Acquisition of flavour preferences in pigs through interactions with conspecifics that had previously consumed flavoured protein solutions.

It is known that pigs can acquire flavour preferences by brief social interactions with conspecifics that previously consumed a flavoured solid feed. However, there is no information about whether a flavoured solution could support flavour preferences through social transmission. Ninety-six pigs (49 days old) were housed in 12 pens (8 pigs/pen). Four animals per pen were randomly selected to act as observers and four as demonstrators. Demonstrator animals were temporarily moved to an empty pen where a protein solution was offered (porcine digestive peptides (PDPs), 4% weight/volume) with the addition of 0.075% aniseed (six pens) or garlic (six pens) powdered artificial flavours for 30 min. Afterwards, demonstrators were returned to interact with observer animals for 30 min. A choice test (30 min) between aniseed and garlic PDP was performed for each observer group after the interaction. Observers showed a higher intake of solutions previously consumed by their demonstrator conspecifics (648 v. 468 ml; SEM 61.36, P < 0.05). As with flavoured solid feeds, protein solutions containing artificial flavours can create preferences in pigs for those flavours through social transmission from conspecifics.

Expression of host defense peptides in the intestine of Eimeria-challenged chickens.

Avian coccidiosis is caused by the intracellular protozoan Eimeria, which produces intestinal lesions leading to weight gain depression. Current control methods include vaccination and anticoccidial drugs. An alternative approach involves modulating the immune system. The objective of this study was to profile the expression of host defense peptides such as avian beta-defensins (AvBDs) and liver expressed antimicrobial peptide 2 (LEAP2), which are part of the innate immune system. The mRNA expression of AvBD family members 1, 6, 8, 10, 11, 12, and 13 and LEAP2 was examined in chickens challenged with either E. acervulina, E. maxima, or E. tenella. The duodenum, jejunum, ileum, and ceca were collected 7 d post challenge. In study 1, E. acervulina challenge resulted in down-regulation of AvBD1, AvBD6, AvBD10, AvBD11, AvBD12, and AvBD13 in the duodenum. E. maxima challenge caused down-regulation of AvBD6, AvBD10, and AvBD11 in the duodenum, down-regulation of AvBD10 in the jejunum, but up-regulation of AvBD8 and AvBD13 in the ceca. E. tenella challenge showed no change in AvBD expression in any tissue. In study 2, which involved challenge with only E. maxima, there was down-regulation of AvBD1 in the ileum, AvBD11 in the jejunum and ileum, and LEAP2 in all 3 segments of the small intestine. The expression of LEAP2 was further examined by in situ hybridization in the jejunum of chickens from study 2. LEAP2 mRNA was expressed similarly in the enterocytes lining the villi, but not in the crypts of control and Eimeria challenged chickens. The lengths of the villi in the Eimeria challenged chickens were less than those in the control chickens, which may in part account for the observed down-regulation of LEAP2 mRNA quantified by PCR. Overall, the AvBD response to Eimeria challenge was not consistent; whereas LEAP2 was consistently down-regulated, which suggests that LEAP2 plays an important role in modulating an Eimeria infection.

Avoidance but not aversion following sensory preconditioning with flavors: a challenge to stimulus substitution.

After two neutral stimuli have been paired (AB), directly conditioning a response to one of them (A) will also be reflected in a change in responding to the other (B). Standard accounts of this sensory preconditioning effect assume that it is mediated by a memory involving the stimulus that was directly conditioned (i.e., A). The reliance on this shared pathway implies that sensory preconditioning (involving B) and direct conditioning (involving A) should support qualitatively similar patterns of responding. In three experiments, directly pairing A with lithium chloride (LiCl) delivery resulted in both a reduction in consumption of A (i.e., avoidance) and a reduction in the size of licking clusters it elicits (i.e., aversion). In contrast, the sensory preconditioning effect resulted in a reduction in the consumption of B but no change in the nature of the licking response that it elicited; and a similar dissociation was observed after trace conditioning. These dissociations involving direct conditioning and sensory preconditioning, observed over a range of flavor concentrations and different doses of LiCl, undermine standard accounts of sensory preconditioning that are based on the assumption of stimulus substitution.

