Effect of the probiotic Enterococcus faecium NCIMB10415 on cell numbers of total Enterococcus spp., E. faecium and E. faecalis in the intestine of piglets

Sows and their piglets were fed a diet supplemented with or without the probiotic E. faecium NCIMB10415 (also known as SF68). Piglets were sacrificed 14, 28, 35 and 56 days after birth and DNA from intestinal segments was extracted and purified. A real time PCR assay was used to distinguish Enterococcus spp. (16s rDNA based), E. faecium (Efaafm gene), E. faecalis (Efaafs gene) as well as the probiotic strain (unique plasmid sequence). Extracts of autoclaved sow feces inoculated with E. faecium and E. faecalis cultures were used to calibrate real time PCR results. The probiotic strain was detected in 14 day old suckling piglets before the piglets had access to the starter diet. In piglets of the probiotic group, probiotic E. faecium cell counts were always a significant proportion of total E. faecium cells in stomach digesta (4-20%), however only a small fraction of the total Enterococcus spp. cell number on day 14 and 28 in all intestinal segments (0.1-0.7%). Compared to control samples, the probiotic E. faecium strain significantly (p < or = 0.05) decreased the amount of total Enterococcus spp. and E. faecalis cells in the colon of 14 day old suckling piglets as well as in jejunum and colon samples one week after weaning. E. faecium cell counts were not modified on any sampling day or intestinal segment. This study showed that the presence of probiotic E. faecium NCIMB10415 coincided with reduced total E. faecalis, but not total E. faecium cell numbers in the intestine of piglets. In view of unchanged cell numbers and ratios in sow feces, modifications must have taken place within the intestine of suckling piglets.     

Adhesion of Lactobacillus plantarum 423 and Lactobacillus salivarius 241 to the intestinal tract of piglets, as recorded with fluorescent in situ hybridization (FISH), and production of plantaricin 423 by cells colonized to the ileum

AIMS: To determine which intestinal section of pre and postweaned piglets are colonized by Lactobacillus plantarum 423 and Lactobacillus salivarius 241, and follow production of plantaricin 423 in a gastro-intestinal model. METHODS AND RESULTS: Lactobacillus plantarum 423 and Lact. salivarius 241, single or in combination, were administered to 1-, 14- and 28-day-old (postweaned) piglets. According to results obtained by fluorescent in situ hybridization (FISH), Lact. plantarum 423 adhered strongly to the ileum and posterior colon and Lact. salivarius 241 to the duodenum in preweaned piglets. High numbers of strain 241 were recorded in the duodenum and posterior colon of postweaned piglets, whereas strain 423 remained localized to the ileum. Lowering in Enterococcus faecalis cell numbers were recorded when preweaned piglets were challenged with strain 241. Plantaricin 423 was produced for 96 h in the ileum section of a gastro-intestinal model. CONCLUSIONS: Lactobacillus plantarum 423 and Lact. salivarius 241 adhere to different sections of the intestinal tract, depending on the piglet's age. Ent. faecalis were inhibited in vivo, probably by plantaricin 423. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescent in situ hybridization proved valuable in the detection of probiotic bacteria adhered to the intestine. This is the first report of bacteriocin production in a model simulating the porcine gastro-intestinal tract.     

Effect of bacterial monoassociation on brush-border enzyme activities in ex-germ-free piglets: comparison of commensal and pathogenic Escherichia coli strains

This study was designed to investigate the effect of monoassociation of germ-free piglets with Escherichia coli strains on the development of intestinal brush-border enzyme activities. Piglets were delivered by hysterectomy, reared for seven days under germ-free conditions and fed milk formula diet. One group was maintained germ-free, the other four groups were monoassociated on day eight with one of four E. coli strains: non-pathogenic O86 or O83 and G58-1, or pathogenic 933D. The development of brush-border digestive enzyme functions in the small intestine was evaluated after 15 days. Germ-free controls exhibited slower developmental declines of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and delayed increases of sucrase and glucoamylase compared to conventionally grown animals. Association of germ-free piglets with the non-pathogenic E. coli strains O86 and O83 resulted in increased enterocyte differentiation along the length of the small intestine, accompanied by declining activities of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and elevated activities of maturational markers such as sucrase and glucoamylase. Maturational changes also occurred along the villus-crypt axis, as revealed by histochemical localization of aminopeptidase N on the villi tips in piglets colonized with E. coli O83. Interestingly, colonization with the pathogenic E. coli strain 933D stimulated changes in the main differentiation enzyme markers lactase, sucrase and glucoamylase to an extent comparable with those produced by the non-pathogenic and probiotic E. coli strains. In conclusion, germ-free piglets represent a valuable tool to study the consequences of colonization of the immature sterile gut with defined strains of bacteria.     

