Toward rational control of<Emphasis Type="Italic"> Escherichia coli</Emphasis> O157:H7 by a phage cocktail

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.     

Analysis of the humoral immune response to immunoselected phage-displayed peptides by a microarray-based method

We describe a novel approach for high-throughput analysis of the immune response in cancer patients using phage-based microarray technology. The recombinant phages used for fabricating phage arrays were initially selected via the use of random peptide phage libraries and breast cancer patient serum antibodies. The peptides displayed by the phages retained their ability to be recognized by serum antibodies after immobilization. The recombinant phage microarrays were screened against either breast cancer or healthy donor serum antibodies. A model-based statistical method is proposed to estimate significant differences in serum antibody reactivity between patients and normals. A significant tumor effect was found with most of the selected phage-displayed peptides, suggesting that recombinant phage microarrays can serve as a tool in monitoring humoral responses towards phage-displayed peptides.     

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

Identification of phage-displayed peptides which bind to the human HnRNPA1 protein-specific monoclonal antibody.

We have screened a peptide phage display library to examine if monoclonal antibody-binding phages could be isolated from the library and thereby predict the antigenic epitopes of the antibodies from the isolated phages. The library was screened for high-avidity binding to monoclonal antibodies by an affinity purification technique called biopanning. Among the monoclonal antibodies examined, the human hnRNPA1 protein-specific monoclonal antibody 9H10 showed selective binding of phages. After two rounds of the biopanning, twelve clones of high-avidity-binding phages were chosen and their inserts were sequenced. Nucleotide sequence comparison of the 12 clones showed that there were 5 different species, with two species containing four members, implying that they were predominantly selected by the biopanning. The amino acid sequences of the inserts of the 12 clones were compared with that of the human hnRNPA1 protein in order to find the putative epitope of the human hnRNPA1 protein for 9H10. The C-terminal region of the human hnRNPA1 protein shows significant homology with the peptide sequences of the selected phage clones. These results show that this peptide phage display library can be useful in defining the epitope of some monoclonal antibodies.     

The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria

The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages.     

重组T4噬菌体gp37基因可扩大其在沙门氏菌中的宿主范围

[目的]将T4噬菌体WG01宿主决定区的gp37基因片段,与另一株T4噬菌体QL01的相应基因进行同源重组,从而获得嵌合噬菌体并进行宿主谱分析,为阐明T4噬菌体的宿主谱形成机制以及快速筛选针对特定病原菌的噬菌体奠定了基础。[方法]通过同源重组的方法将WG01 gp37上的8个基因片段分别替换给QL01,用沙门氏菌作为宿主菌筛选嵌合噬菌体,并对嵌合噬菌体进行宿主谱、最佳感染复数、一步生长曲线和遗传稳定性测定。[结果]本研究共获得了5株嵌合噬菌体(QWA、QWC、QWF、QWG、QWFG)。宿主谱试验结果表明,与噬菌体QL01相比,嵌合噬菌体对21株沙门宿主菌分别可以多裂解7、8、4、10和9株菌,即嵌合噬菌体都获得了相对较宽的宿主谱,其中QWG的沙门氏菌宿主菌拓宽最多。生物学特性试验结果表明,嵌合噬菌体QWG生物学特性稳定。嵌合噬菌体QWG经连续传代培养20代,测序分析第1代和第20代嵌合噬菌体尾丝蛋白基因在传代过程中的稳定性,测序结果表明,嵌合噬菌体改造部分的基因能稳定遗传。[结论]用基因改造的方法可以产生宿主谱拓宽且能稳定遗传的嵌合噬菌体,为快速筛选针对特定病原菌的噬菌体提供了可能。     

Isolation and characterization of phage–host systems from the Baltic Sea ice

In search for sea ice bacteria and their phages from the Baltic Sea ice, two ice samples were collected from land-fast ice in a south-west Finland coastal site in February and March 2011. Bacteria were isolated from the melted sea ice samples and phages were screened from the same samples for 43 purified isolates. Plaque-producing phages were found for 15 bacterial isolates at 3 °C. Ten phage isolates were successfully plaque purified and eight of them were chosen for particle purification to analyze their morphology and structural proteins. Phage 1/32 infecting an isolate affiliated to phylum Bacteroidetes (Flavobacterium sp.) is a siphovirus and six phages infecting isolates affiliated to γ-Proteobacteria (Shewanella sp.) hosts were myoviruses. Cross titrations between the hosts showed that all studied phages are host specific. Phage solutions, host growth and phage infection were tested in different temperatures revealing phage temperature tolerance up to 45 °C, whereas phage infection was in most of the cases retarded above 15 °C. This study is the first to report isolation and cultivation of ice bacteria and cold-active phages from the Baltic Sea ice.     

