In vitro screening of plant lectins and tropical plant extracts for anthelmintic properties.

Lectins are plant secondary metabolites (PSM) found in many forages and which may confer anthelmintic properties to gastrointestinal parasites through disrupting the development of parasitic larvae throughout its life cycle. In experiment 1, the ability of the plant lectins jacalin (JAC), concanavalin A (Con A), phytohemagglutinin E2L2 (PHA-E2L2), phytohemagglutinin L4 (PHA-L4), phytohemagglutinin E3L (PHA-E3L), kidney bean albumin (KBA), Robinia pseudoacacia agglutinin (RPA), Maackia amurensis lectin (MAA), Maclura pomifera agglutinin (MAA), Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA) and Galanthus nivalis agglutinin (GNA) to disrupt the feeding of the first stage larvae (L(1)) of the sheep gastro-intestinal nematodes (GIN) Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis was investigated using a larval feeding inhibition test (LFIT). Only PHA-E3L, WGA and Con A had a potent effect on disrupting larval feeding of all of the three species of GIN investigated. The lectin concentration required to inhibit feeding in 50% of L(1) (IC50) was 7.3±1.2, 8.3±1.4 and 4.3±1.7 µg/ml for PHA-E3L; 59.1±32.4, 58.7±11.9 and 8.1±7.0 µg/ml for Con A and 78.9±11.2, 69.4±8.1 and 28.0±14.1 µg/ml for WGA for T. circumcincta, H. contortus and T. colubriformis larvae, respectively (P=0.006). The addition of the lectin inhibitors fetuin, glucose/mannose or N-acetylglucosamine for PHA-E3L, Con A and WGA, respectively, caused an increase in the proportion of larvae that had fed at all concentrations for PHA-E3L only. In experiment 2, the effect of extracts from the tropical plants Azadiractha indica, Trichanthera gigantea, Morus alba, Gliricidia sepium and Leucaena leucocephala on the feeding behaviour of H. contortus L(1,) was examined. A. indica, T. gigantea and M. alba failed to inhibit 50% of larvae from feeding at concentrations up to 10mg plant extract per ml. In contrast, both G. sepium and L. leucocephala demonstrated a dose-dependent effect on larval feeding with respective IC50 estimates (mean±s.e.) of 0.015 mg/ml ±0.001 and 3.465 mg/ml ±0.144, effects which were partly reversed by the inclusion of either the tannin inhibitor polyethylene glycol or the lectin inhibitor Fetuin. These studies demonstrate that plant lectins can have an inhibitory effect on the feeding behaviour of first stage larvae of ovine GIN in vitro. Moreover they also provide novel evidence that lectins may contribute to the anthelmintic properties of some tropical forage plant extracts, such as G. sepium and L. leucocephala.

Effects of sainfoin (Onobrychis viciifolia) extract and monomers of condensed tannins on the association of abomasal nematode larvae with fundic explants.

Tannin-rich forages offer an alternative to anthelmintic chemicals to control gastrointestinal nematodes. However, the mode of action of such bioactive plants still needs to be assessed. Previous studies have shown that extracts of tannin-rich plants interfere with the first phase of host invasion, i.e., the exsheathment of infective larvae (L3s). In the current study, we examined the hypothesis that exposure to tannins could also affect the second phase of larval establishment, i.e., the tissue association/penetration of the exsheathed L3s into the digestive mucosae. An in vitro direct challenge technique using fundic explants was applied in this study. The main parasite model was Haemonchus contortus. The objectives were to verify: (i) whether a modification of the association/penetration of L3s with the mucosae occurred after contact with sainfoin extract; (ii) whether this is a dose-dependent phenomenon; (iii) whether tannins were responsible for these effects; (iv) whether these effects were dependent on the parasite species; and (v) how the biochemical structure of tannins might influence these effects. Following 3h contact with sainfoin extract at 1,200 microg/ml, the penetration of exsheathed L3s of H. contortus and Teladorsagia circumcincta into fundic explants was significantly reduced. Moreover, a dose-response relationship was found for H. contortus. For both nematodes, the changes were totally alleviated after addition of polyvinyl polypyrrolidone, an inhibitor of tannins, to the sainfoin extract, suggesting that tannins play a major role in the observed effects. Comparison of results obtained with different monomers of condensed tannins confirms a relationship between structure and activity, the prodelphinidin monomers and galloyl-derivatives being more effective than the procyanidin monomers. Combined with the delay or the inhibition of larval exsheathment previously shown, these effects could explain how tanniniferous plants reduce the establishment of infective larvae in small ruminants.

