Isolation and characterisation of sex-specific transcripts from Oesophagostomum dentatum by RNA arbitrarily-primed PCR.

In light of the lack of molecular data on the sexual differentiation, maturation and interaction of parasitic nematodes of livestock, the present study investigated sex-specific gene expression in the nodule worm, Oesophagostomum dentatum (Strongylida). Using the technique of RNA arbitrarily-primed polymerase chain reaction (RAP-PCR), 31 expressed sequence tags (ESTs) differentially-displayed between the sexes were cloned. Northern blot analysis proved ten ESTs to be expressed exclusively in males (adults and fourth-stage larvae), while two were expressed solely in female stages. None of the ESTs were expressed in infective third-stage larvae. Sequence analysis and subsequent database searches revealed two male-specific ESTs to have significant similarity to Caenorhabditis elegans (predicted) proteins, a protein containing an EGF-like cysteine motif and a serine/threonine phosphatase. Another two male-specific ESTs had similarity to non-nematode sequences. The two female-specific ESTs had similarity to vitellogenin-5 and endonuclease III (predicted) from C. elegans. The remaining ESTs had no similarity to any nucleic acid or protein sequences contained in the databases. The isolation and characterisation of sex-specific ESTs from O. dentatum provides a unique opportunity for studying the reproductive biology of parasitic nematodes at the molecular level, with a view toward novel approaches for parasite control.

Experimental Oesophagostomum dentatum infections in the pig: worm populations resulting from trickle infections with three dose levels of larvae.

This paper describes the worm populations in pigs experimentally infected by trickle infections with different dose levels of the nodular worm, Oesophagostomum dentatum. Four groups each of 20 helminth naïve pigs, 10-12 weeks old, were inoculated with 0 (group 1), 100 (group 2), 1000 (group 3), or 10,000 (group 4) infective larvae twice weekly, and the pigs were killed after 10-13 weeks. No overt clinical signs were observed, and only group 4 had slightly lower food conversion rate (P < 0.05) than the controls. Faecal egg counts revealed that the nodular worms in pigs of groups 2 and 3 had a short prepatent period (3-4 1/2 weeks) and a fairly stable egg output, while the worms in the pigs of group 4 had prepatent periods of 3-10 weeks and low, unstable egg excretion. The mean worm burdens increased with the dose rate (group 2: 929 worms; group 3: 7467 worms; group 4: 19,847 worms), but detailed analyses of the worm populations from 10 pigs from each of the infected groups revealed a clear dose-dependency in worm recovery, percentage adult worms, worm lengths and female fecundity, as all these measures declined significantly with increasing dose level. The adult worms seemed to be shorter and less fertile when they were located posteriorly to their predilection site, and especially in group 4 many stunted infertile adults measuring only 2-5 mm were found in the posterior half of the colon, but there were no indications of worm expulsion. Superimposed on the main experiment was a cohort study in which 4 pigs of group 3 were given a single dose of 1000 pyrantel resistant larvae at day 56 (all other larvae were pyrantel sensitive), treated with 28 mg pyrantel per kg body weight at day 85 and killed at day 90. Appropriate control groups were included. The mean establishment of the cohort was similar to previously uninfected controls, but between-animal variation was much higher in the trickle infected group.

Experimental Oesophagostomum dentatum infection in the pig: worm populations resulting from single infections with three doses of larvae.

This report describes the effect of different dose levels of infection upon worm burdens and development and fecundity of the parasites. Three groups each of 40, 9-week-old, helminth naive pigs were inoculated once with either 2000 (group A), 20,000 (group B), or 200,000 (group C) infective third stage larvae of Oesophagostomum dentatum. Subgroups of 5 pigs from each major group were killed 3, 6, 11, 14, 18, 25, 34 and 47 days post inoculation (p.i.) and the large intestinal worm burdens were determined. Faecal egg counts were determined at frequent intervals after day 13 p.i. There were no overt clinical signs of gastrointestinal helminthosis during the experiment. Faecal egg counts became positive in groups A and B at around day 19 p.i., whereas most pigs in the high dose group C did not have positive egg counts until day 27-33 p.i. and some pigs remained with zero egg counts until the end of the study. Throughout the experiment the worm populations in group C consisted mainly of immature larval stages, while those in groups A and B were predominantly adult stages after days 14-18. Adult worms from the low dose group A were significantly longer than those from group C. At high population densities, stunted development of worms and reduced fecundity among female worms were found. Furthermore, there was a tendency for the distribution of the worms within the intestine to be altered with increasing population size.

