Genome-wide association analysis for susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in US Holsteins.

Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP and concurrently modifying management to reduce the likelihood of infection. The current study was motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. Two separate GWAS analyses were conducted, the first using 897 genotyped Holstein artificial insemination sires with phenotypes derived from incidence of MAP infection among daughters based on milk ELISA testing records. The second GWAS analysis was a case-control design using US Holstein cows phenotyped for MAP infection by serum ELISA or fecal culture tests. Cases included cows positive for either serum ELISA, fecal culture, or both. Controls consisted of animals negative for all tests conducted. A total of 376 samples (70 cases and 306 controls) from a University of Minnesota Johne's management demonstration project and 184 samples (76 cases and 108 controls) from a Michigan State University study were used. Medium-density (sires) and high-density (cows) genotype data were imputed to full genome sequence for the analyses. Marker-trait associations were analyzed using the single-step (ss)GWAS procedure implemented in the BLUPF90 suite of programs. Evidence of significant genomic contributions for susceptibility to MAP infection were observed on multiple chromosomes. Results were combined across studies in a meta-analysis, and increased support for genomic regions on BTA7 and BTA21 were observed. Gene set enrichment analysis suggested pathways for antigen processing and presentation, antimicrobial peptides and natural killer cell-mediated cytotoxicity are relevant to variation in host susceptibility to MAP infection, among others. Genomic prediction was evaluated using a 5-fold cross-validation, and moderate correlations were observed between genomic breeding value predictions and daughter averages (∼0.43 to 0.53) for MAP infection in testing data sets. These results suggest that genomic selection against susceptibility to MAP infection is feasible in Holstein cattle.

An ovine hepatorenal fibrocystic model of a Meckel-like syndrome associated with dysmorphic primary cilia and TMEM67 mutations.

Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.

Role of conditioned taste aversion in the development of activity anorexia.

Activity anorexia (AA) occurs when rats are restricted to one meal period (60-90 min) each day and have unlimited access to a running wheel the rest of the time. This AA procedure also contains the conditions necessary for conditioned taste aversion (CTA). The food eaten during the meal period is the conditioned stimulus paired with the unconditioned stimulus produced by wheel running. Thus, CTA to food may account in part for the decreased eating in AA. To test this possibility, male rats were subjected to the AA procedure. They had daily 90-min exposures to their familiar chow and spent the rest of the day in running wheels (experimental condition) or home cages (control condition). A second, concurrent experiment had the same procedure except that novel, rather than familiar, food was used. In both experiments, AA occurred; the rats allowed to wheel run ate less than those in the control condition. Several days after AA training, a two-food preference test assessed whether CTA occurred. Wheel running induced CTA when food was novel but not when it was familiar. Since AA is typically studied with procedures using familiar food, the present findings indicate that CTA plays little or no role in AA.

Preadaptation to the feeding schedule does not eliminate activity-based anorexia in rats.

To test whether activity-based anorexia (ABA) still occurs after preadaptation to the feeding schedule, 20 rats were first exposed to a feeding schedule of one 90-min meal per day until adaptation occurred (measured by maintenance of stable body weight). Then, during ABA training, half the rats (wheel group) were confined in running wheels except during the daily meal, and half (cage group) were not. Wheel running suppressed feeding--that is, food intake in the wheel group was less than that in the cage group. Also, the rats in the wheel group lost weight, whereas those in the cage group did not. Wheel running increased over days. Thus, the defining characteristics of ABA were evident in rats that were not subjected to ABA training until after they had become well adapted to the feeding schedule. These findings support the view that the suppression of feeding produced by wheel running triggers the vicious circle of ABA. They also cast doubt on the hypothesis that activity-induced interference with adaptation to the feeding schedule plays a key role in causing ABA.

Naloxone attenuates the conditioned place preference induced by wheel running in rats.

Pairings, during which an episode of wheel running is followed by confinement in a distinctive place, produce conditioned place preference (CPP) in rats. This finding indicates that wheel running has a rewarding effect that outlasts the activity itself. In two similar experiments, we tested the hypothesis that this rewarding effect of wheel running is mediated by endogenous opioids. During a paired trial, the rats in the naloxone group were first allowed to wheel run for 2 h, then injected with naloxone (0.5 or 0.1 mg/kg in Experiments 1 and 2, respectively), and 10 min later placed in a distinctive chamber. During an unpaired trial, these rats were confined in an adjoining chamber without wheel running. Naloxone was injected before placement in both chambers, so that if naloxone-induced conditioned place aversion occurred, it would have counteracting effects on performance during the preference test. The rats in the saline group were similarly treated, except that saline was injected instead of naloxone. CPP occurred in the saline group, but not in the naloxone group. Thus, naloxone attenuated the CPP induced by wheel running. This finding supports the hypothesis that the rewarding effect of wheel running is mediated by endogenous opioids.