The effect of dietary polyunsaturated fatty acids (PUFA) on infection with the nematodes Ostertagia ostertagi and Cooperia oncophora in calves.

Diet-induced changes in the polyunsaturated fatty acid (PUFA) content of immune cells influences the immune phenotype that develops following infection. The aim of this study was to examine the effect of manipulating dietary PUFA supply on tissue fatty acids composition and immunity to a mixed infection with an abomasal and an intestinal nematode parasite in calves. Calves (n=24) were allocated into two treatment groups and fed 25 g/day of either fish oil (n-3 group) or a binary mixture of palm/rapeseed oil (normal group) as a supplement in milk replacer. Within each treatment group eight calves were infected with 2000 L3 Ostertagia ostertagi and Cooperia oncophora, three times per week for 8 weeks, the remaining calves were pair-fed uninfected controls. Faecal egg counts (FEC) were carried out twice weekly. At slaughter, the whole gut was removed intact for worm counts and tissue samples were taken for fatty acid analysis. Samples of abomasum, duodenum and mid-gut were also collected for immunohistological analysis. FEC were not significantly influenced by oil supplement but tended to remain higher in the palm/rapeseed oil-fed group (normal infected). The number of intestinal immature worms was significantly (p<0.05) higher in the n-3 group. Mucosal mast cell (MMC) and eosinophil numbers were significantly increased (p<0.05) by infection and were significantly lower (p<0.05) in the intestinal tissue of the fish oil supplemented and infected group (n-3 infected group). These results suggest that feeding an n-3 PUFA-rich supplement (fish oil) can influence cellular mediators of immunity to nematode infection. This is the first report of the establishment of patency and the subsequent development of immunity to a mixed infection with O. ostertagi and C. oncophora in calves undergoing early rumen development. The trend in the FEC, MMC and eosinophil numbers in the n-3 group suggests that decreasing the dietary n-6/n-3 PUFA ratio may be a worthwhile immunonutritional strategy for potentiating the immune response to nematode parasite infection in the calf.

Testing for direct anthelmintic effects of bioactive forages against Trichostrongylus colubriformis in grazing sheep.

The aim of the present study was to investigate potential direct anthelmintic effects of forages that contain plant secondary metabolites (PSM) towards the intestinal nematode Trichostrongylus colubriformis. For this purpose, we introduced an experimental design, which enabled us to investigate the direct anthelmintic effects of PSM-rich forages, without significant interference of possible indirect immunological effects of PSM. Sixty parasite naive sheep were infected with 8000 T. colubriformis L(3) on day 1 of the experiment. On day 28, sheep entered one of the experimental plots, which consisted of four PSM-rich forages and one control: Lotus pedunculatus (lotus), Hedysarium coronarium (sulla), Onobrychis viciifolia (sainfoin), Cichorium intybus (chicory) and Lolium perenne/Trifolium repens (grass/clover). On day 35 of the experiment, all sheep were re-infected with 8000 T. colubriformis L(3) and were killed on day 42. By day 42, all parasites of the primary infection would have been recovered as adults, whereas those of the secondary challenge would have only developed to the fourth stage larvae within a week (i.e. days 35-42). The first batch of larvae would enable us to investigate possible direct anthelmintic effects of PSM-rich forages against adult T. colubriformis, whereas the second one whether grazing on the PSM-rich forages could affect the establishment of the incoming infective larvae. Sheep grazing on lotus tended to have a lower FEC compared to sheep grazing on grass/clover (P = 0.06), whereas daily faecal output was higher in sheep grazing lotus compared to those grazing on the other forages (P < 0.05). As a consequence, daily egg output was similar in all sheep. Grazing on the PSM-rich forages for a period of 2 weeks did not affect the immature and adult parasite populations. Although the present experimental design enabled us to test the experimental hypothesis, the lack of evidence on a direct anthelmintic effect of PSM-rich forages can not be considered conclusive, as the composition of PSM-rich forages is variable within and across grazing seasons.

The consequences of short-term grazing of bioactive forages on established adult and incoming larvae populations of Teladorsagia circumcincta in lambs.

