Implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry.

AIMS: The present study describes the implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry. METHODS AND RESULTS: Real-time PCR with Salmonella invA-specific primers and a standard bacteriological method was applied to detect Salmonella in tetrathionate enrichment cultures of 492 intestinal homogenates and 27 drag swabs from 47 poultry flocks. The number of positive individual samples by real-time PCR and culture method was 65 (12.5%) and 35 (6.8%), respectively. The number of Salmonella-positive flocks was 13 (27.7%) by both methods. PCR detection required 25 min for up to 32 samples. Melting curve analysis revealed the Tm for Salmonella-specific PCR product as 87 +/- 1 degrees C. CONCLUSIONS: Implementation of real-time PCR to tetrathionate broth enrichment step reduces the Salmonella detection time to 18 h and 25 min. Isolation of Salmonella should be carried out with PCR to determine the serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR is a powerful tool in rapid and accurate Salmonella monitoring in poultry companies, together with standard bacteriology.

Detection of salmonellae in chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis.

This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonella genus-specific 281-bp PCR product was detected when Salmonella strains but not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml(-1), respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml(-1), respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella could not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harbor Salmonella by both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detect Salmonella with the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased birds, the total time for PCR and detection is reduced to 2 h since the 18-h PE is not required. These results indicate that TTB enrichment, bacterial lysis, and genus-specific capillary PCR combined with capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identify Salmonella-infected flocks.

Glycyl-glutamine inhibits the respiratory depression, but not the antinociception, produced by morphine.

Glycyl-glutamine (Gly-Gln; beta-endorphin(30-31)) is an endogenous dipeptide that is synthesized through the posttranslational processing of beta-endorphin in brain stem regions that control respiration and autonomic function. This study tested the hypothesis that Gly-Gln administration to conscious rats will prevent the respiratory depression caused by morphine without affecting morphine antinociception. Rats were administered Gly-Gln (1-100 nmol) or saline (10 microl) intracerebroventricularly followed, 5 min later, by morphine (40 nmol icv). Arterial blood gases and pH were measured immediately before Gly-Gln and 30 min after morphine injection. Gly-Gln pretreatment inhibited morphine-induced hypercapnia, hypoxia, and acidosis significantly. The response was dose dependent and significant at Gly-Gln doses as low as 1 nmol. In contrast, Gly-Gln (1-300 nmol) had no effect on morphine-evoked antinociception in the paw withdrawal test. When given alone to otherwise untreated animals, Gly-Gln did not affect nociceptive latencies or blood gas values. These data indicate that Gly-Gln inhibits morphine-induced respiratory depression without compromising morphine antinociception.

Localization of pro-opiomelanocortin mRNA transcripts and peptide immunoreactivity in rat heart.

OBJECTIVE: alpha-Melanocyte-stimulating hormone (alpha-MSH), beta-endorphin and other pro-opiomelanocortin-(POMC) derived peptides have been detected in the heart, but it is uncertain whether they are synthesized by cardiomyocytes or by cardiac nerves innervating the heart. The objective of this study was to determine whether POMC peptides are synthesized by cardiomyocytes. METHODS: Pro-opiomelanocortin peptides were localized in rat heart by immunohistochemistry using antisera against alpha-MSH, beta-endorphin and alpha N-acetyl-beta-endorphin, the predominant POMC peptides found in heart. Pro-opiomelanocortin mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) using primers that discriminate between full-length POMC mRNA and a 5' truncated POMC transcript that is presumed to be non-functional. RESULTS: alpha-Melanocyte-stimulating hormone, beta-endorphin and alpha N-acetyl-beta-endorphin immunoreactivities were localized in atrial myocytes, particularly in the atrial appendages, but not to a significant extent in ventricular myocytes. Cardiac nerves were not immunostained. Atrial natriuretic peptide (ANP) immunoreactivity was similarly distributed in the adult heart. In neonatal heart, POMC-peptide and ANP immunoreactivities were present in both atrial and ventricular myocytes. RT-PCR amplification showed that full-length POMC mRNA transcripts were present in both atrial and ventricular tissue and provide evidence that 5' truncated POMC mRNA is expressed in heart. CONCLUSIONS: These results support the hypothesis that cardiomyocytes synthesize POMC peptides.

The effects of choline on body temperature in conscious rats.

