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Two experiments were conducted to evaluate the in vitro and in vivo effects of a novel direct-fed microbial (DFM) containing Lactobacillus animalis LA-51, Propionibacterium freudenreichii PF-24, Bacillus licheniformis CH-200, and Bacillus subtilis King (BOVAMINE DEFEND® Plus) against Clostridium perfringens pathogenic strains. In Experiment 1 (in vitro), an agar diffusion assay was performed to qualitatively evaluate the in vitro inhibitory effects of the DFM against C. perfringens types A and C. Including the DFM in the tested yielded inhibition zones with greater than three ring diameters in a 96-well plate. In Experiment 2 (in vivo), twenty 1-day-old beef calves were allocated to control (n = 10) or DFM (n = 10) for 21 days. All calves were orally challenged with 1.0 × 108 colony forming units of C. perfringens type A strain S-107 per head. The procedures such as general health scores, body weight, and fecal sample collections were performed following the C. perfringens challenge. Daily feeding of DFM significantly reduced the incidence of diarrhea while improving general impression and appearance scores of calves. Overall, these results highlight the ability of the DFM containing L. animalis LA-51, P. freudenreichii PF-24, B. licheniformis CH-200, and B. subtilis (BOVAMINE DEFEND® Plus) to inhibit C. perfringens types A and C under different experimental settings.

Optimization and support of health and performance of preweaning dairy calves is paramount to any dairy operation, and natural solutions, such as probiotics, may help to achieve such a goal. Two experiments were designed to evaluate the effects of direct-fed microbial (DFM) Enterococcus faecium 669 on performance of preweaning dairy calves. In experiment 1, twenty 4-d-old Holstein calves [initial body weight (BW) 41 ± 2.1 kg] were randomly assigned to either (1) no probiotic supplementation (CON; n = 10) or (2) supplementation with probiotic strain E. faecium 669 during the preweaning period (DFM; n = 10) at 2.0 × 1010 cfu/kg of whole milk. Full individual BW was analyzed every 20 d for average daily gain (ADG) and feed efficiency (FE) determination. In experiment 2, thirty 4-d-old Holstein calves (initial BW 40 ± 1.9 kg) were assigned to the same treatments as in experiment 1 (CON and DFM). The DFM supplementation period was divided into period I (from d 0 to 21) and II (from d 22 to 63), with weaning occurring when animals were 67 d of age. During the entire experimental period, DFM was mixed into the whole milk at a rate of 1.5 × 1010 and 2.5 × 109 cfu/kg of whole milk/calf per day for periods I and II, respectively (6-time reduction). Full individual BW was taken every 21 d. As a routine of the experiment, calves were monitored daily, and diarrhea cases were evaluated using a daily 3-point fecal score. For both experiments, all data were analyzed using calf as the experimental unit. In experiment 1, DFM-supplemented calves were heavier on d 40 (+ 4.5 kg) and 60 (+ 6.5 kg) and had a greater ADG (+ 118 g) versus CON. In experiment 2, supplementation with DFM significantly tended to reduce diarrhea occurrence. Treatment × day and treatment × week interactions were observed for BW, ADG, and gain-to-feed ratio. Dairy calves supplemented with DFM were 1.8 and 3.5 kg heavier on d 42 and at weaning, respectively, and had a greater ADG from d 21 to 42 (+ 52 g) and 42 to 63 (+ 77 g) and gain-to-feed ratio from d 42 to 63 (+ 8.6%). In summary, supplementation of E. faecium 669 to dairy calves improved preweaning performance, even when the dose of the DFM was reduced by 6- to 8-times. Additionally, initial promising results were observed on diarrhea occurrence, but further studies are warranted.