The objective of this study was to investigate the consequences of short-term grazing on bioactive forages on (i) the viability and fecundity of established adult Teladorsagia circumcincta population and (ii) the establishment and development of incoming T. circumcincta infective larvae. Forty-eight, parasite naive, 3-month old, grazing lambs were artificially infected with 8000 infective larvae of T. circumcincta on day 1 of the experiment. On day 21 p.i., lambs were allocated to one of three bioactive forage grazing treatments; chicory (Cichorium intybus), sulla (Hedysarum coronarium), lotus (Lotus pedunculatus), and the control grass/clover (Lolium perenne/Trifolium repens) forage. On day 28 of the experiment a second dose of 8000 T. circumcincta infective larvae was administered to the lambs to investigate the effects of forages on the ability of infective larvae to establish within the host. All animals were slaughtered for worm recovery on day 35, while liveweight gain, feacal egg counts (FEC) and total worm egg output were monitored regularly throughout the experiment. Although FEC or total egg output were similar among the groups, adult worm burdens at slaughter were significantly affected (P<0.05) by forage treatment during the 2 week grazing period. Lambs grazing chicory had the lowest adult worm burdens and significantly lower numbers of male worms compared to those grazing on grass/clover (P<0.01), while the lambs grazing on sulla or lotus had similar adult populations to grass/clover fed animals. The results from the worm recoveries of the second dose (immature worm burdens) were affected by physiologically and/or immunologically mediated mechanisms, which reduced larval establishment in all treatments. Nevertheless, immature worm burdens at slaughter were similar between chicory, sulla and grass/clover group, while the immature worm recoveries from the lotus group were significantly higher (P<0.05) compared to those from lambs grazing grass/clover. Overall, the results of the present study support the view that chicory can be a promising candidate species in pasture management practices to control T. circumcincta burdens.

The effects of condensed tannins supplementation of foods with different protein content on parasitism, food intake and performance of sheep infected with Trichostrongylus colubriformis.

The aims of the present study were to investigate (1), the potential anthelmintic properties and (2), the nutritional consequences of commercially available condensed tannins on parasitised sheep fed, ad libitum, either a high- or a low-protein food. For this purpose, forty-eight previously parasite-naïve sheep (n 12) were infected with 2000 Trichostrongylus colubriformis larvae/d for a 67-d experimental period. Two experimental foods were made: a low (L), formulated to be inadequate in meeting the requirements of growing sheep for metabolisable protein (MP), and based on wheat, citrus pulp, and oatfeed; a high (H), expected to be above the requirements of growing sheep for MP, based on similar ingredients but supplemented with protected soyabean meal. Two additional foods were made by adding 60 g Quebracho (a condensed tannins (CT) extract)/kg fresh matter to foods L and H (foods LQ and HQ respectively). This level of Quebracho supplementation has been previously shown to reduce the level of parasitism in restrictedly fed, parasitised sheep. The experiment was divided into two periods: period 1 (P1, day 1-38) and period 2 (P2, day 39-67), each one associated with different phases of an intestinal parasitic infection. Six sheep from each group were slaughtered at the end of P1, and the remaining sheep were slaughtered at the end of P2 (day 67). Although faecal egg counts (FEC; number of parasite eggs/g faeces) and total egg output were reduced in sheep offered the supplemented foods during P1 (P<0.05), worm burdens on day 38 were unaltered. Neither Quebracho supplementation nor food protein content during P2 affected FEC and worm burdens. Food intake and performance were higher in sheep offered food HQ compared with sheep offered food H (P<0.05); no differences were observed in sheep offered foods LQ and L throughout the experiment. The previously shown anthelmintic properties of CT were not observed following ad libitum intake of either low- or high-protein foods supplemented with Quebracho extract. Higher levels of CT supplementation may be required to reduce parasitism and consequently improve the performance of parasitised sheep, when fed ad libitum. Supplementation with CT conferred advantages on the performance of parasitised sheep on a high- but not on a low-protein food.

Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies.