Selection of fecal enterococci exhibiting tcrB-mediated copper resistance in pigs fed diets supplemented with copper

Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ～194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance.     

Growth and virulence of Candida albicans after oral inoculation in the chick with a monoflora of either Escherichia coli or Streptococcus faecalis

Balish, Edward (Syracuse University, Syracuse, N.Y.), and A. W. Phillips. Growth and virulence of Candida albicans after oral inoculation in the chick with a monoflora of either Escherichia coli or Streptococcus faecalis. J. Bacteriol. 91:1744-1749. 1966.-Bacterial protection against intestinal infection by Candida albicans was investigated in chicks with a monoflora of either Escherichia coli or Streptococcus faecalis. These animals were obtained by orally inoculating germ-free chicks (3 days old) with pure cultures of bacteria. Each bacterial species was established in large numbers in the gut of separate groups of animals within 24 hr of inoculation; these numbers were similar in chicks examined 34 days later, at which time all animals were killed. The numbers of bacteria from contents of the crop, small intestine, and ceca were similar in chicks with the E. coli monoflora. Comparable results were obtained in chicks with the S. faecalis monoflora, except for decreased numbers in the duodenum and jejunum. Some of the monoflora chicks (7 days old) were transferred into separate isolators, orally inoculated with C. albicans, and observed for 34 days. All chicks grew well and appeared healthy. However, examinations at autopsy revealed severe crop infections in chicks with a diflora containing S. faecalis. Preferential growth of hyphae (C. albicans) occurred in the lesions and throughout the gut. The numbers of S. faecalis in the gut were comparable to those found in unchallenged animals. Agglutinins against C. albicans were not detected in our test or control chicks. Chicks with a diflora containing E. coli and C. albicans had a few microscopic crop lesions containing small numbers of hyphae. C. albicans was well established in the gut of these animals, largely as the yeast form. The numbers of E. coli in the gut were similar to those in control chicks. Thus, it was concluded that E. coli provided protection against crop infection by C. albicans. In crop contents from unchallenged animals, chicks with S. faecalis monoflora were about pH 5, whereas birds with E. coli monoflora were about pH 7. The challenge did not greatly change the former value, and the latter was slightly decreased. In the crop of unchallenged birds, negative E(h) values were found in chicks with S. faecalis and positive E(h) values in those with E. coli. Challenge did not greatly change these values. These data on pH and E(h) were related to conditions for morphogenesis of C. albicans and virulence. No major difference in the concentrations of serum proteins was seen in chicks with E. coli or S. faecalis after challenge with C. albicans. Possible mechanisms of the protective effect of E. coli are discussed.     

Inhibition of adhesion of food-borne pathogens to Caco-2 cells by Lactobacillus strains

AIMS: To select adhesive strains among strains of Lactobacillus and to apply them to inhibit adhesion of food-borne pathogens. METHODS AND RESULTS: Twelve Lactobacillus strains (10 from intestine) were examined for adhesion using Caco-2 cell cultures. The two most adhesive strains, Lactobacillus crispatus JCM 8779 and Lact. reuteri JCM 1081, were used to test antiadhesion activity against enterotoxigenic Escherichia coli, Salmonella typhimurium and Enterococcus faecalis strains. Adhesion of the pathogens was inhibited by both Lactobacillus strains. Adhesion of Ent. faecalis was especially strongly inhibited by JCM 8779. Although antimicrobial activity was not detected in the culture supernatant fluid by agar well diffusion assay, the supernatant fluid obtained from the harvested JCM 8779 cell suspension showed bactericidal activity against Ent. faecalis. CONCLUSION: The strong antiadhesion activity of JCM 8779 against Ent. faecalis appears to be due to the combined effect of both bactericidal activity and competition for attachment site. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that Lact. crispatus produces a bactericidal substance.     