Abstract Background Kisspeptin is a neuropeptide known for its role in the hypothalamic regulation of the reproductive axis. Following the recent description of kisspeptin and its 7-TM receptor, GPR54, in the dorsal root ganglia and dorsal horns of the spinal cord, we examined the role of kisspeptin in the regulation of pain sensitivity in mice. Results Immunofluorescent staining in the mouse skin showed the presence of GPR54 receptors in PGP9.5-positive sensory fibers. Intraplantar injection of kisspeptin (1 or 3 nmol/5 μl) induced a small nocifensive response in naive mice, and lowered thermal pain threshold in the hot plate test. Both intraplantar and intrathecal (0.5 or 1 nmol/3 μl) injection of kisspeptin caused hyperalgesia in the first and second phases of the formalin test, whereas the GPR54 antagonist, p234 (0.1 or 1 nmol), caused a robust analgesia. Intraplantar injection of kisspeptin combined with formalin enhanced TRPV1 phosphorylation at Ser800 at the injection site, and increased ERK1/2 phosphorylation in the ipsilateral dorsal horn as compared to naive mice and mice treated with formalin alone. Conclusion These data demonstrate for the first time that kisspeptin regulates pain sensitivity in rodents and suggest that peripheral GPR54 receptors could be targeted by novel drugs in the treatment of inflammatory pain.

Abstract Group II metabotropic glutamate receptors (mGluRs) couple to the inhibitory G-protein Gi. The group II mGluRs include two subtypes, mGlu2 and mGlu3, and their pharmacological activation produces analgesic effects in inflammatory and neuropathic pain states. However, the specific contribution of each one of the two subtypes has not been clarified due to the lack of selective orthosteric ligands that can discriminate between mGlu2 and mGlu3 subtypes. In this study we used mGlu2 or mGlu3 knock-out mice to dissect the specific role for these two receptors in the endogenous control of inflammatory pain and their specific contribution to the analgesic activity of mixed mGlu2/3 receptor agonists. Our results showed that mGlu2-/- mice display a significantly greater pain response compared to their wild type littermates. Interestingly the increased pain sensitivity in mGlu2-/- mice occurred only in the second phase of the formalin test. No differences were observed in the first phase. In contrast, mGlu3-/- mice did not significantly differ from their wild type littermates in either phase of the formalin test. When systemically injected, a single administration of the mGlu2/3 agonist, LY379268 (3 mg/kg, ip), showed a significant reduction of both phases in wild-type mice and in mGlu3-/- but not in mGlu2-/- mice. However tolerance to the analgesic effect of LY379268 (3 mg/kg, ip) in mGlu3-/- mice developed following 5 consecutive days of injection. Taken together, these results demonstrate that: (i) mGlu2 receptors play a predominant role over mGlu3 receptors in the control of inflammatory pain in mice; (ii) the analgesic activity of mixed mGlu2/3 agonists is entirely mediated by the activation of the mGlu2 subtype and (iii) the development of tolerance to the analgesic effect of mGlu2/3 agonists develops despite the lack of mGlu3 receptors.

Abstract L-acetylcarnitine (LAC), a drug utilized for the treatment of neuropathic pain in humans, has been shown to induce analgesia in rodents by up-regulating the expression of metabotropic glutamate receptor 2 (mGlu2) in dorsal root ganglia (DRG). We now report that LAC-induced upregulation of mGlu2 expression in DRG cultures involves transcriptional activation mediated by nuclear factor-kappaB (NF-κB). A single application of LAC (250 μM) to DRG cultures induced a transient increase in mGlu2 mRNA, which was observable after 1 hour and was no longer detectable after 1 to 4 days. In contrast, LAC treatment had no effect on mGlu3 mRNA expression. Pharmacological inhibition of NF-κB binding to DNA by caffeic acid phenethyl ester (CAPE) (2.5 μg/ml for 30 minutes) reduced the constitutive expression of mGlu2 and mGlu3 mRNA after 1–4 days and reduced the constitutive expression of mGlu2/3 protein at 4 days. This evidence combined with the expression of p65/RelA and c-Rel in DRG neurons indicated that expression of mGlu2 and mGlu3 is endogenously regulated by the NF-κB family of transcription factors. Consistent with this idea, the transient increase in mGlu2 mRNA induced by LAC after 1 hour was completely suppressed by CAPE. Furthermore, LAC induced an increase in the acetylation of p65/RelA, a process that enhances the transcriptional activity of p65/RelA. These results are consistent with the hypothesis that LAC selectively induces the expression of mGlu2 by acting as a donor of acetyl groups, thus enhancing the activity of the NF-κB family of transcription factors. Accordingly, we show that carnitine, which has no effect on pain thresholds, had no effect on p65/RelA acetylation and did not enhance mGlu2 expression. Taken together, these results demonstrate that expression of mGlu2 and mGlu3 mRNA is regulated by the NF-κB transcriptional machinery, and that agents that increase acetylation and activation of NF-κB transcription factors might induce analgesia via upregulation of mGlu2 in DRG neurons.

