Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Levadura que expresa una fago-endolisina reduce la presencia endógena de Clostridium perfringens Ex vivo en fluidos intestinales de pollos de engorde de 21 días. La fago endolisina PlyCP41, cuando se purifica a partir de Escherichia coli, exhibe actividad lítica contra Clostridium perfringens (Cp) in vitro. La actividad anticlostridial de la endolisina PlyCP41 expresada en levadura transgénica (Saccharomyces cerevisiae) se verificó en solución salina amortiguada con fosfato mediante experimentos de mezclas con cultivos de C. perfringens y suspensiones de levadura transgénica, seguido de cultivos de diluciones en serie y recuentos de colonias en placas de triptosa sulfito cicloserina (TSC; indicador para C. perfringens). La levadura transgénica que contenía PlyCP41 dio como resultado una reducción de log10 4.5 (99.997%; P <0.01) en el cultivo de C. perfringens. Además, este ensayo de dilución en serie en placas se utilizó para demostrar que las suspensiones de levadura transgénica podrían reducir el contenido de C. perfringens endógeno en fluidos de tres regiones gastrointestinales diferentes (proximal, medial y distal) de pollos de engorde de 21 días de edad. El tratamiento con levadura transgénica de las suspensiones intestinales dio como resultado una reducción de log10 de 1.19, 4.53 y 1.28 en las suspensiones intestinales proximal, medial y distal (90% a 99.99 % de C. perfringens endógena; P < 0.01), respectivamente, en comparación con los controles no tratados. Estos resultados indican que la fago-endolisina PlyCP41 expresada en S. cerevisiae es eficaz para reducir el contenido endógeno de C. perfringens en los fluidos gastrointestinales de pollos de engorde. Los estudios futuros medirán el efecto contra C. perfringens in vivo mediante la administración de levadura transgénica a pollos de engorde en el alimento.

The poultry-associated microbiome: network analysis and farm-to-fork characterizations.

Microbial communities associated with agricultural animals are important for animal health, food safety, and public health. Here we combine high-throughput sequencing (HTS), quantitative-PCR assays, and network analysis to profile the poultry-associated microbiome and important pathogens at various stages of commercial poultry production from the farm to the consumer. Analysis of longitudinal data following two flocks from the farm through processing showed a core microbiome containing multiple sequence types most closely related to genera known to be pathogenic for animals and/or humans, including Campylobacter, Clostridium, and Shigella. After the final stage of commercial poultry processing, taxonomic richness was ca. 2-4 times lower than the richness of fecal samples from the same flocks and Campylobacter abundance was significantly reduced. Interestingly, however, carcasses sampled at 48 hr after processing harboured the greatest proportion of unique taxa (those not encountered in other samples), significantly more than expected by chance. Among these were anaerobes such as Prevotella, Veillonella, Leptrotrichia, and multiple Campylobacter sequence types. Retail products were dominated by Pseudomonas, but also contained 27 other genera, most of which were potentially metabolically active and encountered in on-farm samples. Network analysis was focused on the foodborne pathogen Campylobacter and revealed a majority of sequence types with no significant interactions with other taxa, perhaps explaining the limited efficacy of previous attempts at competitive exclusion of Campylobacter. These data represent the first use of HTS to characterize the poultry microbiome across a series of farm-to-fork samples and demonstrate the utility of HTS in monitoring the food supply chain and identifying sources of potential zoonoses and interactions among taxa in complex communities.

Comparison of live Eimeria vaccination with in-feed salinomycin on growth and immune status in broiler chickens.

Coccidiosis vaccines and anticoccidial drugs are commonly used to control Eimeria infection during commercial poultry production. The present study was conducted to compare the relative effectiveness of these two disease control strategies in broiler chickens in an experimental research facility. Birds were orally vaccinated with a live, attenuated vaccine (Inovocox), or were provided with in-feed salinomycin (Bio-Cox), and body weights, serum levels of nitric oxide (NO) and antibodies against Eimeria profilin and Clostridium perfringens PFO proteins, and intestinal levels of cytokine gene transcripts were measured. Vaccinated chickens had increased body weights, greater NO levels, and higher profilin and PFO antibody levels compared with salinomycin-fed birds. Transcripts for interleukin-6 (IL-6), tumor necrosis factor superfamily 15, and interferon-γ were increased, while mRNAs for IL-4 and IL-10 were decreased, in immunized chickens compared with salinomycin-treated chickens. In conclusion, vaccination against avian coccidiosis may be more effective compared with dietary salinomycin for increasing body weight and augmenting pro-inflammatory immune status during commercial poultry production.