Pairings of a distinctive chamber with the aftereffect of wheel running produce conditioned place preference.

Wheel running reinforces behavior that precedes it. Also, wheel running can produce activity anorexia, a marked suppression of feeding in food-restricted rats. Some authors propose that the activity anorexia effect is produced by activation of the same reward system that mediates the reinforcing effect. One hypothesis is that such activation persists after wheel running stops and results in a rewarding aftereffect that suppresses feeding. Alternatively, such activation may give rise to an opponent process, an aversive aftereffect that suppresses feeding. The method of place conditioning was used to test whether the aftereffect of wheel running is rewarding or aversive. Food-deprived rats received pairings of a distinctive chamber with the aftereffect of wheel running. In Experiment 1, 2 h in a running wheel followed by 30 min in a distinctive chamber produced conditioned place preference. In Experiment 2, 22-22.5 h in a running wheel was followed by 30 min in the chamber and then a 60-min feeding test. Wheel running suppressed feeding and produced conditioned place preference. The conditioned place preference indicates that the aftereffect of wheel running is reinforcing rather than aversive. This finding supports the idea that the activation of the reward system persists after wheel running stops, thereby suppressing food intake.

Activity in the circular alley does not produce the activity anorexia syndrome in rats.

The activity anorexia syndrome is characterized by reduced food intake and body weight compared to control levels and increasing levels of physical activity. To induce it, food-restricted rats are confined in running wheels except during the daily meal. We tested whether activity in a flat circular alley also produces the activity anorexia syndrome. In Experiment 1, food-restricted rats were maintained in alleys, wheels, or home cages (control condition). In Experiment 2, they were maintained in alleys, wheels, novel cages, or home cages. The novel cage was added to control for the possibility that the alley might produce an anorectic effect simply because it was a new living space. The alley did not produce the activity anorexia syndrome whereas the wheel did. Although weight loss was greater in the alley than home-cage condition, the alley produced weak, inconsistent suppression of feeding. Moreover, the suppression produced by the alley may have stemmed simply from living in a novel environment. Finally, in contrast to wheel running, alley activity decreased over days. Alley activity, unlike wheel running, may not be reinforcing. Likely, a physical activity must be reinforcing to produce the activity anorexia syndrome. Implications for anorexia nervosa were discussed.

Chlordiazepoxide attenuates activity-induced anorexia and weight loss in rats.

In Experiment 1, the effect of repeated injections of 2.5, 5.0, or 10.0 mg/kg of chlordiazepoxide (CDP) on food intake and body weight was studied in rats on an activity anorexia (AA) regimen. For several days before CDP testing began, rats lived in activity wheels and had one 60-min meal per day. During CDP testing, this regimen continued except that each rat was injected with an appropriate dose of CDP or saline 30 min before each meal. CDP enhanced food intake; 5.0 mg/kg seemed most effective. However, the CDP-induced increase in eating did not noticeably stem weight loss. In Experiment 2, after several days of AA training, CDP (5.0 mg/kg) was tested under less severe conditions; food remained restricted, but access to the wheels was discontinued. Rats given CDP ate more and gained more weight than controls. These findings suggest that benzodiazepines such as CDP may help in treating anorexia nervosa and other anorectic conditions in humans.

Wheel running simultaneously induces CTA and facilitates feeding in non-deprived rats. Conditioned taste aversion.

Previous findings indicate that wheel running can have either an aversive or an appetitive effect. That is, wheel running for 30 min induces conditioned taste aversion (CTA) in rats trained while hungry and thirsty but facilitates feeding in non-deprived rats. In Experiment 1, wheel running was also found to be effective in producing CTA in non-deprived rats. Therefore, Experiment 2 tested whether wheel running produces the aversive and appetitive effects simultaneously. During each of four training trials, two groups of non-deprived rats were given a flavored solution to drink for 10 min. Then those in the wheel group were put in running wheels for 30 min whereas those in the cage group spent 30 min in small cages. Finally, all rats were given a 60-min feeding test. After the first trial, the wheel group drank less flavored solution than the cage group during each of the remaining trials. The wheel group also ate more than the cage group on each feeding test. These results indicate that wheel running produces CTA and facilitates eating at the same time. A role for the mesolimbic dopamine reward system in these effects was considered.

