Caprylic acid reduces Salmonella Enteritidis populations in various segments of digestive tract and internal organs of 3- and 6-week-old broiler chickens, therapeutically.

We investigated the efficacy of feed supplemented with caprylic acid (CA), a natural, 8-carbon fatty acid for reducing Salmonella enterica serovar Enteritidis colonization in commercial broiler chickens. In separate 3- and 6-wk trials, 1-d-old straight-run broiler chicks (n = 70 birds/trial) were assigned to a control group (challenged with Salmonella Enteritidis, no CA) and 2 replicates of 0.7 and 1% CA (n = 14 birds/group). Water and feed were provided ad libitum. On d 1, birds were tested for any inherent Salmonella (n = 2 birds/group). For the 3-wk trial, on d 5, birds were challenged with 8 log(10) cfu of Salmonella Enteritidis of a 4-strain mixture by crop gavage, and after 5 d postchallenge, birds (n = 2 birds/group) were euthanized to ensure Salmonella Enteritidis colonization. Caprylic acid was supplemented the last 5 d before tissue collection (n = 10 birds/group). For the 6-wk trial, on d 25, birds were challenged and confirmed for Salmonella Enteritidis colonization. The birds (n = 10 birds/group) were euthanized for tissue samples after CA supplementation for the last 5 d. Caprylic acid at 0.7 or 1% decreased Salmonella Enteritidis populations in cecum, small intestine, cloaca, liver, and spleen in both 3- and 6-wk trials. Body weight of birds did not differ between the groups (P ≥ 0.05). Further, to elucidate a potential antibacterial mechanism of action of CA, we investigated if CA could reduce Salmonella Enteritidis invasion of an avian epithelial cell line and expression of invasion genes hilA and hilD. The cell invasion study revealed that CA reduced invasive abilities of all Salmonella Enteritidis strains by ~80% (P < 0.05). Gene expression studies indicated that CA downregulated (P < 0.001) Salmonella invasion genes hilA and hilD. These results suggest that supplementation of CA through feed could reduce Salmonella Enteritidis colonization in broiler chicken and potentially reduces the pathogen's ability to invade intestinal epithelial cells by downregulating key invasion genes, hilA and hilD.

Monitoring gastrointestinal intraluminal PCO2: problems with airflow methods.

UNLABELLED: Gastrointestinal intraluminal PCO2 (PiCO2) information is used to assess the adequacy of trauma patient resuscitation and to assist in choosing resuscitative interventions. Therefore, determining the limitations and potential caveats of different PiCO2 monitoring systems is clinically important. This study compared two PCO2 monitoring systems. The airflow device adds and then removes air samples to quantitate PCO2, whereas the fiber-optic device does not. METHODS: Airflow (TRIP Tonometer/Tonocap) and fiber-optic (Neotrend) systems were used. In vitro they were compared with each other and to two end-tidal CO2 monitors measuring the PCO2 of humidified air containing 5% and then 10% CO2. In vivo the two systems' catheters were surgically juxtaposed in 15 dogs' stomachs; paired PiCO2 readings were taken throughout hemorrhage and resuscitation. RESULTS: In vitro, paired PCO2 values from the airflow and fiber-optic devices correlated with each other (r = 0.99) and with end-tidal values (r = 0.99 with airflow, r = 0.95 with fiber-optic). In vivo, paired values differed significantly (P < 0.0001), correlating poorly for two devices simultaneously measuring the same variable (r = 0.61). Fiber-optic PiCO2 values were higher than airflow values (mmHg +/- SEM): 69.3 +/- 4.8 vs. 61.3 +/- 5.6 at the start of hemorrhage, 141.3 +/- 12.9 vs. 87.7 +/- 7.9 by end of hemorrhage, and 104.3 +/- 9.6 vs. 82.8 +/- 7.0 by end of resuscitation for fiber-optic and airflow, respectively. CONCLUSIONS: Despite agreement in vitro, airflow methods can influence PiCO2 values obtained in vivo. Passive sensing methods used to monitor PiCO2, such as fiber-optic methods, are preferable because they neither deliver O2 to, nor remove CO2 from the local microenvironment.

A novel Tn10 tetracycline regulon system controlling expression of the bacteriophage T3 gene encoding S-adenosyl-L-methionine hydrolase.

To study the effects of in vivo DNA methylation, we have developed an inducible system to control the intracellular concentration of S-adenosyl-L-methionine (AdoMet). The product of the bacteriophage T3 AdoMet hydrolase-encoding gene (amh), which degrades AdoMet to L-homoserine and 5'-methylthioadenosine, was employed to lower AdoMet concentrations in vivo. The amh gene was placed downstream from the inducible tetA promoter of the Tn10 tetracycline regulon substituting for most of the tetA gene. Unlike in the original isolates [Hughes et al., J. Bacteriol. 169 (1987) 3625-2632], this promoter allows controlled expression. These constructs are stable and can be induced in a dose-dependent manner. The system is maximally induced 2-3 h after addition of the inducer, autoclaved chlortetracycline (cTc). DNA methylation in vivo was assessed in this model system by BamHI cleavage of plasmid DNA isolated from cells cotransformed by two compatible plasmids, one carrying the inducible amh gene, the other M.BamHII methyltransferase encoding gene. The induction of amh decreased the intracellular pool of AdoMet which M.BamHII requires as a cofactor. Under these conditions, there is a decrease in DNA methylation. The unmethylated DNA is assayed by BamHI cleavage. This system will be useful for studying transcription, DNA replication, gene repair and other cellular phenomena affected by methylation.