Molecular characterization of podoviral bacteriophages virulent for Clostridium perfringens and their comparison with members of the Picovirinae.

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.

Complete genome sequence of the podoviral bacteriophage ΦCP24R, which is virulent for Clostridium perfringens.

Bacteriophage ΦCP24R was isolated from raw sewage from a waste treatment plant, and lytic activity was observed against a type A Clostridium perfringens isolate. Electron microscopy revealed a small virion (44-nm-diameter icosahedral capsid) with a short, non-contractile tail, indicative of a member of the family Podoviridae. The phage had a linear, double-stranded DNA genome of 18,919 base pairs (bp) with 41 bp inverted terminal repeats and a type B DNA polymerase, which are characteristics of members of the subfamily Picovirinae. Out of 22 predicted genes in the genome, ten had significant sequence similarity to proteins of known function. Three distinct genes with lytic domains were identified, including a zinc carboxypeptidase domain that has not been previously reported in viruses. The ΦCP24R genome described herein is only the second Clostridium perfringens podovirus genome reported to date.

Effects of in ovo vaccination and anticoccidials on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters.

The present study reports the effects of various field anticoccidial programs on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters. The programs included in ovo vaccination and various medications with either chemicals, ionophores, or both. In general, serum samples from these chickens showed anticoccidial antibody titers when tested at days 7 and 14 post hatch with the peak response at day 43. Serum anticoccidial titers were highest in birds fed a non-medicated diet compared with those vaccinated or fed medicated diets. Total number of Eimeria oocysts and the composition of Eimeria spp. present in the litter samples from different treatment groups varied depending on the type of anticoccidial program. Oocyst counts in general ranged from 3.7×10(3) to 7.0×10(4) per g of litter. Importantly, both morphological and molecular typing studies revealed four major predominant Eimeria spp., E. acervulina, E. maxima, E. praecox, and E. tenella in the litter samples. Collectively, these results indicate that the field anticoccidial programs influenced the type and abundance of Eimeria spp. present in the litter samples and also modulated host immune response to Eimeria.

Genome-wide differential gene expression profiles in broiler chickens with gangrenous dermatitis.

Gangrenous dermatitis (GD) is a disease of poultry characterized by necrosis of the skin and severe cellulitis of the subcutaneous tissues caused by infection with Clostridium septicum (CS) and/or Clostridium perfringens (CP) type A. While GD causes significant morbidity, mortality, and economic loss to the poultry industry, the fundamental mechanisms underlying this host-pathogen interaction are relatively unknown. This study used comparative global gene expression microarray analysis of GD-affected and clinically healthy chickens from a recent GD outbreak to glean insights into the molecular and cellular changes associated with this disease process. Histopathologic and immunohistochemical analyses confirmed extensive muscle damage and prominent leukocyte infiltration in the skin of GD-affected birds but not in healthy controls. The levels of mRNAs in the skin and underlying muscle corresponding to 952 microarray elements were altered in GD-afflicted birds compared with healthy controls, with 468 being increased and 484 decreased. From these, a subset of 386 genes was identified and used for biologic function and pathway analyses. The biologic functions that were most significantly associated with the differentially expressed genes were "inflammatory response" and "cellular growth and proliferation" classified under the categories of "disease and disorders" and "molecular and cellular functions," respectively. The biologic pathway that was most significantly associated with the differentially expressed genes was the nuclear factor-erythroid 2-related factor 2 (NRF2)-mediated oxidative stress pathway. Finally, in vitro infection of chicken macrophages with CS or CP modified the levels of mRNAs encoding interferon (IFN)-alpha, IFN-gamma, interleukin (IL)-1beta, IL-6, IL-12p40, tumor necrosis factor superfamily 15 (downregulated), IL-8, and IL-10 (upregulated), thus confirming the suppressive effect of GD on the chicken immune system.

Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential.

BACKGROUND: Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. RESULTS: Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. CONCLUSIONS: Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

Chicken intestine microbiota following the administration of lupulone, a hop-based antimicrobial.

The use of antibiotic growth promotants in poultry rearing is a public health concern due to antibiotic resistance in bacteria and the harborage of resistance genes. Lupulone, a hop ß-acid from Humulus lupulus, has been considered as a potential feed additive growth promotant. Here, the effect of lupulone was evaluated for its effect on the microbiota of the chicken intestine. The intestinal microbiota of broilers was quantified after the addition of 125 mg L(-1) lupulone to water and challenge with Clostridium perfringens. Microbial DNA was extracted from the broiler midgut and cecal sections and bacterial groups were quantified using real-time PCR. The predominant cecal bacterial groups were Clostridium leptum subgroup 16S rRNA gene Cluster IV, Clostridium coccoides subgroup 16S rRNA gene Clusters XIVa and XIVb and Bacteroides, whereas Lactobacillus, the Enterobacteriaceae family and Enterococcus dominated the midgut. Lupulone at 125 mg L(-1) significantly decreased the C. perfringens subgroup 16S rRNA gene Cluster I, which contains several pathogenic species, in both the midgut and the cecum and Lactobacillus in the midgut. No significant changes were noted in the overall microbiota for the cecum or the midgut. Lupulone warrants further evaluation as a botanical agent to mitigate C. perfringens overgrowth in antibiotic-free reared poultry.