In vitro and in vivo studies were conducted to determine possible direct anthelmintic effects of condensed tannins towards different ovine gastrointestinal nematodes. A larval development/viability assay was used to investigate the effect of a condensed tannin extract (Quebracho) towards larvae of Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus vitrinus. The development to infective larvae and their viability was assessed in all three species and LD 50 values were calculated. The presence of Quebracho extract in the cultures decreased the viability of L3 in all species; the LD 50 were not significantly different for the different species. Forty-eight sheep were allocated to one of eight groups and were infected with a single dose of either 4000 L3 H. contortus (groups 1 and 2) or 5000 L3 T. colubriformis and 5000 L3 Nematodirus battus simultaneously (groups 3-6) or 10,000 L3 of T. circumcincta (groups 7 and 8). From day 28 until day 31 of the experiment, sheep infected with the intestinal species were drenched with Quebracho extract at 4, 8 or 16% w/w of food intake, or remained as undrenched controls; sheep infected with the abomasal species were either drenched with Quebracho extract at 8% w/w of food intake or remained as undrenched controls. All sheep were slaughtered 4 days after the end of the drenching period. Sheep infected with the intestinal species and drenched with 16% w/w Quebracho had lower FEC compared to sheep drenched with 8% w/w (P<0.05), which in turn were lower than in sheep either drenched with 4% Quebracho or which remained undrenched (P<0.05). The lowest intestinal worm burden was recovered from sheep drenched with 8% w/w Quebracho extract (P<0.05). The administration of Quebracho extract at 8% of food intake for 3 days did not affect FEC or worm burdens in sheep infected with the abomasal species compared to controls.

Consequences of long-term feeding with condensed tannins on sheep parasitised with Trichostrongylus colubriformis.

Naive wethers were used to investigate the long-term effects of dietary condensed tannins from Quebracho extract, during an intestinal parasitic infection in sheep. Sheep were allocated to eight groups; seven groups were daily infected with 3000 L(3) Trichostrongylus colubriformis for 10 weeks and the eighth group was the uninfected control. The 10-week experiment was divided into two periods; Period 1 (P(1), week 1-5) corresponded to high worm establishment and acquisition of immunity, whereas Period 2 (P(2), week 6-10) to the established worm population and expression of host immunity. Three experimental foods with similar composition were formulated: Q0, Q3 and Q6. Their difference was in the content of Quebracho extract which was 0, 30 and 60 g per kg fresh matter, respectively. All foods were offered at an allowance of 3.5% of sheep liveweight. During P(1), parasitised sheep were offered one of the three experimental foods and during P(2) they either remained on the same food or changed food according to the design (P(1)-P(2)): Q0-Q0, Q0-Q3, Q0-Q6, Q3-Q0, Q3-Q3, Q6-Q0, Q6-Q6. Control sheep were offered the allowance of Q0 throughout. Sheep that consumed Q3 and Q6 reduced their faecal egg counts (FEC) compared to sheep offered Q0, during both periods (P<0.05). No differences were observed in the FEC between sheep offered Q3 and Q6. The changeover from Q0 in P(1) to either Q3 or Q6 during P(2), was accompanied by a reduction in FEC (P<0.05), whereas an increase in FEC was observed when food changed from Q3 or Q6 to Q0 (P<0.05). Worm burdens and fecundity at the end of the experiment were reduced in sheep offered foods Q3 and Q6 compared to sheep offered Q0. A significant decrease in liveweight gain and in food conversion efficiency of parasitised sheep offered Q3 and Q6 compared to sheep offered Q0, was observed in P(1) (P<0.05) but not in P(2). By the end of the experiment control sheep had achieved higher liveweight and converted food more efficiently than parasitised sheep (P<0.05). In conclusion, evidence for a long-term effect of Quebracho extract, during both the initial establishment and on the established T. colubriformis population in sheep, was provided by the present study. It is suggested that the effect observed was a direct anthelmintic effect of the condensed tannins included in sheep diets.

Temporal effects of protein nutrition on the growth and immunity of lambs infected with Trichostrongylus colubriformis.

