The effects of lactoferrin on the intestinal environment of broiler chickens.

The influence of in-feed lactoferrin (Lf) on bird production, intestinal microbiota, mucosal immune system and gut microarchitecture was assessed in male Cobb 500 broiler chickens. Birds were given one of four diets from day of hatch: Control (basal diet with no additives), ZnB (basal diet + 50 mg/kg zinc bacitracin), Lf 250 mg/kg (basal diet + 250 mg/kg Lf) and Lf 500 mg/kg (basal diet + 500 mg/kg Lf); n = 24 birds/treatment. An apparent metabolisable energy study was performed between d 25-32. Lf did not affect growth rate or feed conversion in the period 0-21 d of age, nor performance or energy metabolism during the 7 d metabolism experiment which commenced at 25 d of age.The profiles of caecal microbial communities were significantly different in birds given ZnB compared with birds given a diet with no additives, or supplemented with 250 mg/kg Lf. Birds given 250 mg/kg Lf also had a different microbial profile compared with birds given 500 mg/kg Lf. In comparison to control birds, Lf treated birds showed some differences in the T cell proportions in caecal tonsil and spleen. No differences in ileal villus height, crypt depth or goblet cell proportions were observed amongst dietary treatments. Whilst Lf had little effect on the measured parameters, the use of an integrated approach to study the influence of novel feed additives may facilitate a greater understanding of the relationships between nutrition, gut health and bird performance.

Comparison of alternatives to in-feed antimicrobials for the prevention of clinical necrotic enteritis.

AIMS: The capacity for Lactobacillus johnsonii and an organic acid (OA) blend to prevent Clostridium perfringens-induced clinical necrotic enteritis (NE) in chickens was studied. METHODS AND RESULTS: Cobb 500 birds were allocated into six groups (n = 25 birds/pen, eight pens/treatment); Unchallenged, Challenged, Antimicrobial (zinc bacitracin (ZnB)/monensin), OA, probiotic Lact. johnsonii and probiotic sham (Phosphate-buffered saline). All birds were challenged with Eimeria spp. and Cl. perfringens except for unchallenged controls. Birds fed antimicrobials were protected from NE development as indicated by maintenance of body weight, low mortality and clostridium levels, and decreased intestinal macroscopic lesion scores compared to challenged controls (P < 0.05). Lactobacillus johnsonii-fed birds had reduced lesion scores, whilst OA-fed birds had decreased Cl. perfringens levels. Both Lact. johnsonii and OA-fed birds had improved feed efficiency between days 0 and 28 compared to challenged controls; however, mortality and body weights were not improved by either treatment. Microbial profiling indicated that the challenge procedure significantly altered the jejunal microbiota. The microbiota of antimicrobial-fed birds was significantly different from all other groups. CONCLUSIONS: Whilst Lact. johnsonii and OA altered specific intestinal parameters, significant protection against NE was not observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus johnsonii and OA did not prevent NE; however, some improvements were evident. Other related treatments, or combinations of these two treatments, may provide greater protection.

Dietary omega-3 polyunsaturated fatty acid does not influence the intestinal microbial communities of broiler chickens.

The capacity for n-3 polyunsaturated fatty acids (PUFA) to improve broiler chicken growth, influence the intestinal microbial communities, and modify the PUFA content of meat was studied. Male Cobb 500 chickens were fed 1 of 4 diets from hatch: control (standard diet with no additives), ZnB (standard diet with added antibiotics), 2% SALmate (standard diet with 2% SALmate, which is composed of 42% fish oil and 58% starch), and 5% SALmate (standard diet with 5% SALmate). A 7-d energy metabolism study was conducted between d 15 and 22 posthatch. Birds were killed at d 25 and intestinal samples were collected to assess microbial communities by terminal restriction fragment length polymorphism and Lactobacillus PCR-denaturing gradient gel electrophoresis. Diet did not affect BW, feed intake, feed conversion, or ileal digestible energy (P > 0.05). Apparent ME was greater in ZnB-fed birds compared with all other diets (P < 0.05). Breast tissue levels of eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, and total n-3 PUFA were elevated significantly in 2% SALmate- and 5% SALmate-fed chickens compared with control and ZnB diets (P < 0.05). No significant differences in overall microbial communities were observed in the ileum or cecum as assessed by terminal RFLP (P > 0.05). Birds fed 2% SALmate had a significantly different cecal Lactobacillus species profile compared with birds fed the control diet (P < 0.05); however, no differences were observed in birds fed 5% SALmate compared with birds fed all other diets. In addition to the expected increase in breast tissue n-3 fatty acid levels, a low level of dietary n-3 PUFA also altered the intestinal Lactobacillus species profiles. However, n-3 PUFA supplementation did not alter the overall microbial communities or broiler performance.