Bacillus subtilis-based direct-fed microbials augment macrophage function in broiler chickens.

The present study was conducted to evaluate the function of Bacillus subtilis-based direct-fed microbials (DFMs) on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis in broiler chickens. DFMs used in this study were eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple strain DFM product (Avicorr™) containing equal amount of Bs2084, LSSAO1 and 15AP4. NO concentrations were monitored in plasma and in the supernatants from the peripheral blood-derived monocytic cells (PBMC)-derived macrophages stimulated by either chicken recombinant interferon gamma (IFNγ) or lipopolysaccharide (LPS) from Escherichia coli or Salmonella typhi. In addition, phagocytosis of fluorescent beads and green fluorescent protein (GFP)-labeled Salmonella by PBMC-derived macrophage was assayed. Plasma NO levels were significantly higher in groups given 3AP4 or Bs27 diets compared with the control group at days 7 and 14. NO production by PBMC-derived macrophages stimulated with IFNγ or LPS was apparent, although the effect was strain-dependent. Phagocytosis of fluorescent beads or GFP-labeled Salmonella by macrophages was augmented in groups on DFM-supplemented diets compared with those fed the control diet. This study describes the immunomodulatory effects of Bacillus-based DFMs on innate immunity in broiler chickens.

Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions.

Poultry intestinal material, sewage and poultry processing drainage water were screened for virulent Clostridium perfringens bacteriophages. Viruses isolated from broiler chicken offal washes (O) and poultry feces (F), designated ΦCP39O and ΦCP26F, respectively, produced clear plaques on host strains. Both bacteriophages had isometric heads of 57 nm in diameter with 100-nm non-contractile tails characteristic of members of the family Siphoviridae in the order Caudovirales. The double-strand DNA genome of bacteriophage ΦCP39O was 38,753 base pairs (bp), while the ΦCP26F genome was 39,188 bp, with an average GC content of 30.3%. Both viral genomes contained 62 potential open reading frames (ORFs) predicted to be encoded on one strand. Among the ORFs, 29 predicted proteins had no known similarity while others encoded putative bacteriophage capsid components such as a pre-neck/appendage, tail, tape measure and portal proteins. Other genes encoded a predicted DNA primase, single-strand DNA-binding protein, terminase, thymidylate synthase and a transcription factor. Potential lytic enzymes such as a fibronectin-binding autolysin, an amidase/hydrolase and a holin were encoded in the viral genomes. Several ORFs encoded proteins that gave BLASTP matches with proteins from Clostridium spp. and other Gram-positive bacterial and bacteriophage genomes as well as unknown putative Collinsella aerofaciens proteins. Proteomics analysis of the purified viruses resulted in the identification of the putative pre-neck/appendage protein and a minor structural protein encoded by large open reading frames. Variants of the portal protein were identified, and several mycobacteriophage gp6-like protein variants were detected in large amounts relative to other virion proteins. The predicted amino acid sequences of the pre-neck/appendage proteins had major differences in the central portion of the protein between the two phage gene products. Based on phylogenetic analysis of the large terminase protein, these phages are predicted to be pac-type, using a head-full DNA packaging strategy.

Recombinant expression of two bacteriophage proteins that lyse clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal active domains.

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins that are responsible for disease symptoms and pathogenesis in a variety of animals, humans, and poultry. The organism is the third leading cause of human foodborne bacterial disease, and C. perfringens is the presumptive etiologic agent of necrotic enteritis among chickens, which in the acute form can cause increased mortality among broiler flocks. Countries that have complied with the ban on antimicrobial growth promoters (AGP) in feeds have had increased incidences of C. perfringens-associated necrotic enteritis in poultry. To address this issue, new antimicrobial agents, putative lysins from the genomes of bacteriophages, are identified. Two putative phage lysin genes (ply) from the clostridial phages phiCP39O and phiCP26F were cloned and expressed in Escherichia coli , and the resultant proteins were purified to near homogeneity. Gene and protein sequencing revealed that the predicted and chemically determined amino acid sequences of the two recombinant proteins were homologous to N-acetylmuramoyl-l-alanine amidases. The proteins were identical in the C-terminal putative cell-wall binding domain, but only 55% identical to each other in the presumptive N-terminal catalytic domain. Both recombinant lysins were capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays. The observed reduction in turbidity was correlated with up to a 3 log cfu/mL reduction in viable C. perfringens on brain-heart infusion agar plates. However, other member species of the clostridia were resistant to the lytic activity by both assays.