The aim of this research was to determine whether metabolisable protein supply during the early period of infection with Trichostrongylus colubriformis influenced resilience and the later stages in the development and magnitude of host resistance in previously nematode-naïve lambs. Eighty TexelxGreyface lambs were fed pelleted feeds calculated to provide grossly different amounts of metabolisable protein. Sixty of the lambs received a trickle infection of T. colubriformis and 20 lambs were kept as uninfected controls. There were four initial groups, namely infected or uninfected and fed either a moderate or a high protein feed. After 5weeks of infection, a further four groups were established by changing the feed of half of the animals fed the moderate protein feed to the high protein feed and of half of the animals fed high protein to the moderate protein feed. Live weight gain and feed conversion ratio were greatest for lambs fed the high protein feed and were reduced by infection. Faecal egg counts, worm burdens and per capita fecundity of adult female nematodes were unaffected by changes to metabolisable protein supply. Decreasing metabolisable protein supply following 5weeks of infection reduced live weight gain without any effect on resistance to T. colubriformis. Haematological variables, indicative of improved resistance, were also largely unaffected by metabolisable protein supply. It is concluded that the requirements of immune function probably had priority over those of growth and that the metabolisable protein supply provided by the moderate protein feed was sufficient to account for the requirements for the expression of immunity. It is probable that the potential for metabolisable protein supply to enhance resistance to infection from T. colubriformis is dependent on the levels and magnitude (i.e. in relation to maintenance requirements) of metabolisable protein supply being compared and the demand of other competing physiological functions.

A novel zinc finger-containing RNA-binding protein conserved from fruitflies to humans.

The Drosophila lark gene encodes an essential RNA-binding protein of the RNA recognition motif (RRM) class that is required during embryonic development. Genetic analysis demonstrates that it also functions as a molecular element of a circadian clock output pathway, mediating the temporal regulation of adult emergence in the fruitfly. We now report the molecular characterization of a human gene with significant similarity to lark. Based on fluorescence in situ hybridization and radiation hybrid mapping, the human gene has been localized to chromosome region 11q13; it is closely linked to several identified genes including the locus of Bardet-Biedl syndrome type 1. The lark-homologous human gene expresses a single 1.8-kb size class of mRNA in most or all tissues including brain. Additional database searches have identified a mouse counterpart that is virtually identical to the human protein. Similar to lark protein, both mammalian proteins contain two copies of the RRM-type consensus RNA-binding motif. Unlike most RRM family members, however, the Drosophila and mammalian proteins also contain a retroviral-type (RT) zinc finger that is situated 43 residues C-terminal to the second RRM element. Within a 184-residue segment spanning the RRM elements and the RT zinc finger, the human and mouse proteins are 61% similar to the Drosophila lark sequence. These common sequence features and comparisons among a large collection of RRM proteins suggest that the human and mouse proteins represent homologues of Drosophila lark.

The pathophysiology and development of immunity during long-term subclinical infection with Trichostrongylus colubriformis of sheep receiving different nutritional treatments.

The objective of this study was to investigate the relationship between protein nutrition, the pathophysiology, and acquisition and expression of immunity in long-term subclinical intestinal parasitism in sheep. Growing sheep were either uninfected controls or parasitised for 27 weeks with a daily dose of 2500 larvae of Trichostrongylus colubriformis, whilst they were given access to: (1) a low protein food, (2) a high protein food, or (3) a choice between the two foods, where they were allowed to select their diet. Blood samples were taken weekly for determination of serum albumin, total protein, Ca, P, urea and fructosamine concentrations. At the end of the study all sheep received a single (secondary) challenge infection (30,000 T. colubriformis L3) after treatment with anthelmintic to assess their immune status. The concentrations of sheep-mast cell proteinases (SMCP) in intestinal tissue, the number of circulating eosinophils and the total worm numbers recovered from the intestinal tract were used to investigate the effects of previous nutrition on the acquisition and expression of immunity. From the biochemical variables measured over 27 weeks, only serum fructosamine was affected by the interaction between feeding treatment and parasitism: fructosamine concentrations declined only in the parasitised animals on the low protein food during Weeks 6-15 of infection. This casts doubt on the usefulness of plasma fructosamine levels as an indicator of gastrointestinal parasitism, due to its being influenced by the nutritional environment. Total protein, albumin, calcium and phosphorus concentrations in the serum were affected by parasitism, but independently of feeding treatment. During the period of secondary challenge eosinophil numbers and SMCP concentrations were higher in the parasitised animals, reflecting the animals immune responsiveness. The numbers of worms recovered from the intestine of previously parasitised sheep were low; all three indicators of the development of acquired immunity were unaffected by previous nutritional treatment of the sheep. The results do not support the view that the pathophysiology of long term subclinical intestinal parasitism and the expression of acquired immunity induced by a trickle infection could be affected by the feeding treatment of the sheep (protein nutrition).

Chronic (1-year) oral toxicity study of erythritol in dogs.