Recombination between bacteriophage T4 and plasmid pBR322 molecules containing cloned T4 DNA.

Reciprocal recombination between T4 DNA cloned in plasmid pBR322 and homologous sequences in bacteriophage T4 genomes leads to integration of complete plasmid molecules into phage genomes. Indirect evidence of this integration comes from two kinds of experiments. Packaging of pBR322 DNA into mature phage particles can be detected by a DNA--DNA hybridization assay only when a T4 restriction fragment is cloned in the plasmid. The density of the pBR322 DNA synthesized after phage infection is also consistent with integration of plasmid vector DNA into vegetative phage genomes. Direct evidence of plasmid integration into phage genomes in the region of DNA homology comes from genetic and biochemical analysis of cytosine-containing DNA isolated from mature phage particles. Agarose gel electrophoresis of restriction endonuclease-digested DNA, followed by Southern blot analysis with nick-translated probes, shows that entire plasmid molecules become integrated into phage genomes in the region of T4 DNA homology. In addition, this analysis shows that genomes containing multiple copies of complete plasmid molecules are also formed. Among phage particles containing at least one integrated copy, the average number of integrated plasmid molecules is almost ten. A cloning experiment done with restricted DNA confirms these conclusions and illustrates a method for walking along the T4 genome.

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells.

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.

Late events in T4 bacteriophage DNA replication. III. Specificity of DNA reinitiation as revealed by hybridization to cloned genetic fragments.

Through the use of the technique of hybridization to cloned genes, the site specificity of the reinitiation of T4 DNA replication was examined at late times after infection, when a large amount of DNA had accumulated in the infected cell. Replication was examined under two conditions; (i) when there was recombination but the repair of the recombinants was inhibited, and (ii) when recombination was followed by covalent joining. When no covalent repair of recombinant was allowed, reinitiation occurred in the areas known to be also involved in the initiation of replication of the parental molecule: thus late reinitiation, if covalent joining is prevented, is site specific. When there was covalent joining, reinitiation displayed no apparent site specificity. The results are discussed in light of the possibility that at late times after infection recombinant intersections act as primers. The similarity of the model proposed to the "break-and-copy" model for lambda phage and the fitness of the proposed model to the genetic phenomena described by others are emphasized.

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas.

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.

In vivo expression of the rII region of bacteriophage T4 present in chimeric plasmids.

The expression of the T4 rII genes in uninfected cells has been examined by use of recombinant plasmids. Hybridization analysis of pulse-labelled RNA prepared from cells carrying pTB101, a plasmid that contains the end of T4 gene 60 and the beginning of gene rIIA, shows that about 0.7% of the labelled RNA is rII specific. By contrast, only 0.02% of pulse-labelled RNA prepared from cells carrying plasmid pTB301, which probably contains the middle-mode rIB promoter, may be rII specific. When separated strands of T4 DNA were used for hybridization, we found that the pTB101 transcripts have a strand specificity identical to that of the rIIA transcripts made during phage infection. The same strand specificity was observed irrespective of the orientation of the inserted DNA in the vector. This result argues that the transcripts initiate within the inserted DNA rather than somewhere else on the plasmid. We also found that essentially none of the pulse-labelled pTB101 RNA would hybridize to the DNA of a T4 deletion mutant that lacks the rIIA gene. This suggests that little of the gene 60 DNA of the plasmid is being transcribed. In addition to the rII transcript, a new protein of 56,000 Daltons molecular weight is found in cells carrying pTB101. Fingerprint analysis of the protein shows that it is specified by the rIIA gene of the plasmid. Taken together, these results indicate that transcription of the plasmid rIIA gene initiates at or near the beginning of the gene.

Differential amplification of specific areas of phage T4 genome as revealed by hybridization to cloned genetic segments.

Under various conditions, specific genetic areas of the phage T4 DNA molecule are preferentially and repeatedly replicated, resulting in the amplification of these areas. These areas are found to lie in the vicinity of the known origins of DNA replication.

Origins of phage T4 DNA replication as revealed by hybridization to cloned genes.

[3H]Thymidine-labeled progeny DNA was isolated after infection of Escherichia coli with two different bacteriophage T4 mutants. These strands were isolated shortly after the initiation of DNA replication and hybridized to 15 different (EcoRI) T4 restriction fragments cloned in plasmid vectors. Uniformly labeled T4 [32P]DNA extracted from phage particles was cohybridized as a normalizing reference. The results obtained lead to the conclusions that, among the loci tested, initiation occurs predominantly in the area of genes 50-5 and less prominently in the area of genes 25-29. However, our data do not support the idea of initiation in the area of genes 40-43. In contrast, this area displays the least replication among the genes tested.

Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes.

Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56, denA and denB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA with EcoRI have been cloned using the vector plasmid pCR1. Clones containing T4 DNA were identified by hybridization with radioactive early and late T4 RNA. A simple marker rescue technique is described for the genetic identification of the cloned T4 fragments. Some of the T4-hybrid plasmids which contain entire T4 genes can complement temperature sensitive and amber mutants of T4.