Re-assessing causal accounts of learnt behavior in rats.

Rats received either a common-cause (i.e., A→B, A→food) or a causal-chain training scenario (i.e., B→A, A→food) before their tendency to approach the food magazine during the presentation of B was assessed as a function of whether it was preceded by a potential alternative cause. Causal model theory predicts that the influence of an alternative cause should be restricted to the common-cause scenario. In Experiment 1, responding to B was reduced when it occurred after pressing a novel lever during the test phase. This effect was not influenced by the type of training scenario. In Experiment 2, rats were familiarized with the lever prior to test by training it as a potential cause of B. After this treatment, the lever now failed to influence test responding to B. In Experiment 3, rats given common-cause training responded more to B when it followed a cue that had previously been trained as a predictor of B, than when it followed another stimulus. This effect was not apparent in rats that received causal-chain training. This pattern of results is the opposite of that predicted by causal model theory. Thus, in three experiments, the presence of an alternative cause failed to influence test responding in manner consistent with causal model theory. These results undermine the application of causal model theory to rats, but are consistent with associative analyses.

"Causal reasoning" in rats: a reappraisal.

It has recently been argued that rats engage in causal reasoning and they do so in a way that is consistent with Bayes net theories (Blaisdell, Sawa, Leising & Waldmann, 2006). This argument was based upon the finding that the tendency of cues to elicit approach to a food-well was reduced when their presentation was contingent on lever pressing. There is, however, an alternative interpretation of the critical experimental findings that is based on the simple principle of response competition: wherein lever pressing interferes with the tendency to approach the food well. Here the authors replicated Experiments 1 and 2a of Blaisdell et al. (2006) and found reciprocal patterns of lever pressing and food well approach during the critical cues. These results lend direct support for an interpretation in terms of response competition while providing evidence contrary to Bayes net theories, and are readily interpreted within the theoretical framework provided by traditional associative learning theory.

Inhibitory associations contribute to perceptual learning in humans.

Perceptual learning in humans was examined in 2 experiments. In Experiment 1, participants received intermixed exposure to 2 similar compounds (AX, BX, AX, BX, . . .) and blocked exposure to a 2nd pair of similar compounds (CY, CY, . . . , DY, DY, . . .). Aversions established to AX and CY generalized less to BX than to DY. In Experiment 2, 1 pair of compounds was presented in a forward order (i.e., AX3BX), whereas the 2nd pair of compounds was presented in a backward order (i.e., DY-->CY). Aversions established to AX and CY generalized less to BX than to DY. These results indicate that inhibitory associations contribute to perceptual learning in humans and thereby establish a fundamental similarity between the mechanisms that underlie perceptual learning in humans and rats.

Characterization of a blood activated chitinolytic system in the midgut of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi.

We characterized a cDNA from Phlebotomus papatasi, PpChit1, which encodes a midgut specific chitinase and show the presence of a functional, blood-induced chitinolytic system in sand flies. PpChit1 is detected only in the midgut and is regulated by blood feeding. A recombinant protein (rPpChit1) produced in HEK 293-F cells exhibited a similar activity profile to that found in the native protein against several specific substrates, including an oligomeric glycol chitin and synthetic 4-methyl-umbelliferone labelled substrates. Western blotting showed that the native protein is recognized by mouse polyclonal antibodies against rPpChit1. Additionally, the rPpChit1 and the native chitinase displayed similar retention times in a HPLC size fractionation column. When added to rPpChit1 or to midgut lysates, PpChit1 sera reduced chitinolytic activity by 65-70%.

Reinforcer devaluation in palatability-based learned flavor preferences.

Rats exposed to simultaneous compounds of 1 neutral flavor with dilute (2%) sucrose and a 2nd flavor with dilute (2%) maltodextrin subsequently consumed both flavors in preference to a 3rd flavor that was never paired with a palatable taste. Brief training exposure under ad lib food and water minimized the post-ingestive effects of nutrients, emphasizing the contribution of palatability to these preferences. Devaluation of sucrose or maltodextrin by pairing with illness (Experiment 1) or sensory-specific satiety (Experiment 2) selectively reduced the preference for the flavor previously paired with the devalued reinforcer. Such reinforcer-specific devaluation effects suggest that palatability-based learned flavor preferences are under-pinned by a Pavlovian process whereby the cue flavor is associated with the taste of the concurrently consumed palatable reinforcer.