The chronic oral toxicity of erythritol was examined by feeding erythritol at dietary levels of 0 (controls), 2, 5, or 10% to groups of four male and four female dogs for 53 weeks. Erythritol was well tolerated at all dose levels without evidence of diarrhea. Water consumption was slightly higher in the high-dose group than in controls. Body weights and weight gains were not affected by treatment. There were no clinically relevant changes in hematological or clinicochemical parameters attributable to treatment. In particular, plasma electrolyte concentrations remained unaffected. Evaluation of a number of urinary parameters (including electrolytes and renal enzymes) was hampered by widely varying urine volumes among individual dogs; however, the available data did not indicate treatment-related effects on the urinary excretion of electrolytes (K+, Na+, Mg2+, and Pi) or enzymes (gamma-glutamyltranspeptidase, N-acetyl glucosaminidase, and lactate dehydrogenase). Quantitation of erythritol in the urine demonstrated that 50 to 80% of the ingested dose was absorbed and excreted in the urine. Analysis of terminal organ weights did not reveal treatment-related differences. No histopathological changes attributable to treatment were observed in the kidneys or in any other organ or tissue examined. It was concluded that daily erythritol consumption of up to 3.5 g/kg body wt was well tolerated by dogs.

Purification and characterization of oil-bodies (oleosomes) and oil-body boundary proteins (oleosins) from the developing cotyledons of sunflower (Helianthus annuus L.)

Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of M(r) 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of alpha-helical proteins with alpha-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.

Changes in brain and spinal cord water content during recurrent experimental autoimmune encephalomyelitis in female Lewis rats.

Regional changes in percent water content, a measure of regional levels of edema, were determined in female Lewis rats during key stages of recurrent experimental autoimmune encephalomyelitis (rEAE). The changes in percent water content of the spinal cord and brainstem closely paralleled the clinical and, to a lesser extent, histological course of rEAE (increasing during exacerbations and decreasing during remissions), whereas the percent water content of the forebrain, thalamus/midbrain, hypothalamus, and cerebellum remained constant and equal to control levels at all stages of the disease process. These results suggest that edema formation and resolution in the brainstem and spinal cord may be significant determinants of the transient and recurrent course of neurological dysfunction exhibited by rats with rEAE.

Effects of dietary molybdenum on nematode and host during Haemonchus contortus infection in lambs.

The addition of molybdenum (0.05 mmol kg-1 dry-matter) to the diet of lambs given a trickle infection of Haemonchus contortus larvae (500 third stage larvae d-1 over six weeks) reduced mean faecal egg counts (epg) from 3952 to 2312 +/- 402 by 32 days (P less than 0.02) and greatly reduced the mean number of worms recovered from the abomasum 14 days after infection ceased (907 compared with 4167: P less than 0.01). Infection reduced haemoglobin concentrations less in lambs given molybdenum although the difference was small relative to the reduction in worm burden. Lambs not given molybdenum had low intraepithelial mast cell counts in the abomasal mucosa and less abomasal hypertrophy than expected from abomasal parasitism. Molybdenum did not consistently reduce the copper status of the host or the parasite. Previous exposure to molybdenum greatly reduced protein but not proteinase activity in, or secreted by, adult worms cultured for eight hours. It is suggested that molybdenum either increased the inflammatory response which preceded worm rejection or that it indirectly enhanced that reaction by reducing the effectiveness of copper-dependent, anti-inflammatory enzymes in the gastrointestinal mucosa.

Effects of dietary molybdenum on nematode and host during Trichostrongylus vitrinus infection in lambs.

The addition of molybdenum (0.05 mmol kg-1 dry matter) to the diet of lambs exposed for four weeks to a trickle (2500 third stage larvae per day) infection with Trichostrongylus vitrinus reduced the number and length of adult worms retrieved from the small intestine 11 days later: both effects were particularly marked in female worms from female lambs (P less than 0.01). Worms from lambs given molybdenum contained less proteinase enzyme activity and secreted less proteinases in culture irrespective of the sex of the host. Pathogenicity was not attenuated by molybdenum. Damage to the intestinal mucosa was severe in both dietary groups but infected females given molybdenum developed lower plasma albumin concentrations and lighter dressed carcases than those not given molybdenum. Neither the effects on the parasite nor those on the host could be attributed simply to molybdenum-induced copper depletion, using conventional measures of copper status. Molybdenum may be toxic to T vitrinus but may also facilitate or enhance the inflammatory process limiting larval establishment or increasing parasite rejection.