This study investigated the impact of L. animalis 506 on gut barrier integrity and regulation of inflammation in vitro using intestinal epithelial cell lines. Caco-2 or HT29 cell monolayers were challenged with enterotoxigenic E. coli (ETEC) or a ruminant isolate of Salmonella Heidelberg in the presence or absence of one of six probiotic Lactobacillus spp. strains. Among these, L. animalis 506 excelled at exerting protective effects by significantly mitigating the decreased transepithelial electrical resistance (TEER) as assessed using area under the curve (AUC) (p < 0.0001) and increased apical-to-basolateral fluorescein isothiocyanate (FITC) dextran translocation (p < 0.0001) across Caco-2 cell monolayers caused by S. Heidelberg or ETEC, respectively. Similarly, L. animalis 506 and other probiotic strains significantly attenuated the S. Heidelberg- and ETEC-induced increase in IL-8 from HT29 cells (p < 0.0001). Moreover, L. animalis 506 significantly counteracted the TEER decrease (p < 0.0001) and FITC dextran translocation (p < 0.0001) upon challenge with Clostridium perfringens. Finally, L. animalis 506 significantly attenuated DON-induced TEER decrease (p < 0.01) and FITC dextran translocation (p < 0.05) and mitigated occludin and zona occludens (ZO)-1 redistribution in Caco-2 cells caused by the mycotoxin. Collectively, these results demonstrate the ability of L. animalis 506 to confer protective effects on the intestinal epithelium in vitro upon challenge with enteric pathogens and DON known to be of particular concern in farm animals.

Few data are available evaluating the stability of direct-fed microbials (DFM) following their inclusion in different feed matrices. Therefore, six Exp. evaluated the recovery of bacilli spores (BOVACILLUSTM; Exp. 1 to 3) and an Enterococcus faecium DFM (LACTIFERM®; Exp. 4 to 6) when included in different feed preparations. The Bacillus-based DFM was included into pelleted feed prepared in different temperatures (75 to 95 °C), whereas both DFM were assessed in premix and milk replacer preparations. Bacillus spores and E. faecium recovery was evaluated through standard methodologies and data were reported as log10 colony forming units/gram of feed. The recovery of Bacillus spores was within the expected range and was not impacted by the temperature of pellet preparation (Exp. 1). Bacilli recovery was also stable up to 12 months in the premix and was not impacted by the temperature of milk replacer preparation. Regarding the Exp. with E. faecium (Exp. 4 to 6), its recoveries in the mineral premix and milk powder did not differ from T0 and were not impacted by the conditions of milk replacer preparation. These data are novel and demonstrate the stability of a Bacillus-based and an E. faecium-based DFM when included in different feed matrices often used in dairy production.