Indigestible carbohydrates alter the intestinal microbiota but do not influence the performance of broiler chickens.

AIMS: Prebiotics are a potential alternative to in-feed antimicrobials to improve performance of chickens. We investigated the effects of mannanoligosaccharide (MOS) and fructooligosaccharide (FOS) on growth, performance and the intestinal microbiota. METHODS AND RESULTS: Cobb 500 birds were fed either: Control, starter diet without antimicrobials; ZnB, Control + 50 ppm zinc bacitracin; MOS, Control + 5 g kg(-1) MOS; or FOS, Control + 5 g kg(-1) FOS. An energy metabolism study was conducted and intestinal microbial communities assessed by T-RFLP and Lac PCR-DGGE. Diet did not influence performance. Ileal microbial communities were significantly different in ZnB-fed birds compared to all diets, and FOS-fed chickens compared to Control. MOS-fed chickens had a different caecal profile to ZnB and FOS-fed birds. Consensus Lac PCR-DGGE profiles indicated Lactobacillus communities clustered according to diet with Lactobacillus johnsonii characteristic of ZnB diet. Control and MOS-fed chickens displayed significantly different jejunal Lactobacillus profiles to each other whilst ileal profiles were different between MOS and FOS-fed birds. CONCLUSION: Prebiotics influenced the intestinal microbiota, but did not affect performance. SIGNIFICANCE AND IMPACT OF THE STUDY: In light of pressure for in-feed antimicrobial withdrawal, the impact of alternative compounds on the intestinal microbiota and bird performance is critical to the poultry industry.

Relationship of dietary antimicrobial drug administration with broiler performance, decreased population levels of Lactobacillus salivarius, and reduced bile salt deconjugation in the ileum of broiler chickens.

Straight-run broiler chickens were raised either in floor pens or wire-floored cages (trial 1) or in floor pens only (trials 2, 3, and 4). Birds raised in floor pens had lower BW and feed intakes than those raised in cages. The administration of bacitracin in the feed increased feed intake from d 12 to d 35, decreased the feed conversion ratio during the same period in trial 2, and improved the weight gain of broilers from d 0 to 10 in trial 3. The concentrations of conjugated bile salts (taurocholic and taurochenodeoxycholic acids) were higher in the ileal contents of broilers administered the antimicrobials compared with untreated birds. Supplementation of the feed with monensin increased fat digestibility in the ileum of the birds. Although total numbers of bacteria in ileal contents were the same regardless of whether antimicrobials were administered or not, the bacterial community differed qualitatively. Populations of Lactobacillus salivarius were reduced in birds fed antimicrobials relative to untreated broilers. A representative ileal isolate of L. salivarius deconjugated bile salts in pure culture in the laboratory and in the ileal contents of ex-Lactobacillus-free chickens maintained in a protective environment and colonized by the Lactobacillus isolate. These observations provide a link between bile salt deconjugation in the ileum by L. salivarius and decreased weight gain of broilers. Lactobacillus salivarius populations could be targeted in future studies aimed at modification of the ileal bacterial community to achieve growth promotion of broilers without the administration of antimicrobial drugs.

Investigating the effects of commercial probiotics on broiler chick quality and production efficiency.

A study was undertaken to test the effect of 2 commercially available probiotics on the production efficiency of broiler chickens hatched from the same breeder flock at 3 different ages (28, 43, and 57 wk). At each of the 3 breeder flock ages, 1,600 broiler chickens were hatched and randomly allocated to 1 of 4 treatments: 1) no probiotics (control), 2) probiotic 1 administered in the drinking water, 3) probiotic 1 administered as a spray, and 4) probiotic 2 administered in the feed. A coccidiostat was included in the feed, but no other antimicrobial agents were given. Broilers were then reared on straw litter in identical floor pens for a period of 6 wk. There were no significant differences in broiler BW, feed conversion, or mortality between the probiotic treatments and the control group in any of the trials. The 43-wk-old breeder flock had the highest fertility and hatchability and the lowest percentage of chicks culled at hatching. Throughout the broiler production period, the broilers from the 43- and 57-wk-old breeder flocks had higher BW and weight gains than the broilers produced at 28 wk of breeder flock age. Broiler feed conversion over the 6-wk production period decreased as the breeder flock aged. Probiotics had no effect on chick quality or production efficiency in broilers produced by the breeder flock ages examined.

DNA analysis of the genes encoding acidocin LF221 A and acidocin LF221 B, two bacteriocins produced by Lactobacillus gasseri LF221.