Isolation of Lambda Transducing Phage with the bio Genes Inserted between Lambda Genes P and Q

Plaque-forming, biotin-transducing phages were constructed with the bio genes inserted between lambda genes P and Q. These phages were isolated for the eventual aim of fusing the lambda Q gene to the bio operon. The following steps were used to construct these phages: A defective temperature-sensitive lysogen was constructed with the bio genes adjacent to and to the left of lambda genes beta NcI857OPQSRA. Heat-resistant survivors were screened for deletions with endpoints in the bio operon and to the right of lambda P and to the left of lambda A. Five of approximately 1,600 heat-resistant survivors had these properties. Two had the gene order bioAB .... lambda QSRA. When these two strains were lysogenized with lambda cI857b221 and heat induced, the desired transducing phages were obtained. We characterized these phages and studied one in detail. Two-thirds of the plaque-forming transducing phages isolated carried the entire bioB gene and only part of the bioA gene, and one-third carried the entire bioA and bioB genes. The phages isolated lost the bio genes upon propagation, indicating that they contain a partial duplication of phage genes. The duplication was shown not to involve the entire lambda Q gene in one of these phages, lambda bioq1b221. A recombinant of this phage, lambda Nam7am53c17b221, failed to form plaques under biotin-derepression conditions. We conclude that if the lambda Q gene was fused to the bio operon in this phage, not enough lambda Q gene product was made to allow phage propagation.     

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.     

Targeting phosphatidylserine on apoptotic cells with phages and peptides selected from a bacteriophage display library

Phosphatidylserine (PS) is a well-characterized biomarker for apoptosis. Ligands that bind to PS can be used for noninvasive imaging of therapy-induced cell death, particularly apoptosis. In this study, we screened a random 12-mer peptide phage library on liposomes prepared from PS. One clone displaying the peptide SVSVGMKPSPRP (designated as PS3-10) bound to PS approximately 4-fold better than its binding to phosphatidylcholine and 18-fold better than to bovine serum albumin in a solid-phase binding assay. In addition, the binding of the corresponding PS3-10 peptide to PS was significantly higher than that of a scrambled peptide. PS3-10 phages, but not a control 4-2-2 phage, bound to aged red blood cells that had PS exposed on their surface. Binding of PS3-10 phages and PS3-10 peptide to TRAIL-induced apoptotic DLD1 cells was 3.2 and 5.4 times higher than their binding to untreated viable cells, respectively. Significantly, immunohistochemical staining confirmed selective binding of PS3-10 phages to apoptotic cells. Our data suggest that panning of phage display libraries may allow the selection of suitable peptide ligands for apoptotic cells and that PS3-10 peptide may serve as a template for further development of molecular probes for in vitro and in vivo imaging of apoptosis.     

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces

AIM: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot. METHODS AND RESULTS: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (approximately 97%) contained phage that infected E. coli B or O157:H7. CONCLUSIONS: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.     

SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages

T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes.     

Cloning of Thermomonospora fusca genes coding for beta 1-4 endoglucanases E1, E2 and E5

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI and fragments in the size range from 4 to 15 kb were isolated, ligated into lambda gtWES.lambda B arms, packaged, and the recombinant phages plated on Escherichia coli. The plaques were screened for carboxymethyl cellulase (CMCase) activity by a gel overlay procedure, and 25 plaques were positive among the 15,000 plaques that were screened. Positive phages were amplified and used to prepare infected E. coli extracts which were assayed for CMCase activity before and after treatment with antisera prepared against five purified T. fusca beta 1-4 endoglucanases (E1-E5). One phage produced an enzyme that was inhibited by E1 antiserum, nine of the phages produced enzymes that were inhibited by E2 antiserum, 14 produced enzymes that were inhibited by E5 antiserum and the enzyme produced by the other phages was not inhibited by any of the five antisera. The DNA insert present in the phage coding for E1 was cut into a number of different fragments which were subcloned into E. coli first using lambda gtWES.lambda B and then plasmid pBR322. The smallest active subclone, pTE12, contained a 3.1-kb insert. The insert present in one of the phages coding for E2 was also subcloned and the smallest active subclone pTE23 contained a 2-kb insert. E. coli HB101 containing plasmid pTE12 or pTE23 produced enzymes that were identical to E1 and E2, respectively, in all the properties tested.     

