, increased when exsheathment took place in tap water rather than buffer.

Growth and development of Haemonchus contortus in jirds, Meriones unguiculatus.

Growth and development of Haemonchus contortus were examined in jirds and were compared to these processes in lambs. Number, sex, size, and stage of development were determined for worms recovered at necropsy at various times postinoculation (PI) from immunosuppressed jirds inoculated with approximately 1,000 exsheathed infective larvae (L3) of H. contortus. In addition, gastric tissue samples from jirds were examined histologically. Parallel studies were done in lambs inoculated with approximately 7,500 L3. Typically, 5-30% of the inoculum established and survived in jirds at reasonably stable numbers to day 14 PI. By day 21 PI, worm numbers in jirds decreased dramatically. Although the parasite was similar in size and development on day 4 PI in jirds and lambs, from day 7 PI on, worms were significantly smaller and less developed in jirds. On histological examination, the parasite was found only in the glandular portion of the stomach of jirds (anatomically similar to its predilection site in the abomasum of lambs), and histological changes were consistent for both host species. Although growth and development of H. contortus are slower and incomplete in jirds, the parasite establishes, grows, and develops (at anatomically comparable sites in both hosts) in this model. Thus, the model appears to provide a useful laboratory host to study H. contortus.

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.

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.

Size of inoculum dose regulates in part worm burdens, fecundity, and lengths in ovine Haemonchus contortus infections.

Density-dependent factors frequently have been shown to regulate population parameters of free-living and parasitic helminths. To test the effects of various infection levels of Haemonchus contortus on fecundity and worm size, lambs were inoculated with 3,000, 10,000, and 30,000 infective larvae. Daily eggs per gram (epg) and daily total fecal production per lamb were monitored continuously. Worms were collected from abomasa on 6, 15, 22, and 30 days postinfection (PI). Female worms were smaller on each day in the high-dose group when compared to female worms from the low-dose group; males in the high-dose group were smaller from days 15 through 30 PI. The high- and medium-dose groups had higher mortality rates on day 30 PI, and fecundity (eggs/female/day) was 78% lower. Daily epg and daily total eggs/lamb/day were lower in the high-dose group. Fecundity and worm size were correlated with the log-transformed dose level but not with adult worm number. Early parasite and/or host responses apparently exert long-term negative effects on growth and reproduction relative to the size of the establishing population of H. contortus.

Evaluation of Haemonchus contortus infections in sexually intact and ovariectomized ewes.

Three experiments were conducted to investigate experimentally the occurrence of periparturient nematode egg rise in ewes and the hormonal modulation of Haemonchus contortus infections. In the first experiment, fall-bred and winter-bred pregnant (n = 4 and 14, respectively) and nonpregnant (n = 5 and 29, respectively) ewes were treated with anthelmintic and were pastured together on fields that were contaminated with H contortus. Three weeks before lambing, all ewes were placed in concrete pens; fecal egg counts for the winter-bred group were obtained on alternate days. Pregnant and lactating ewes had significantly larger numbers (P less than 0.01) of H contortus eggs than did the nonpregnant controls 1 week before and after lambing. Lactating, fall-bred ewes had significantly (P less than 0.01) more adult worms in their abomasum through natural acquisition than the nonpregnant controls. In the second experiment, fall-bred and winter-bred, helminth-free, pregnant (n = 4 and 8, respectively) and nonpregnant (n = 3 and 15, respectively) ewes were inoculated on 5 alternate days, beginning 70 days after breeding with 20,000 infective H contortus larvae. The ewes were maintained on concrete pens throughout pregnancy. Fecal egg counts were significantly (P less than 0.05) greater in pregnant ewes, beginning 1 week before lambing until 1 week after lambing. Abomasums of lactating ewes from both lambing seasons yielded significantly (P less than 0.01) more adult worms at necropsy than nonpregnant ewes. In the third experiment, ewes were ovariectomized (n = 15) or sham-operated (n = 9); half of the control ewes were bred.(ABSTRACT TRUNCATED AT 250 WORDS)

Unilateral orchidectomy of ram lambs: acute, chronic and hCG-stimulated androgen levels.

1. The objectives of this study were to determine whether compensation of androgen secretion occurred acutely, chronically or after hCG-stimulation in unilaterally orchidectomized (ULO) rams. 2. Testosterone (T) concentrations were not significantly different (P greater than 0.10) between ULO and sham-operated ram lambs during the period immediately following ULO. 3. Chronically, testosterone concentrations were not significantly different (P greater than 0.10) between ULO and sham-operated ram lambs. 4. After hCG injection, the testosterone response of chronic ULO ram lambs was approx. half the response of the sham-operated ram lambs. 5. These data indicate that a rapid and sustained compensatory response of basal secretion of testosterone but not hCG-inducible testosterone secretion occurred in the ULO'd ram lambs.

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.

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.

Metabolism of exogenous androgens in rams and wethers: differential clearance rates.

The effect of the presence of the testes on the clearance rates of 5 and 50 mg of testosterone and androstenedione was examined in 8-month old rams and wethers. Castration did not affect (P greater than 0.10) the clearance of androstenedione or testosterone at the 5 mg dosage. Castration had an effect (P less than 0.05) on the clearance of 50 mg of androstenedione and testosterone. These data indicate that reproductive status has a differential effect on the metabolism of exogenously administered steroids.

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.

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.

Longitudinal study of the surge of gonadotropins induced by exogenous hormones in prepuberal gilts.

The transition at puberty to adult hormonal patterns has been hypothesized to involve a shift in the sensitivity of the hypothalamic-hypophysial axis to hormonal stimulation. Prepuberal gilts were treated with GnRH or estradiol-benzoate (EB) to compare responsiveness of the pituitary at 40, 80, 120, and 160 days of age. Mean BW at each was 15.6, 36.3, 56.4, and 70.7 kg, respectively. Gilts were injected with GnRH (625 ng/kg; iv) or EB (600 micrograms/kg; im) or with vehicles. Blood samples were collected at 10-min intervals for 3 h after injection of GnRH or vehicle or 4-h intervals for 96 h after injection with EB or vehicle. Plasma concentrations of LH and FSH were measured by RIA, and data were analyzed by a general linear model for split plot design. Pretreatment basal concentrations of LH and FSH decreased between 40 and 160 days of age from 1.7 to less than 0.7 ng/ml and from 29.9-4.1 ng/ml, respectively. GnRH-induced surges of LH became more synchronous relative to time of injection and decreased in maximum concentration with age. In the youngest gilts, multiple surges of LH were released after a single injection of GnRH. The magnitude of the initial release of LH decreased with age from 5.6-2.7 ng/ml. Gilts treated with EB had two or more surges of LH at all ages. However, the concentration of the surge was greatest and occurred synchronously 12 h earlier in the 160-day-old gilts than at other ages. Therefore, sensitivity to EB was enhanced both in response time and amount of LH released. Concentrations of FSH were highly variable among gilts, and treatment with GnRH or EB induced an adult-like release of gonadotropins only at 160 days of age. Maturation of the surge mechanism involved at least two distinct alterations for LH. First there was a reduction in the amount of LH released in response to GnRH after day 40. Second an increase in the precision (timing) and magnitude of the surge of LH in response to EB was seen as gilts approached puberty.