The effect of experimental trichostrongyle infections of housed young calves on the subsequent course of natural infection on pasture.

The present studies were designed to investigate whether experimental, mixed trichostrongyle infections of stabled calves prior to their first grazing season could confer sufficient immunity to significantly reduce egg excretion after turnout, and thereby prevent loss-producing infections later on. The study comprised four groups, each of seven calves. During spring, two of the groups received two different dose levels of infective larvae twice weekly, and one group received larger larval doses at monthly intervals. One group served as non-experimentally exposed controls. In May all groups grazed separately on similarly contaminated plots. All experimental groups had reduced egg counts, and herbage infectivity of their plots was significantly lower than that of the controls. These findings were reflected in serum pepsinogen levels and in specific serum antibody responses. Additionally, clinical disease was only observed in the control group animals.

Effects of hydrochlorothiazide and amiloride on salt taste and excretion (intake).

The effects of hydrochlorothiazide (HCTZ) and amiloride on salt excretion (intake) and taste were examined in 73 normotensive adults (aged 18 to 56) who were randomly assigned to receive 50 mg/day of HCTZ (n = 24), 5 mg twice daily of amiloride (n = 24) or placebo (n = 25) for 8 weeks. Two and three week placebo periods preceded and followed active treatment, respectively. Relative to baseline, significant increases in sodium excretion (intake) of 31%, 53% and 30% were observed in subjects receiving HCTZ after 2, 4 and 8 weeks of treatment, respectively. An identical follow-up study with 16 subjects replicated the sodium excretion (intake) findings, but failed to reveal the source of the extra dietary sodium. Doubling the dose of amiloride in 11 additional subjects led to higher aldosterone excretion relative to thiazide-treated subjects, but did not increase salt excretion (intake) over a four week period. Only amiloride-treated subjects displayed significant increases in salt taste sensitivity. The increased sodium intake in HCTZ-treated patients may partially offset the desired effects of therapy and exacerbate potassium wasting.

Protracted methemoglobinemia after phenazopyridine overdose in an infant.

Acquired methemoglobinemia may be produced by the ingestion or absorption of certain chemicals and xenobiotics. A case of methemoglobinemia in an 8.5-month old infant who ingested approximately 227 mg/kg of phenazopyridine is presented. Although this adverse event is often reversed with a single dose of methylene blue, this patient required three doses of methylene blue (1 mg/kg) over a 25-hour period. It is suggested that the need for repeated doses of methylene blue in this case was not only related to the large dose of phenazopyridine, but also its metabolites (i.e., aniline), which have the potential to produce methemoglobinemia. This case illustrates the need for close observation and serial monitoring of methemoglobin levels in patients who are at increased risk for the development of protracted methemoglobinemia. Integration of knowledge of developmental pharmacology, drug metabolism, and pharmacodynamic properties are critical determinants in the evaluation and treatment of patients with drug-induced methemoglobinemia.

Salivary changes in solution pH: a source of individual differences in sour taste perception.

The role of saliva in sour taste perception was investigated in a series of 4 experiments. In one pair of experiments, solution pH was measured before and after acetic, citric or hydrochloric acid solutions were mixed with saliva either normally in the oral cavity or after saliva was directly added to solutions. The results showed that large increases in solution pH occurred over a wide range of acid concentrations and that the changes in pH were related to individual salivary flow rates; greater increases in solution pH occurred among those individuals with higher flow rates. The other pair of experiments measured taste threshold and suprathreshold responses to different volumes of acids. The results demonstrated that individuals with high salivary flow rates were less sensitive to the taste of acids and that large volumes of acid were more easily perceived. The pattern of findings suggest that saliva-induced changes in solution pH are important in sour taste perception.

Development of brief methods to classify individuals by PROP taster status.