Perceptual learning in humans: roles of preexposure schedule, feedback, and discrimination assay.

In three experiments, humans received preexposure to two compound flavours (AX and BX: saline-lemon and sucrose-lemon) that were presented either in an intermixed (e.g., AX, BX,...BX, AX,...) or a blocked (e.g., AX, AX,...BX, BX...) fashion. Subsequently, AX was paired with an unpleasant bitter taste, and the discriminability of AX and BX was assessed using the accuracy of same/different judgements and by the extent to which any learned dislike of AX generalized to BX. When participants received feedback about the accuracy of their same/different judgements during preexposure those given intermixed preexposure were more accurate in making these judgements during the test than those given blocked preexposure (Experiments 1 and 2A), however, there was no evidence of any learned dislike in these experiments. In Experiment 2B, in which participants did not receive feedback about the accuracy of their judgements, there was no effect of the preexposure regime on same/different judgements, but there was a learned dislike of AX, and this generalized less to BX in participants given intermixed than in those given blocked preexposure. The beneficial effects of intermixed preexposure are consistent with results from other species (chicks and rats), but the differences created by the presence or absence of feedback place constraints on the analysis of these effects.

Learning about cues in their absence: evidence from flavour preferences and aversions.

A simple behavioural task may involve the presentation of two or more stimuli. Any learning that takes place in such a situation may be analysed in terms of the formation of an association between the central representations of those stimuli. Presumably performance based on this learning can occur because presentation of one stimulus will then activate the representations of other stimuli that were previously presented with it. To examine the role that these representations play in learning in and of themselves requires that the stimuli themselves are absent. A review of a number of flavour preference and aversion studies indicates that an associatively activated stimulus representation can support learning that is both similar to and the opposite of that maintained by the presentation of the stimulus itself. Which occurs is dependent upon the nature of the reinforcer and the temporal relationships between the training events. Although this pattern of results appears suggestive of separate learning rules, a reanalysis raises the possibility of an explanation in terms of a single associative system.

Alternating exposure to two compound flavors creates inhibitory associations between their unique features.

Rats were exposed to two compound solutions, saline-lemon and sucrose-lemon. In Group ALT, trials with one solution alternated with trials with the other. Group BLK received all trials with one solution before any trials with the other. Previous retardation tests had implied that only alternating exposure would establish sucrose as an inhibitor of saline. To provide a complementary summation test for this inhibition, in Experiment 1, all the animals received pairings of peppermint and saline and were tested for consumption of peppermint-sucrose under sodium depletion. Consumption was increased by sodium depletion only in Group BLK. In Experiment 2, a retardation test was used to show that presentation of saline-lemon before sucrose-lemon on each exposure day would establish sucrose as an inhibitor of saline. Neither exposure to sucrose-lemon before saline-lemon nor alternating exposure to sucrose and saline alone had the same effect. These results provide support for an associative theory of perceptual learning that suggests that exposure to complex stimuli aids later discrimination partially as a result of establishing inhibitory associations between their unique elements.

A unique surface membrane anchored purine-salvage enzyme is conserved among a group of primitive eukaryotic human pathogens.

Previously, we isolated and characterized the gene encoding the 3'-Nucleotidase/Nuclease (Ld3'NT/NU) from the human pathogen, Leishmania donovani. This unique cell surface enzyme has been shown to be involved in the salvage of host-derived purines, which are essential for the survival of this important protozoan parasite. In this report, we assessed whether the 3'-Nucleotidase/Nuclease was conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that a Ld3'NT/NU gene homolog was present in each of the visceral and cutaneous Leishmania species tested (i.e. isolates of L. donovani, L. infantum, L. tropica, L. major and L. mexicana, respectively). Further, results of colorimetric assays using 3'-adenosine monophosphate as substrate demonstrated that each of these organisms also expressed significant levels of 3'-nucleotidase enzyme activity. In addition, we showed that a Ld3'NT/NU gene homolog was expressed in each of these Leishmania species as a > 40 kDa 3'-nucleotidase enzyme activity. A Ld3'NT/NU gene homolog was also identified in two Crithidia species (C. fasciculata and C. luciliae) and Leptomonas seymouri but was only marginally detectable in Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens. Cumulatively, results of this study showed that an Ld3'NT/NU homolog was conserved amongst pathogenic Leishmania sp. which suggests that this enzyme must play an critical role in purine salvage for all members of this group of human pathogens.