An outbreak of gangrenous dermatitis in commercial broiler chickens.

The present report describes an outbreak of gangrenous dermatitis (GD) infection in a commercial poultry farm in Delaware involving 34-day-old broiler chickens. In addition to obvious clinical signs, some GD-affected broilers also showed severe fibrino-necrotic enteritis and large numbers of Gram-positive rods in the necrotic tissue. Histopathological findings included haemorrhage, degeneration and necrosis of parenchymatous cells, especially of skin, muscle, and intestine. Immunofluorescence staining revealed Clostridium-like bacilli in the skin and the intestine. Both Clostridium perfringens and Clostridium septicum genomic sequences were identified by polymerase chain reaction in bacterial cultures isolated from the skin, muscle, and intestine, and in the frozen tissues from the GD-affected birds. Serological analysis demonstrated that both affected and clinically healthy birds from the same house had high serum antibody titres against C. perfringens, C. septicum, Eimeria, chick anaemia virus, and infectious bursal disease virus. These results are discussed in the context of the relationship between the different Clostridium spp. and the pathogenesis of GD.

Immunopathology and cytokine responses in commercial broiler chickens with gangrenous dermatitis.

Gangrenous dermatitis (GD) is an emerging disease of increasing economic importance in poultry resulting from infection by Clostridium septicum and Clostridium perfringens type A. Lack of a reproducible disease model has been a major obstacle in understanding the immunopathology of GD. To gain better understanding of host-pathogen interactions in GD infection, we evaluated various immune parameters in two groups of birds from a recent commercial outbreak of GD, the first showing typical disease signs and pathological lesions (GD-like birds) and the second lacking clinical signs (GD-free birds). Our results revealed that GD-like birds showed: reduced T-cell and B-cell mitogen-stimulated lymphoproliferation; higher levels of serum nitric oxide and alpha-1-acid glycoprotein; greater numbers of K55(+), K1(+), CD8(+), and MHC class II(+) intradermal lymphocytes, and increased K55(+), K1(+), CD8(+), TCR1(+), TCR2(+), Bu1(+), and MHC class II(+) intestinal intraepithelial lymphocytes; and increased levels of mRNAs encoding proinflammatory cytokines and chemokines in skin compared with GD-free chickens. These results provide the first evidence of altered systemic and local (skin and intestine) immune responses in GD pathogenesis in chickens.

Effect of Bacillus-based direct-fed microbials on Eimeria maxima infection in broiler chickens.

The effect of dietary Bacillus-based direct-fed microbials (DFMs; eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple-strain DFM product [AVICORR]) on growth performance, intestinal lesions, and innate and acquired immunities were evaluated in broiler chickens following Eimeria maxima (EM) infection. EM-induced reduction of body weight gain and intestinal lesions were significantly decreased by addition of 15AP4 or Bs27 into broiler diets compared with EM-infected control birds. Serum nitric oxide levels were increased in infected chickens fed with Bs27, but lowered in those given Bs2084, LSSAO1, 3AP4 or 15AP4 compared with the infected controls. Recombinant coccidial antigen (3-1E)-stimulated spleen cell proliferation was increased in chickens given Bs27, 15AP4, LSSAO1, 3AP4, or Bs18, compared with the infected controls. Finally, all experimental diets increased concanavalin A-induced splenocyte mitogenesis in infected broilers compared with the nonsupplemented and infected controls. In summary, dietary Bacillus subtilis-based DFMs reduced the clinical signs of experimental avian coccidiosis and increased various parameters of immunity in broiler chickens in a strain-dependent manner.

Predicting Salmonella enterica serotypes by repetitive sequence-based PCR.