The purpose of this study was to develop brief methods for classifying individuals by genetic taste sensitivity to 6-n-propylthiouracil (PROP). Two methods are described, which are modifications of a commonly used, suprathreshold procedure. Eighty-nine adult subjects rated the perceived intensity of solutions of 0.032, 0.32 and 3.2 mmol/l PROP and 0.01, 0.1, 1.0 mol/l sodium chloride (NaCl) (three-solution test), as well as solutions of 0.32 mmol/l PROP and 0.1 mol/l NaCl (one-solution test) using the Labeled Magnitude Scale (LMS). Subjects were classified as PROP nontasters (n=22), medium tasters (n=51) or supertasters (n=16) by the three-solution test. Taster status was independently determined by the one-solution test using numerical cutoff scores, which were determined by calculating the +/-95% confidence interval around the group means for PROP taste intensity. Supertasters gave PROP a rating of > or =51 ("very strong" on the LMS) and nontasters gave PROP a rating of < or =15.5 (approximately "moderate" on the LMS). Medium tasters fell between these two limits. Ninety-one percent of nontasters, 82% of medium tasters and 89% of supertasters were classified in a similar way by the two methods. Agreement between methods was high [coefficient of association (P)=0.74; P < or =.001]. These data suggest that three- and one-solution methods can reliably classify subjects by taste sensitivity to PROP and could provide valuable tools in population-based studies.

Preference of rats for food flavors and texture in nutritionally controlled semi-purified diets.

Preference for nutritionally controlled, semi-purified diets modified by the addition of potent food flavors was determined for Sprague Dawley rats using two-choice diet preference tests. Intake of each food cup was monitored after 1 hr and for each 24 hr period thereafter up to 5 days. Preference was also determined for the flavored diets prepared in three forms differing in texture: powdered, and pellets of two sizes. Rats easily detected minor amounts of the food flavors, and the tests provided a catalog of 12 preferred flavors. Exposure time to the diets altered preference for a minority of flavors; diets initially avoided in the first hour test were likely to become less aversive upon continued exposure. Whether or not a specific flavored diet was preferred, total food intake was not affected during the 5 day period monitored. Rats displayed strong preference for diets of a pelleted texture compared to the same diets in a powdered form.

Effects of pharmacologic reductions in salivary flow on taste thresholds in man.

The effects of short-term salivary flow reductions on human taste thresholds were measured. Recognition and detection thresholds were obtained from 65 subjects during periods of both normal and reduced salivary flow. Decreased salivary flow was achieved by oral administration of either Elavil, Benadryl or atropine. Thresholds were measured for NaCl, citric acid, quinine sulphate and sucrose with a traditional series of aqueous solutions as well as with a series of dry taste stimuli using a filter-paper base. Whole mouth resting flow and stimulated salivary flow were measured before and after taste testing. The pharmacologic agents produced depressions in salivary flow ranging between 30 and 75 per cent of normal levels. The large decreases in flow produced no measurable changes in taste thresholds with the exception that an increased sensitivity to aqueous and dry citric acid stimuli consistently was observed following atropine administration. Changes in salivary bicarbonate levels, produced by atropine, may have mediated the observed shifts in oral sensitivity to citric acid.

Bacterial agglutinin activity in the saliva of human identical and fraternal twins.

The major factor in human saliva responsible for the specific aggregation of oral streptococci is a high molecular-weight glycoprotein (agglutinin). To determine if the level of this glycoprotein in whole and parotid saliva was genetically determined, agglutinin activity for Streptococcus sanguis and Streptococcus mutans in saliva obtained from identical and fraternal twins was compared. Evidence for the heritability of agglutinin activity and also parotid flow rate and total protein was obtained. There was no evidence for a significant genetic contribution to salivary sodium concentration.

The influence of moderate reduction in dietary sodium on human salivary sodium concentration.

Twenty-four healthy subjects were placed for 12-13 weeks on diets that reduced average sodium intake from 145 to 74 m-equiv. Na+/day as determined by multiple 24-h urine collections before and during the diet. Whole-mouth resting and stimulated saliva was collected and analysed for flow rate and sodium concentration several times before and during the low-sodium period. Sodium restriction did not influence salivary flow rates but salivary sodium levels fell 25 per cent for resting and 17 per cent for stimulated saliva. Thus moderate reductions in sodium intake are accompanied by significantly lower salivary sodium levels.

Non-surgical transplantation of Oesophagostomum dentatum to recipient pigs via rectal intubation.