Chlordiazepoxide counteracts activity-induced suppression of eating in rats.

Because benzodiazepines such as chlordiazepoxide increase food intake, the present experiments tested the effect of chlordiazepoxide on food intake in an animal model of anorexia nervosa, called activity anorexia (AA). To induce AA, rats (Rattus norvegicus) were maintained in activity wheels and restricted to a single 60-min feeding period each day. As previously found, this procedure suppressed food intake. After several days of this training, food intake was measured 30 min after the rats were injected with chlordiazepoxide (5 mg/kg) or saline. In 2 experiments, chlordiazepoxide counteracted the suppression of food intake produced by AA. Because benzodiazepines have been found to increase food intake in many mammalian species including primates, the present results suggest that benzodiazepines could be useful in the treatment of anorexia nervosa.

A small amount of wheel running facilitates eating in nondeprived rats.

A high level of dopaminergic activation such as that produced by a high dose of amphetamine suppresses eating, whereas a low level such as that produced by a low dose can have the opposite effect. Like a high dose of amphetamine, a high level of wheel running also suppresses eating. It was hypothesized that this suppression is due to dopaminergic activation produced by wheel running. If so, a relatively small amount of wheel running should produce a low level of dopaminergic activation and thus have the same effect as a low dose of amphetamine: facilitation of eating. In the present experiment, nondeprived rats were allowed to run in a wheel for 30 min before a feeding test. As predicted, a little wheel running facilitated eating in these rats relative to appropriate controls. The present results may be relevant to an understanding of eating disorders in humans.

Wheel running induces conditioned taste aversion in rats trained while hungry and thirsty.

Rats while both hungry and thirsty were given three exposures to either a salt solution or a sour solution followed by a brief period of wheel running. On other occasions, these rats while hungry and thirsty were exposed to the alternate solution without wheel running. Conditioned aversion to the flavor paired with wheel running was evident after two pairings. During final tests with each solution, the rats were thirsty but not hungry. As expected, conditioned aversion to the particular flavor paired with wheel running was still observed despite a change in deprivation state from training to test. These results indicate that wheel running can induce sickness. Because sickness suppresses eating, activity anorexia in rats and some forms of anorexia nervosa in humans may be caused, at least in part, by sickness.

Pairings of a drug or place conditioned stimulus with lithium chloride produce conditioned sickness, not antisickness.

In different experiments, pairings of a drug (pentobarbital or morphine) or place as the conditioned stimulus (CS) with lithium-induced sickness as the unconditioned stimulus (US) were given to rats to produce Pavlovian conditioning. Control rats received unpaired exposures. In the test, each rat was exposed to the CS, injected with lithium, and then offered food. If such pairings produce conditioning of antisickness (i.e., a compensatory response that opposes lithium sickness), then the experimental rats should eat more than the controls. The reverse occurred. Thus, pairings of a drug or place CS with a lithium US resulted in conditioned sickness rather than antisickness.

Conditioned taste preference produced by pairing a taste with a low dose of morphine or sufentanil.

Taste conditioning produced by pairing a taste with low doses of morphine or sufentanil was studied in rats in five experiments. Conditioned taste preferences were obtained with a trace conditioning procedure in which ingestion of a flavored solution was followed by an injection of sufentanil, either 0.25 mcg/kg in experiment 1 or 0.50 mcg/kg in experiment 2. Morphine produced less consistent results than sufentanil. When a similar trace conditioning procedure was used with morphine, a dose of 0.25 mg/kg produced no observable taste conditioning in experiment 3 while 0.42 mg/kg was marginally effective in producing a conditioned taste aversion in experiment 4. In experiment 5, however, conditioning of a taste preference was produced by 0.42 mg/kg morphine with a simultaneous conditioning procedure in which the morphine injection preceded ingestion of the flavored solution. The simultaneous procedure was presumed to facilitate the conditioning of taste preference by minimizing the conditioning of taste aversion.

Repeated exposures intensify rather than diminish the rewarding effects of amphetamine, morphine, and cocaine.