Mediated conditioning and retrospective revaluation with LiCl then flavour pairings.

It is becoming accepted that the associative strength of a cue can change in its absence, despite this being difficult to explain using existing theories of Pavlovian conditioning. To investigate the influence of timing on learning about the representation of an absent cue, lithium chloride (LiCl) or a flavour previously paired with LiCl was presented in a distinctive context that had previously been paired with a neutral target flavour. The former treatment produced an aversion to the target flavour whether the LiCl was presented 10 min before, or immediately after, exposure to the context. However, presenting the flavour associate of LiCl created an aversion to the target flavour only if it had been presented 10 min after LiCl during initial training. This pattern of results cannot be explained in the terms of a simple timing account, and it is proposed that an explanation will require different associative rules operating in simultaneous and successive training schedules.

Evidence for inhibitory associations between the unique elements of two compound flavours.

In each of two experiments, rats were pre-exposed to two flavoured solutions, saline-lemon and sucrose-lemon. For group ALT, trials with one solution alternated with trials with the other. Group BLK received all trials with one solution in a block, before any trials with the other. An associative theory suggests that the alternating, but not the blocked, schedule would establish an inhibitory association between sucrose and saline. To provide a retardation test of this inhibition, some animals in each group were then given a single pairing of saline and sucrose, experienced sodium depletion, and were finally tested for their consumption of sucrose. Sodium depletion increased consumption of sucrose more in group BLK than in group ALT. In groups given no saline-sucrose pairing, sodium depletion had only a small effect on sucrose consumption, which was the same in both groups. After multiple pairings of saline and sucrose, sodium depletion had an equally large effect on sucrose consumption in both ALT and BLK groups. These results imply that alternating pre-exposure to two compound solutions does establish an inhibitory association between their unique elements, and thus provide support for an associative theory of perceptual learning.

Secretory and endocytic pathways converge in a dynamic endosomal system in a primitive protozoan.

Leishmania are a group of primitive eukaryotic trypanosomatid protozoa that are apically polarized with a flagellum at their anterior end. Surrounding the base of the flagellum is the flagellar reservoir that constitutes the site for endocytosis and exocytosis in these organisms. In the present study, we define a novel multivesicular tubular compartment involved in the intracellular trafficking of macromolecules in Leishmania. This dynamic structure appears to subtend the flagellar reservoir and extends towards the posterior end of the cell. Functional domains of several surface-expressed proteins, such as the gp63 glycosyl phosphatidyl inositol anchor and the 3'nucleotidase/nuclease transmembrane domain were fused to green fluorescent protein. These chimeric proteins were found to traffic through the secretory pathway and, while reaching their intended destinations, also accumulated within the intracellular tubular compartment. Using various compounds that are efficient fluid-phase markers used to track endocytosis in higher eukaryotes, we showed that this tubular compartment constitutes an important station in the endocytic pathway of these cells. Based on our functional observations of its role in the trafficking of expressed proteins and endocytosed markers, this compartment appears to have properties similar to endosomes of higher eukaryotes.

Invasive group B streptococcal disease in Maryland nursing home residents.