Choline (75-300 microg) produced dose-dependent hypothermia when injected intracerebroventricularly (i.c.v.). Pre-treatment with the muscarinic receptor antagonist, atropine (10 microg, i.c.v.), blocked the hypothermic effect of choline (150 microg), but the response was only partially attenuated by pre-treatment with the nicotinic receptor antagonist, mecamylamine (20 microg, i.c.v.). Pirenzepine (25 microg), a muscarinic M1 receptor antagonist, or hexahydro-siladifenidol (HHSD) (100 microg), a muscarinic M3 receptor antagonist, also blocked choline-induced hypothermia when injected centrally. Unlike the other muscarinic receptor antagonists, M2-selective 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyri do[2,3-b][1,4]benzodiazepin-6-one (AF-DX116) (10 microg), did not affect choline-induced hypothermia. We also found that choline-induced hypothermia was very sensitive to the ambient temperature. Similar to its effect at room temperature, choline produced dose-dependent hypothermia at 4 degrees C, but this effect was abolished at 32 degrees C. These data suggest that choline produces hypothermia and this effect is mediated by muscarinic receptors.

Cyclo(Gly-Gln) inhibits the cardiorespiratory depression produced by beta-endorphin and morphine.

Glycyl-L-glutamine (Gly-Gln; beta-endorphin 30-31) is an endogenous dipeptide that is synthesized through the post-translational processing of beta-endorphin. Previously, we showed that Gly-Gln inhibits the hypotension and respiratory depression produced by central beta-endorphin administration. In this study, we tested whether cyclo(Gly-Gln), a non-polar, cyclic Gly-Gln derivative, was similarly effective following intracerebro-ventricular (i.c.v.) or intra-arterial (i.a.) administration to pentobarbital-anesthetized rats pretreated with beta-endorphin (0.5 nmol i.c.v.). Intracerebroventricular cyclo(Gly-Gln) (0.3, 0.6 or 1.0 nmol) injection produced a dose-dependent inhibition of beta-endorphin-induced hypotension, but not bradycardia, with a potency similar to that of Gly-Gln. Cyclo(Gly-Gln) (5 mg/kg) was also effective following i.a. injection and significantly attenuated the fall in arterial pressure elicited by i.c.v. beta-endorphin, consistent with evidence that cyclic dipeptides permeate the blood-brain barrier; i.a. Gly-Gln was ineffective. Intra-arterial cyclo(Gly-Gln) (5 mg/kg) and i.c.v. Gly-Gln (10 nmol) also attenuated the hypotension and respiratory depression induced by morphine (50 or 100 nmol i.c.v.). Cyclo(Gly-Gln) (0.5, 5.0 or 50.0 mg/kg i.a.) had no effect on arterial pressure or heart rate when given alone. These findings indicate that cyclo(Gly-Gln) is a biologically active peptide capable of reversing the cardiorespiratory depression produced by beta-endorphin or morphine.

Beta-endorphin-induced cardiorespiratory depression is inhibited by glycyl-L-glutamine, a dipeptide derived from beta-endorphin processing.

Glycyl-L-glutamine (Gly-L-Gln), or beta-endorphin-(30-31) [beta-End-(30-31)], is synthesized through the post-translational processing of beta-End-(1-31). Evidence that gly-L-gln is a prominent end product of beta-End-(1-31) processing in cardioregulatory regions of rat brain prompted us to investigate whether it modulates the cardiorespiratory depression induced by central beta-End-(1-31) injection. As shown previously, beta-End-(1-31) (0.5 nmol) lowered mean arterial pressure (MAP) and HR when administered i.c.v. to pentobarbital-anesthetized rats. Gly-L-gln (0.3, 0.6, 1.0 and 10.0 nmol) produced a dose-related inhibition of beta-End-(1-31)-induced hypotension, but not bradycardia, when injected i.c.v. 15 min after beta-End-(1-31). This effect was not attributable to hydrolysis, because equimolar amounts of L-glycine and L-glutamine were ineffective. A comparable response was observed when gly-L-gln was administered to urethane-anesthetized rats and when it was injected before beta-End-(1-31). Gly-L-gln also attenuated the respiratory depressant effect of beta-End-(1-31), significantly inhibiting beta-End-(1-31)-induced hypoxia and hypercapnia. Gly-L-gln (1, 10 and 100 nmol) was inactive when injected alone, however, and produced no significant variation from base-line MAP or HR values. These results demonstrate that gly-L-gln inhibits beta-End-(1-31)-induced cardiorespiratory depression, consistent with accumulating evidence that gly-L-gln functions as a neuromodulator.