It is commonly believed that repeated exposures diminish the pleasurable effects of drugs and hence that pleasure must have only a minor role in addiction. In six experiments with rats, repeated exposures to amphetamine, morphine, or cocaine were found to enhance the drug-induced rewarding effect as measured by conditioned place preference. Thus, sensitization to the rewarding effect, rather than tolerance, was obtained. Also, cross-sensitization was obtained; exposures to amphetamine enhanced the rewarding effect of morphine and vice versa; similarly, exposures to morphine enhanced the rewarding effect of cocaine. These findings support a new theory: drugs of abuse are addictive because repeated exposures sensitize the central reward mechanism so that drug taking produces a progressively greater reinforcing effect each time it occurs.

The hedonic effects of amphetamine and pentobarbital in goldfish.

Goldfish were confined in a distinctive chamber while drugged with amphetamine in Experiment A or pentobarbital in Experiment P. During a later test, the goldfish in Experiment A showed a preference for the chamber associated with amphetamine, whereas those in Experiment P showed an aversion to the chamber associated with pentobarbital. Thus, amphetamine produced a rewarding effect while pentobarbital was aversive. The mechanism of pentobarbital's aversive effect is unknown. However, there is convincing evidence that amphetamine produces a rewarding effect in rats, monkeys and humans by increasing the synaptic concentration of dopamine in the central reward system. Since the goldfish brain has cells containing dopamine, the same mechanism is likely to be responsible for amphetamine's rewarding effect in goldfish. This similarity suggests that the central reward systems of such diverse species as goldfish, rats, monkeys, and humans have a common evolutionary origin.

Enhancement of conditioned preference for a place paired with amphetamine produced by blocking the association between place and amphetamine-induced sickness.

Rats learn to prefer a place that has been paired with the rewarding effect of amphetamine. Since amphetamine is also known to produce an aversive effect, called here sickness, pairings of a place with amphetamine should produce a place-sickness association as well as the place-reward association that underlies the conditioned place preference. The purpose of the present experiments was to enhance the conditioned place preference produced by place-amphetamine pairings by blocking the place-sickness association. In Experiments 1 and 2, the taste of saccharin was paired with sickness induced by amphetamine or by lithium, respectively. The saccharin taste was presented prior to each pairing of a white chamber with amphetamine to block the place-sickness association. In Experiment 3, a brief placement in a distinctive cage that had previously been paired with lithium-induced sickness preceded each pairing of the white chamber with amphetamine. Blocking of the place-sickness association occurred as evidenced by the reliable enhancement of conditioned place preference obtained in each of the three experiments.

Conditioned slowing of stomach emptying produced by Pavlovian pairings of a drug CS or a place with lithium chloride.

Lithium chloride, in common with other drugs with emetic effects, prolongs stomach emptying. In different experiments, a drug state induced by a low dose of pentobarbital in Experiment 1 and morphine in Experiment 2 or a distinctive place (Experiments 3, 4) was the conditioned stimulus paired with lithium chloride as the unconditioned stimulus. In each case, Pavlovian conditioning occurred and the conditioned response mimicked lithium's unconditioned effect on stomach emptying.

Formation of associations of colored and flavored food with induced sickness in five avian species.

Domestic ducks, geese, pigeons, quail, and chickens were given colored, flavored, or colored and flavored food and then injected with lithium chloride. Each species showed learning of color and taste aversions. Flavor facilitated the formation of color aversions in ducks, geese, and pigeons but not in quail or chickens. Color interfered with the formation of flavor aversions in quail and chickens but not in the other three species. These findings indicate that all birds can probably associate both colored and flavored food with induced sickness and that colored food is more easily associated with induced sickness than is colored water. Moreover, these findings suggest that the capacity to associate colored and flavored food with induced sickness and the interaction between color and flavor in food vary between species. Birds, such as quail and chickens, that eat relatively tasteless food rely more on color than on flavor cues when forming learned food aversions. Birds that can select their food on the basis of taste, such as ducks and geese, rely more on flavor than on color when forming aversions to food. Birds, such as pigeons, that are initially raised on tasty food before switching to predominantly tasteless food show tendencies that are similar to those of ducks and geese.

The painlike effect of gallamine and naloxone differs from sickness induced by lithium chloride.

In rats, the conditioned place and taste aversions produced by 31.8 mg/kg lithium chloride were compared with those produced by 10 mg/kg gallamine in Experiment 1 and 20 mg/kg or 2.5 mg/kg naloxone in Experiments 2 and 3, respectively. Lithium produced stronger taste aversions than gallamine or the two doses of naloxone, but gallamine and naloxone each produced stronger place aversions than lithium. These findings support the distinction between two kinds of drug-induced aversive effects, having different associative properties. One effect, called sickness, is more associable with taste than with place cues; the second effect is, like pain, more associable with place than with taste.