Direct-fed microbial products (DFM) are probiotics that can be used advantageously in ruminant production. The in vitro gas production technique (IVGPT) is a method to simulate rumen fermentation and can be used to measure degradation, gas production, and products of fermentation of such additives. However, inter-laboratory differences have been reported. Therefore, tests using the same material were used to validate laboratory reproducibility. The objective of this study was to assess the effect of adding two DFM formulations on fermentation kinetics, methane (CH4) production, and feed degradation in two different basal feeds while validating a newly established IVGPT laboratory. Six treatments, with three replicates each, were tested simultaneously at the established IVGPT lab at the University of Copenhagen, and the new IVGPT lab at Chr. Hansen Laboratories. Maize silage (MS) and grass silage (GS) were fermented with and without the following DFM: P1: Ligilactobacillus animalis and Propionibacterium freudenreichii (total 1.5 × 107 CFU/mL), P2: P1 with added Bacillus subtilis and B. licheniformis (total 5.9 × 107 CFU/mL). The DFM were anaerobically incubated in rumen fluid and buffer with freeze-dried silage samples for 48 h. Total gas production (TGP: mL at Standard Temperature and Pressure/gram of organic matter), pH, organic matter degradability (dOM), CH4concentration (MC) and yield (MY), and volatile fatty acid (VFA) production and profiles were measured after fermentation. No significant differences between the laboratories were detected for any response variables. The dOM of MS (78.3%) was significantly less than GS (81.4%), regardless of the DFM added (P1 and P2). There were no significant differences between the effects of the DFM within the feed type. MS produced significantly more gas than GS after 48 h, but GS with DFM produced significantly more gas at 3 and 9 h and a similar gas volume at 12 h. Both DFM increased TGP significantly in GS at 48 h. There was no difference in total VFA production. However, GS with and without probiotics produced significantly more propionic acid and less butyric acid than MS with and without probiotics. Adding P2 numerically reduced the total methane yield by 4–6% in both MS and GS. The fermentation duration of 48 h, used to determine maximum potential dOM, may give misleading results. This study showed that it is possible to standardize the methodology to achieve reproducibility of IVGPT results. Furthermore, the results suggest that the P2 DFM may have the potential to reduce CH4 production without affecting organic matter degradation.

Metabotropic glutamate (mGlu) receptors are considered as candidate drug targets for the treatment of schizophrenia. These receptors form a family of eight subtypes (mGlu1 to −8), of which mGlu1 and −5 are coupled to Gq/11, and all other subtypes are coupled to Gi/o. Here, we discuss the possibility that selective ligands of individual mGlu receptor subtypes may be effective in controlling the core symptoms of schizophrenia, and, in some cases, may impact mechanisms underlying the progression of the disorder. Recent evidence indicates that activation of mGlu1 receptors inhibits dopamine release in the meso-striatal system. Hence, selective positive allosteric modulators (PAMs) of mGlu1 receptors hold promise for the treatment of positive symptoms of schizophrenia. mGlu5 receptors are widely expressed in the CNS and regulate the activity of cells that are involved in the pathophysiology of schizophrenia, such as cortical GABAergic interneurons and microglial cells. mGlu5 receptor PAMs are under development for the treatment of schizophrenia and cater the potential to act as disease modifiers by restraining neuroinflammation. mGlu2 receptors have attracted considerable interest because they negatively modulate 5-HT2A serotonin receptor signaling in the cerebral cortex. Both mGlu2 receptor PAMs and orthosteric mGlu2/3 receptor agonists display antipsychotic-like activity in animal models, and the latter drugs are inactive in mice lacking mGlu2 receptors. So far, mGlu3 receptors have been left apart as drug targets for schizophrenia. However, activation of mGlu3 receptors boosts mGlu5 receptor signaling, supports neuronal survival, and drives microglial cells toward an antiinflammatory phenotype. This strongly encourages research of mGlu3 receptors in schizophrenia. Finally, preclical studies suggest that mGlu4 receptors might be targeted by novel antipsychotic drugs, whereas studies of mGlu7 and mGlu8 receptors in animal models of psychosis are still at their infancy.