Lactobacillus gasseri LF221, an isolate from the feces of a child, produces two bacteriocins. Standard procedures for molecular techniques were used to locate, clone and sequence the fragments of LF221 chromosomal DNA carrying the acidocin LF221 A and B structural genes, respectively. Sequencing analysis revealed the gene of acidocin LF221 A to be an open reading frame encoding a protein composed of 69 amino acids, including a 16-amino-acid N-terminal extension. The acidocin LF221 B gene was found to encode a 65-amino-acid bacteriocin precursor with a 17-amino-acid N-terminal leader peptide. DNA homology searches showed similarities of acidocin LF221 A to brochocin B, lactococcin N and thermophilin B, whereas acidocin LF221 B exhibited some homology to lactacin F and was virtually identical to gassericin X. The peptides encoded by orfA1 and orfB3 showed characteristics of class II bacteriocins and are suspected to be the complementary peptides of acidocin A and B, respectively. orfA3 and orfB5 are proposed to encode putative immunity proteins for the acidocins. Acidocin LF221 A and acidocin LF221 B are predicted to be members of the two-component class II bacteriocins, where acidocin LF221 A appears to be a novel bacteriocin. L. gasseri LF221 is being developed as a potential probiotic strain and a food/feed preservative. Detailed characterization of its acidocins is an important piece of background information useful in applying the strain into human or animal consumption. The genetic information on both acidocins also enables tracking of the LF221 strain in mixed populations and complex environments.

Molecular characterization of a new abortive infection system (AbiU) from Lactococcus lactis LL51-1.

This study reports on the identification and characterization of a novel abortive infection system, AbiU, from Lactococcus lactis. AbiU confers resistance to phages from the three main industrially relevant lactococcal phage species: c2, 936, and P335. The presence of AbiU reduced the efficiency of plaquing against specific phage from each species as follows: 3.7 x 10(-1), 1.0 x 10(-2), and 1.0 x 10(-1), respectively. abiU involves two open reading frames, abiU1 (1,772 bp) and abiU2 (1,019 bp). Evidence indicates that AbiU1 is responsible for phage resistance and that AbiU2 may downregulate phage resistance against 936 and P335 type phages but not c2 type phage. AbiU appeared to delay transcription of both phage 712 and c2, with the effect being more marked on phage c2.

Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri.

O-antigen modification (serotype conversion) in Shigella flexneri, which is an important virulence determinant, is conferred by temperate bacteriophages. Several serotype-converting phages have been isolated and preliminary characterization has identified the genes involved in O-antigen modification, and has also provided insight into the molecular biology of these phages.

Functional analysis of the gene encoding immunity to lactacin F, lafI, and its use as a Lactobacillus-specific, food-grade genetic marker.

Lactacin F is a two-component class II bacteriocin produced by Lactobacillus johnsonii VPI 11088. The laf operon is composed of the bacteriocin structural genes, lafA and lafX, and a third open reading frame, ORFZ. Two strategies were employed to study the function of ORFZ. This gene was disrupted in the chromosome of NCK64, a lafA729 lafX ORFZ derivative of VPI 11088. A disruption cassette consisting of ORFZ interrupted with a cat gene was cloned into pSA3 and introduced into NCK64. Manipulation of growth temperatures and antibiotic selection resulted in homologous recombination which disrupted the chromosomal copy of ORFZ with the cat gene. This ORFZ mutation resulted in loss of immunity to lactacin F but had little effect on production of LafX, which is not bactericidal without LafA. Expression of ORFZ in this ORFZ- background rescued the immune phenotype. Expression of ORFZ in a bacteriocin-sensitive derivative of VPI 11088 also reestablished immunity. These data indicate that ORFZ, renamed lafI, encodes the immunity factor for the lactacin F system. The sensitivity of various Lactobacillus strains to lactacin F was further evaluated. Lactacin F inhibited 11 strains including several members of the A1, A2, A3, A4, B1, and B2 L. acidophilus homology groups. Expression of lafI in bacteriocin-sensitive strains L. acidophilus ATCC 4356, L. acidophilus NCFM/N2, L. fermentum NCDO1750, L. gasseri ATCC 33323, and L. johnsonii ATCC 33200 provided immunity to lactacin F. Furthermore, it was shown that lactacin F production by VPI 11088 could be used to select for L. fermentum NCDO1750 transformants containing the recombinant plasmid encoding LafI. The data demonstrate that lafI is functional in heterologous hosts, suggesting that it may be a suitable food-grade genetic marker for use in lactobacillus species.