Detection of Escherichia coli with fluorescent labeled phages that have a broad host range to E. coli in sewage water

Escherichia coli is used as an indicator microorganism in public health. The conventional way to detect E. coli requires several days to produce a result, because it requires incubation of cells. Therefore a rapid and sensitive detection method is needed. T4e-/GFP phage, characterized by suppression of lysozyme and fusion of GFP (green fluorescent protein) to its SOC (small outer capsid) protein, was constructed, and it was shown to be able to detect E. coli K12 sensitively within several hours. However, because the host range of T4 phage to E. coli present in sewage water and sea water is narrow, this phage cannot be used to detect E. coli in environmental water. Two phages named IP008 and IP052, which have a broad host range to E. coli present in sewage influent, were screened from sewage influent. Mixture of these two phages produced clear plaques on 50% of E. coli screened from sewage influent. To use these phages as a tool for detection of E. coli, gfp was inserted into gene e, which encodes a lytic enzyme, and thus lytic-activity-suppressed phages were constructed (IP008e-/GFP and IP052e-/GFP). However, the fluorescent intensity of E. coli cells infected with IP008e-/GFP and IP052e-/GFP was not enough for visualization of the cell. Therefore, in addition to the insertion of gfp into gene e, fusion of GFP to SOC of IP008e-/GFP and IP052e-/GFP was conducted to produce IP008e-/2xGFP and IP052e-/2xGFP. E. coli cells infected with IP008e-/2xGFP and IP052e-/2xGFP showed much stronger fluorescence intensity than E. coli cells infected by IP008e-/GFP and IP052e-/GFP. It is anticipated that, using these GFP-labeled phages, a broad range of E. coli present in sewage influent water can be detected rapidly.     

BLyS对噬菌体随机12肽库的筛选

用B淋巴细胞刺激因子（BLyS）对噬菌体随机12肽库进行亲和淘洗,3轮筛选后阳性噬菌体得到富集。用ELISA鉴定噬菌体克隆,多个阳性克隆测序后得到了同一个小肽序列(RHKIQLRQNIIT)。将该小肽与GST融合,在大肠杆菌中进行表达及纯化,ELISA实验进一步验证了其具有与BLyS特异结合的活性。该小肽有可能成为其天然受体的拮抗剂。      

Molecular ecology of Streptococcus thermophilus bacteriophage infections in a cheese factory.

A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control.     

Comparative study of temperate bacteriophages isolated from Yersinia

170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enterocolitica. The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. mollaretii. The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain.     

Combining phage display and screening of cDNA expression libraries: a new approach for identifying the target antigen of an scFv preselected by phage display

A potential method for identifying new tumor-specific antibody structures as well as tumor-associated antigens is by selecting scFv phage libraries on tumor cells. This phage display technique involves multiple rounds of phage binding to target cells, washing to remove non-specific phage and elution to retrieve specific binding phage. Although the binding properties of an isolated tumor-specific scFv can be evaluated by ELISA, FACS and immunohistochemistry, it still remains a challenge to define the corresponding antigen. Here, we provide evidence that the target antigen of a given scFv displayed on phages can be detected in an immobilized lambda phage cDNA expression library containing thousands of irrelevant clones. The library contained CD30-negative breast-cancer specific cDNA as well as human CD30 receptor cDNA. The interaction of anti-CD30 scFv phages and their target antigen after blotting onto nitrocellulose filters was documented under defined conditions. Screening of different ratios between CD30 receptor and breast cancer specific clones (1:1 and 1:200) revealed that the CD30 antigen could be detected by anti-CD30 scFv phages using at least 5x10(12) plaque forming units of filamentous phages per blot. These investigations demonstrate that it is possible to detect the target antigen of a preselected scFv displayed on filamentous phages in lambda phage cDNA expression libraries.     

Isolation and characterization of Campylobacter bacteriophages from retail poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4 degrees C.