Between 1991 and 1995, among 999 nonpregnant adult Maryland residents with group B Streptococcus (GBS) isolated from a normally sterile site, 84 resided in nursing homes (NHs). The age-adjusted annual incidence of GBS infection (per 100,000 population) among those > or = 65 years old was 72.3 for NH residents and 17.5 for community residents (relative risk, 4.1; P < 0.001). Thirty-four case patients resided in 11 NHs with > or = 2 cases; 1 NH had 8 case patients within 22 months. Six of 8 case patients from 3 NHs had serotype V GBS. Molecular subtyping of several isolates identified 2 case patients in 1 NH with identical subtype patterns. NH residents have a markedly higher incidence of invasive GBS than do community residents > or = 65 years old and may serve as a target group for immunization when GBS vaccines become available. Further evaluation of intra-NH transmission of GBS is warranted.

Molecular characterization of a hyperinducible, surface membrane-anchored, class I nuclease of a trypanosomatid parasite.

The 3'-nucleotidase/nuclease (3'-NT/NU) is a surface enzyme unique to trypanosomatid parasites. These organisms lack the pathway for de novo purine biosynthesis and thus are entirely dependent upon their hosts to supply this nutrient for their survival, growth, and multiplication. The 3'-NT/NU is involved in the salvage of preformed purines via the hydrolysis of either 3'-nucleotides or nucleic acids. In Crithidia luciliae, this enzyme is highly inducible. For example, in these organisms purine starvation triggers an approximately 1000-fold up-expression of 3'-NT/NU activity. In the present study, we cloned and characterized a gene encoding this intriguing enzyme from C. luciliae (Cl). Sequence analysis showed that the Cl 3'-NT/NU deduced protein possessed five regions, which we defined here as being characteristic of members of the class I nuclease family. Further, we demonstrated that the Cl 3'-NT/NU-expressed protein possessed both 3'-nucleotidase and nuclease activities. Moreover, we showed that the dramatic up-expression of 3'-NT/NU activity in response to purine starvation of C. luciliae was concomitant with the approximately 100-fold elevation in steady-state mRNA specific for this gene. Finally, results of our nuclear run-on analyses demonstrated that such up-regulation in 3'-NT/NU enzyme activity was mediated at the posttranscriptional level.

Structurally conserved soluble acid phosphatases are synthesized and released by Leishmania major promastigotes.

Previously it was reported that promastigotes of virtually all pathogenic Leishmania species, except Leishmania major, release a structurally conserved soluble acid phosphatase (AcP) activity during their growth in vitro (P. S. Doyle and D. M. Dwyer, Exp. Parasitol. 77, 435-444 1993). In the current study we used a highly sensitive fluorogenic detection method to demonstrate that soluble AcPs were in fact produced by promastigotes of several different strains of L. major. These L. major AcP activities were readily immunoprecipitated with a rabbit antibody previously generated against the L. donovani AcP. Results of metabolic labeling and immunoprecipitations demonstrated that AcPs produced by the L. majors strains examined had an apparent molecular mass of approximately 77 kDa. Results of Southern hybridization analysis with an L. donovani AcP gene probe showed that the AcP gene loci were conserved in the L. major strains examined. Taken together, these results indicate that the AcP enzyme has been structurally and functionally conserved throughout the evolution of pathogenic species of Leishmania. Such conservation suggests that the AcPs play a functional role in the growth and survival of this group of important human pathogens.

Pathogenic leishmania secrete antigenically related chitinases which are encoded by a highly conserved gene locus.

Recently, we identified and characterized a single-copy chitinase gene (LdCht1) from Leishmania donovani, a protozoan pathogen of humans. It has been hypothesized that this parasite enzyme plays a critical role in the survival of all Leishmania species within their sandfly vectors and for their transmission to humans. Thus, in the current study, pulse-field gel electrophoresis and Southern hybridization with the LdCht1 gene probe were used to demonstrate that this chitinase gene has been conserved across species lines of various pathogenic Leishmania. Further, immunoprecipitation and enzyme activity assays using an anti-LdCht1-peptide serum were used to show that the chitinases produced and released by this group of parasites possessed both highly conserved antigenic epitopes and enzyme activities. Results of these studies demonstrate that the chitinase gene locus and enzyme activity have been conserved across species lines among this group of human pathogens.

Dissection of the functional domains of the Leishmania surface membrane 3'-nucleotidase/nuclease, a unique member of the class I nuclease family.

Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3'NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of the Ld3'NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. Using Ld3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3'NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of the Ld3'NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family.