This experimental was designed to evaluate a new transplantation method, which employs a non-invasive rather than a surgical technique for transplanting Oesophagostomum dentatum worms to recipient pigs. Four groups of pigs were used. Group A (four pigs) served as a donor group, and these pigs were each inoculated with 6000 L3 of O. dentatum. Groups B, C, and D (five pigs each) served as helminth naive recipient pigs. On Day 28 post inoculation, the donor pigs were slaughtered, and the worms recovered by an agar-gel technique. Within 3-4 h after slaughter of the donor animals, a mean of 357 worms (male/female = 1.0) were transferred to each of the sedated recipient pigs via a PVC tube inserted approximately 50 cm up into the rectum and colon descendens. The infection was then monitored by weekly faecal egg counts and by killing the recipient pigs at Week 1 (group B), Week 2 (group C), and Week 3 (group D) post transplantation. The majority of the recovered worms were found in the proximal third of the colon, i.e., the normal predilection site of O. dentatum. The mean worm recovery for groups B and D was 85%, whereas from group C it was only 23%. The faecal egg counts were positive throughout the experiment, although low in group C at the time of slaughter 2 weeks post transplantation. The male/female ratios changed from an initial 1.0 to 1.5 group C, whereas there was little or no change in groups B and D. The reason for the deviating results in group C are obscure. This method is less traumatic to animals when compared with surgical transfer techniques, is rapid to perform, and will allow studies on a larger scale.

Concurrent Ascaris suum and Oesophagostomum dentatum infections in pigs.

The aim of this study was to examine interactions between Ascaris suum and Oesophagostomum dentatum infections in pigs with regard to population dynamics of the worms such as recovery, location and length; and host reactions such as weight gain, pathological changes in the liver and immune response. Seventy-two helminth-naïve pigs were allocated into four groups. Group A was inoculated twice weekly with 10000 O. dentatum larvae for 8 weeks and subsequently challenge-infected with 1000 A. suum eggs, while Group B was infected with only 1000 A. suum eggs; Group C was inoculated twice weekly with 500 A. suum eggs for 8 weeks and subsequently challenge-infected with 5000 O. dentatum larvae, whereas Group D was given only 5000 O. dentatum larvae. All trickle infections continued until slaughter. Twelve pigs from Group A and B were slaughtered 10 days post challenge infection (p.c.i.) and the remaining 12 pigs from the each of the four groups were slaughtered 28 days p.c.i.. No clinical signs of parasitism were observed. The total worm burdens and the distributions of the challenge infection species were not influenced by previous primary trickle-infections with the heterologous species. Until day 10 p.c.i. the ELISA response between A. suum antigen and sera from the O. dentatum trickle infected pigs (Group A) pigs were significantly higher compared to the uninfected Group B. This was correlated with a significantly higher number of white spots on the liver surface both on Day 10 and 28 p.c.i. in Group A compared to Group B. The mean length of the adult O. dentatum worms was significantly reduced in the A. suum trickle infected group compared to the control group. These results indicate low level of interaction between the two parasite species investigated.

Attempts to immunize cattle against Ostertagia ostertagi infections employing 'normal' and 'chilled' (hypobiosis-prone) third stage larvae.

Immunity to Ostertagia ostertagi infections in calves develops slowly and only becomes manifest towards the end of a grazing season in which they have been exposed to the parasite. In an attempt to hasten the onset of immune reactions, three immunization protocols were set up. Twenty four heifers were allocated into four groups. Beginning in January, animals in two of the groups were inoculated with four 1-monthly increasing dosages of either 'normal' or 'chilled' (hypobiosis-prone) larvae, those in the third group received a single large infection with 'chilled' larvae and those in the fourth group served as non-infected controls. All animals were turned out on a common pasture in late April. Development of immunity was evaluated through determinations of faecal egg counts, live weight gains, serum pepsinogen levels and specific serum antibody responses of three isotypes (IgG1, IgG2 and IgA). Significantly reduced egg excretions in the immunized groups were apparent early in the season, indicating that the immunizations had, in this respect, been efficacious. The 'chilled' and 'normal' larvae seemed equally efficient given as multiple and single infections. A single large dosage of 'chilled' larvae seemed to have adverse effects. Only moderate antibody responses were elicited probably because of low challenge infection level on pasture. Considerable variation in responses existed between and within the four groups, for which reason conclusions regarding correlations between antibody isotype responses and immune effects on parasites could not be made.

Recovery of Oesophagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar-gel.

Four groups with three pigs in each group were inoculated with Oesophagostomum dentatum larvae (L3 larvae). Groups 1 and 3 were inoculated with 20,000 larvae, and Groups 2 and 4 with 200,000 larvae. On Days 11 and 34, respectively, Groups 1 and 2 and Groups 3 and 4 were slaughtered, and the contents from the large intestines collected. Subsamples of intestinal contents were mixed with agar to a final concentration of 1% agar and allowed to set. The worms were allowed to migrate from the agar-gel into 38 degrees C 0.9% saline overnight. Then the worms were collected on a sieve (38 microns mesh) and counted. The worms retained in the agar-gel were counted after pouring the melted agar through a sieve (38 microns mesh). The results showed that more than 95% of the worms migrated out of the agar-gel, and subsequently were available for counting in an almost clean suspension. Additionally the method yielded a high worm recovery; all stages were recovered. The recovery percentage was not significantly affected by either the dose of parasites or the time interval from slaughtering to start of incubation (37-128 min).