Larvicidal effects of grain sorghum (Sorghum bicolor) seedling extracts upon Culex pipiens larvae.

A study of laboratory and field reared 2nd and 3rd instar Culex pipiens larvae suggests that extracts from 2 varieties of Sorghum bicolor seedlings are significant (P less than 0.05) larvicides under laboratory conditions. These plant extracts contain the organic cyanogen dhurrin and were calibrated to produce 90% mortality in 2nd instar Culex pipiens larvae at 0.82 ppm and 90% mortality in 3rd instar larvae at 1.12 ppm. A preliminary behavioral assessment of late 3rd instar larvae exposed to 1.42 ppm suggests that these plant extracts produce 80% mortality after only 4-5 h of contact. Plant extracts appear stable when stored at up to 32 degrees C in a closed container. Once the extracts are infused in water and exposed to air, however, they biodegrade after 24 h. These laboratory results emphasize the need for field tests against natural populations of Culex pipiens and nontarget organisms.

Drosophila GABAergic systems: sequence and expression of glutamic acid decarboxylase.

A mammalian glutamic acid decarboxylase (GAD) cDNA probe has been utilized to isolate Drosophila cDNA clones that represent a genomic locus in chromosome region 64A. Deletion analysis indicates that this chromosomal locus encodes an enzymatically active GAD protein. The in vitro translation of cRNA representing a Drosophila cDNA clone yields a 57-kDa protein that can be immunoprecipitated by an anti-GAD antiserum. A GAD-immunoreactive protein of the same size can also be detected in Drosophila head extracts. The nucleotide sequence derived from two overlapping Drosophila cDNA clones predicts a 57,759-dalton protein composed of 510 residues that is 53% identical to mammalian GAD. Sequence comparisons of mammalian and Drosophila GAD identify two highly conserved regions (greater than or equal to 70% identity), one of which encompasses a putative co-factor-binding domain. Transcriptional analyses show that expression of the Drosophila Gad gene commences early in embryonic development (4-8 h) and continues in all later developmental stages. A 3.1-kb class of mRNA is detected throughout embryogenesis, in all three larval stages, in pupae, and in adults. This transcript class has a widespread distribution in the adult CNS. A smaller 2.6-kb transcript is expressed in a developmentally regulated manner; it is detected only in embryos and pupae.

Influence of two levels of concurrent infection with Ostertagia circumcincta and Trichostrongylus vitrinus on the growth performance of lambs.

Groups of seven lambs were infected on five days each week for 11 weeks with either 1000 or 2000 Trichostrongylus vitrinus larvae alone or concurrently with 1500 or 2500 Ostertagia circumcincta larvae and their growth performance compared to that of worm-free controls. The cumulative liveweight gain of the infected groups was significantly lower than that of the controls but the gain of the concurrently infected lambs did not differ significantly from the respective T vitrinus group. There were no significant differences in the numbers of T vitrinus or O circumcincta recovered from the single and concurrently infected groups, although there was a tendency for lower mean T vitrinus burdens at the higher level of ostertagia/trichostrongylus infection. The distribution of T vitrinus along the small intestine was similar in single and concurrently infected lambs. The reason for the lack of a marked additive effect on growth rate with concurrent infection is discussed in relation to changes in the concentration of two plasma constituents and lesions in the gastrointestinal tract.

Prescribing postpartum iron supplementation: a survey of practicing obstetricians.

Supplemental iron (excluding the amount in prenatal vitamins) is routinely recommended post partum by standard obstetric textbooks. We surveyed practicing obstetricians in Maryland to examine their indications for and prescription of postpartum iron supplements. The survey was returned by 201 physicians (33% response rate). Iron was prescribed by 25% of the physicians always, 12% never, and 63% depending upon the patient. An average of 37% of postpartum patients were given iron. The most frequently cited indication for prescribing iron was the postpartum hematocrit level (used by 90% of the physicians), followed by high blood loss (41%), operative delivery (15%), mean corpuscular volume (12%), and toxemia (9%). The postpartum iron dosage (325 mg) was prescribed as a single daily dose by 22%, twice daily by 37%, and three times daily by 32%. These practices do not correspond to the current recommendation that postpartum iron should always be prescribed.