Utilization of the leucocin A export system in Leuconostoc gelidum for production of a Lactobacillus bacteriocin.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 (ATCC 11506) and is active against various lactobacilli and Enterococcus faecalis. The genetic determinants encoding the lactacin F peptides, LafA and LafX, are organized in a chromosomal operon comprised of genes lafA, lafX, and ORFZ. The lactacin F operon was introduced into Leuconostoc (Lc.) gelidum UAL187-22 which produces leucocin A. Leucocin A, a plasmid-encoded bacteriocin, inhibits E. faecalis, Listeria monocytogenes, and other lactic acid bacteria. The culture supernatant of the Leuconostoc transformant containing the lactacin F operon inhibited both lactacin F-and leucocin A-sensitive indicators. Concurrent expression of both bacteriocins did not alter the production of native leucocin A. Additive inhibitory effects due to the presence of both bacteriocins were not observed. An isogenic derivative of UAL187-22, which has lost the leucocin-encoding plasmid, was unable to produce active lactacin F when transformed with the appropriate recombinant plasmid. The ability of Lc. gelidum UAL187-22 to produce lactacin F demonstrates that the export system for leucocin A is capable of producing both bacteriocins simultaneously.

Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 and is active against five other Lactobacillus species and Enterococcus faecalis. The genetic determinants encoding the lactacin F complex are organized in a 1-kb polycistronic operon which comprises three genes, lafA, lafX, and ORFZ (encoding the putative immunity protein). The lafA and lafX genes encode the bacteriocin precursors with N-terminal extensions characterized by a Gly-Gly-1*Xaa+1 cleavage site (*). The Gly-Gly motif is conserved in several other bacteriocins, including carnobacteriocins A, BM1, and B2. Carnobacterium piscicola LV17 produces carnobacteriocins which are active against Listeria monocytogenes and other lactic acid bacteria. In this study, the lactacin F operon was introduced into C. piscicola LV17. The transformants produced lactacin F concurrently with the carnobacteriocins. When the lafA and lafX genes were separated and cloned individually into LV17, production of either LafA or LafX by C. piscicola LV17 was detected by complementation with L. johnsonii clones producing LafX or LafA, respectively. Transformants of C. piscicola LV17 which produced lactacin F, LafA, or LafX, in combination with the carnobacteriocins, were assayed for an increased and expanded inhibitory spectrum. The recombinant organisms were only active against lactacin F- and carnobacteriocin-sensitive strains. A plasmidless derivative of LV17 which does not produce the carnobacteriocins failed to produce lactacin F, LafA, or LafX when transformed with the appropriate recombinant plasmids. The ability of C. piscicola LV17 to produce lactacin F demonstrates that the machinery for the carnobacteriocins is capable of processing and exporting bacteriocins from both systems.

Expansion of bacteriocin activity and host range upon complementation of two peptides encoded within the lactacin F operon.

Lactacin F is a membrane-active bacteriocin produced by Lactobacillus johnsonii VPI11088 (Laf+). The genetic determinants encoding lactacin F are organized in a 1-kb polycistronic operon composed of a promoter (P(laf)), three genes (lafA, lafX, and ORFZ), and a functional rho-independent transcription terminator. Two Laf- derivatives of VPI11088, designated NCK64 and NCK65, were characterized. NCK64 contained a frameshift mutation in the lafA gene causing premature termination of translation. NCK65 harbored a 10-kb chromosomal deletion covering the laf operon. When the lafA gene was cloned independently and expressed in NCK65, bacteriocin activity was limited to L. helveticus 87, only one of the six known lactacin F-sensitive (Lafs) indicators. When lafX was introduced into NCK65, no bacteriocin activity against any of the sensitive strains was detected. Genetic combination of lafA and lafX, in cis or in trans, restored bacteriocin activity against all Lafs indicators. When two NCK65 clones containing either lafA or lafX were plated slightly apart on agar plates, fully active lactacin F was present in the intervening area where the two excreted gene products, LafA and LafX, diffused together. The genetic analysis revealed that the interaction of two bacteriocinogenic peptides encoded within the laf operon is likely to participate in the formation of poration complexes in the membranes of susceptible bacteria.

Psychiatric implications of religious conversion.

Some of the literature concerning religious phenomena and conversion experience is reviewed. Three cases have been described in which there are several common factors leading to religious conversion - a specific unconscious conflict, a conscious conflict with which the individual is actively struggling, adolescence, and a fundamentalist religious belief. The cases presented are examples of each of three possible solutions to the patient's conflictual dilemma. A central feature of the psychodynamics in these cases is the resolution of an (Edipal conflict through delayed identification with the parent of the opposite sex.