The effect of three distinct sex ratios at two Oesophagostomum dentatum worm population densities.

Helminth-free donor pigs were inoculated with infective larvae of Oesophagostomum dentatum. Five weeks later, the donor pigs were killed to recover the adult worms. By nonsurgical rectal transplantation, 6 groups of helminth-free recipient pigs were then given different female-to-male ratios (FMR) of O. dentatum worms (10% females [F], 50% F, 90% F) at either high (300 worms) or low (30 worms) doses. Fecal egg excretions were measured once weekly following transplantation until the pigs were killed 4 wk posttransplantation to assess the worm burdens and their location. There was a strong and significant correlation between numbers of male and female worms located in the different sections of the large intestine, suggesting that the worm sex as well as physiological factors in the pig intestine may govern the location of the worms. There were no significant differences in fecal egg counts or female worm fecundity between any of the groups. However at the low dose level, the female worm fecundity was markedly lower in the L10% F group than in the 2 other groups, thus suggesting for low worm densities an upper threshold for the FMR above which O. dentatum females produce fewer eggs.

Ultrastructure and development of the body cuticle of Oesophagostomum dentatum (Strongylida, Nematoda).

The ultrastructure, development, and morphogenesis of the body cuticle of adult and juvenile Oesophagostomum dentatum (Strongylida, Nematoda) were investigated by light microscopy, scanning electron microscopy, and transmission electron microscopy. The cuticle of the first 3 juvenile stages consists of a trilaminate epicuticle, an amorphous layer, and a radially striated layer. In the last juvenile stage and the adult worm, the radially striated layer is replaced by a fibrous layer with 3 sublayers of giant fibers and a basal amorphous layer. The new forming cuticle of the 3rd juvenile stage exhibits the epicuticle and amorphous material and, at a later time, the epicuticle and the radially striated layer; finally, amorphous material appears between these layers. Our finding of a structural change in the cuticular morphology between the 3rd and 4th juvenile stage harmonizes with earlier reports about the Strongylida. Such a change occurs at different ontogenetic stages or seems to be missing in other nematodes. Morphogenetic events such as the formation of the radial striation layer from amorphous precursor material agrees with previous observations on strongylids.

Serological assays for the identification of Oesophagostomum dentatum infections in pigs.

Oesophagostomum dentatum antigen preparations of third (L3) or fourth (L4) stage larvae were characterised by Western blotting. Panels of sera obtained from pigs infected with O dentatum and Ascaris suum, respectively, reacted with the same bands of L3 antigen. In contrast high specificity against a characteristic band, was observed when L4 extract was employed as antigen. To establish an enzyme-linked immunosorbent assay (ELISA), a panel of homologous and heterologous sera was tested against O dentatum L4 extract. The best combined specificity and sensitivity was obtained when horseradish peroxidase (HRP) goat anti swine IgG conjugate was used rather than HRP rabbit anti swine Ig conjugate. Testing series of sera from pigs infected with single doses of either 2000, 20,000 or 200,000 infective larvae by the ELISA, a significant dose dependency in the antibody response was observed between the low and high dosage groups. This assay may be useful in future studies of the immune-mechanisms against nodular worm infections in pigs.

Ostertagia ostertagi in neonatal calves: establishment of infection in ruminating and non-ruminating calves.

An experiment was carried out to study the role of the ruminal function in the establishment of Ostertagia ostertagi in neonatal calves. Three groups of calves were fed either milk only (groups A and C), or hay and concentrate in addition to milk (group B) from birth. At the time of infection, ruminal function was negligible in groups A and C, whereas it was well developed in group B. Calves of groups A and B were each given 25,000 normal ensheathed infective larvae of O ostertagi and those of group C were given 25,000 infective larvae exsheathed in vitro. Daily faecal egg output and post mortem worm counts 28 days after infection were higher in calves with well developed ruminal function than those having only negligible ruminal function. In the latter group, exsheathed larvae established at a lower rate than did ensheathed larvae. The results suggest that the degree of development of the ruminal function influences the establishment of O ostertagi.