Repetitive extragenic palindromic sequence-based PCR (rep-PCR) utilizing a semi-automated system, was evaluated as a method to determine Salmonella serotypes. A group of 216 Salmonella isolates belonging to 13 frequently isolated serotypes and one rarer serotype from poultry were used to create a DNA fingerprint library with the DiversiLab System software. Subsequently, a blinded set of 44 poultry isolates were fingerprinted and queried against the library in an attempt to putatively assign a serotype designation to each Salmonella isolate. The query isolates were previously typed employing standard serological techniques. Utilizing pair-wise similarity percentages as calculated by the Pearson correlation coefficient, the predicted serotype of 28 isolates matched the serological typing result. For eight isolates, rep-PCR results were interpreted as one of two very closely-related serotypes, Hadar and the rarer Istanbul. Traditional serological assays have difficulty distinguishing between these groups, and sequencing interspacer regions of the rrfH gene was unable to differentiate among isolates of these two serovars. Six of the remaining isolates resulted in no match to the database (similarity values <95%) and these indeed proved to be serotypes not included in the original library. The two remaining samples proved discrepant at the 95% similarity threshold, however examination of electropherograms clearly indicated fingerprint variability between query and library samples, suggesting an expanded rep-PCR library will be necessary for increased utility. Since serological assays can take several days to weeks to provide information, the DiversiLab System holds promise for more rapid serotype classification for members of this group.

Antimicrobial activity of chicken NK-lysin against Eimeria sporozoites.

NK-lysin is an antimicrobial and antitumor polypeptide that is considered to play an important role in innate immunity. Chicken NK-lysin is a member of the saposin-like protein family and exhibits potent antitumor cell activity. To evaluate the antimicrobial properties of chicken NK-lysin, we examined its ability to reduce the viability of various bacterial strains and two species of Eimeria parasites. Culture supernatants from COS7 cells transfected with a chicken NK-lysin cDNA and His-tagged purified NK-lysin from the transfected cells both showed high cytotoxic activity against Eimeria acervulina and Eimeria maxima sporozoites. In contrast, no bactericidal activity was observed. Further studies using synthetic peptides derived from NK-lysin may be useful for pharmaceutical and agricultural uses in the food animal industry.

Diverse antimicrobial killing by Enterococcus faecium E 50-52 bacteriocin.

An effective bacteriocin was identified and characterized. Lactic acid bacteria were screened against Campylobacter jejuni. One bacteriocin producer, Enterococcus faecium (NRRL B-30746), was studied. The isolate was grown, and the bacteriocin was purified to single-band homogeneity. Biochemical traits indicated that the peptide was a Class IIa bacteriocin, and it was named E 50-52. The bacteriocin had a molecular weight of 3339.7 and an isoelectric point of 8.0. The minimal inhibitory concentrations of E 50-52 against C. jejuni, Yersinia spp., Salmonella spp., Escherichia coli O157:H7, Shigella dysenteriae, Morganella morganii, Staphylococcus spp., and Listeria spp. ranged from 0.025 to 32 microg/mL. In therapeutic broiler trials, oral treatment with E 50-52 reduced both C. jejuni and Salmonella enteritidis by more than 100,000-fold in the ceca, and systemic S. enteritidis was reduced in the liver and spleen. The wide range of antibacterial activity of bacteriocin E 50-52 against pathogens provides a promising alternative to antibiotics.

Formation of biofilm at different nutrient levels by various genotypes of Listeria monocytogenes.

Strains of Listeria monocytogenes differ in their ability to form biofilms. The objectives of this study were to determine whether genetically related strains have similar biofilm-forming capacities and what effect nutrient concentration has on the ability of different strains to produce biofilms. Biofilms of 30 strains of L. monocytogenes, obtained from a variety of sources were grown on stainless steel in tryptic soy broth (TSB) or in a 1:10 dilution of TSB (DTSB) for 24 h at 32 degrees C. The amount of biofilm formed was determined with image analysis after cells were stained with bisBenzimide H 33258 (Hoechst 33258). The strains were genetically subtyped by repetitive element sequence-based PCR (rep-PCR) with the primer set rep-PRODt and rep-PROG5. Data were analyzed with an analysis of variance and Duncan's multiple range test. Eleven strains produced the same amount of biofilm in both media. Fourteen strains produced more biofilm in TSB than in DTSB. Five strains produced more biofilm in DTSB than in TSB. Serotype 4b strains produced more biofilm in TSB than did serotype 1/2a strains, whereas serotype 1/2a strains produced more biofilm in DTSB than did serotype 4b strains. Growth in DTSB resulted in decreased biofilm accumulation for serotype 4b strains. There was no correlation between genetic subtype and the amount of biofilm accumulation. These results indicate that strains of serotype 1/2a and serotype 4b differ in the regulation of their biofilm phenotype. The poor biofilm accumulation of serotype 4b isolates when grown in DTSB could be a factor in the predominance of serogroup 1/2 strains in food processing plants, where nutrients may be limited.

Quantitative detection of Clostridium perfringens in the broiler fowl gastrointestinal tract by real-time PCR.

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10(2) CFU/g of ileal material, but only about 10(4) CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.