A strain of Enterococcus faecium (18C23) inhibits adhesion of enterotoxigenic Escherichia coli K88 to porcine small intestine mucus

Few studies, if any, have addressed the adhesion of enterococci to the intestinal mucosa and their interference with the adhesion of pathogens, although more than 60% of probiotic preparations in the market contain strains of enterococci. The objective of this study was to investigate if Enterococcus faecium 18C23 has the ability to inhibit the adhesion of Escherichia coli K88ac and K88MB to the small intestine mucus of piglets. Approximately 9% of E. faecium 18C23 organisms adhered to the small intestine mucus, and the adhesion was found to be specific. Living E. faecium 18C23 culture efficiently inhibited the adhesion of E. coli K88ac and K88MB to the piglet intestine mucus. Inhibition of the adhesion of E. coli K88ac to the small intestine mucus was found to be dose dependent. Inhibition of >90% was observed when 10(9) CFU or more of living E. faecium 18C23 culture per ml was added simultaneously with E. coli to immobilized mucus. The substances from both the 18C23 cells and the spent culture supernatant contributed to the inhibition of adhesion of E. coli K88 to the small intestine mucus receptors. The inhibiting effect was not solely a pH effect since considerable inhibitory action was demonstrated after neutralizing the mixture or spent culture supernatant to pH 7.0. Part of the inhibition of adhesion of E. coli K88ac by E. faecium 18C23 or its supernatant might occur through steric hindrance.     

Escherichia coli Nissle 1917 for probiotic use in piglets: evidence for intestinal colonization

Aims:  This study was prompted to investigate the intestinal localization and colonization of orally administered Escherichia coli Nissle 1917 (EcN) in piglets.
Methods and Results:  EcN was fed to ten EcN-negative piglets (3 months) over seven consecutive days. Faecal samples were collected repeatedly and tested for EcN-DNA by a combined culture/PCR assay and for viable EcN by culture methods, respectively. EcN-DNA was detectable in faeces of all piglets within the first 24 h after it was added to the feed. After the administration of EcN had been stopped, the presence of EcN-DNA in faecal samples indicated that all piglets shedded EcN with their faeces intermittently through up to 33 days. In addition, E. coli strains indistinguishable from EcN by all markers tested (rdar colony morphotype, multiplex PCR and GEI II-PCR analyses, Xba I-pattern, K5 phage susceptibility) were isolated from faecal samples and from mucosal swabs taken at euthanasia at the end of the experiment.
Conclusions:  EcN colonizes the intestine and persists in conventionally reared piglets for at least 4 weeks upon oral administration.
Significance and Impact of the Study:  Results of this study have implications for efficacy and safety assessments of EcN as a probiotic strain for use in pigs.     

Transgenic milk containing recombinant human lactoferrin modulates the intestinal flora in piglets

Lactoferrin (LF) is a beneficial multifunctional protein in milk. The objective of this study was to determine whether bovine transgenic milk containing recombinant human lactoferrin (rhLF) can modulate intestinal flora in the neonatal pig as an animal model for the human infant. We fed 7-day-old piglets (i) ordinary whole milk (OM), (ii) a 1:1 mixture of OM and rhLF milk (MM), or (iii) rhLF milk (LFM). LFM provided better average daily mass gain than OM (P = 0.007). PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis revealed that the LFM piglets exhibited more diversity of the intestinal flora than the OM group. Except for the colon in the LFM group, an increasing trend in microbial diversity occurred from the duodenum to the colon. Fecal flora was not different across different ages or different treatment groups, but a cluster analysis showed that the fecal flora of OM- and MM-fed piglets had a higher degree of similarity than that of LFM-fed piglets. Based on culture-based bacterial counts of intestinal content samples, concentrations of Salmonella spp. in the colon and of Escherichia coli throughout the intestine were reduced with LFM (P < 0.01). Concentrations of Bifidobacterium spp. in the ileum and of Lactobacillus spp. throughout the intestine were also increased with LFM (P ≤ 0.01). We suggest that rhLF can modulate the intestinal flora in piglets.     

The growth performance,intestinal digestive and absorptive capabilities in piglets with different lengths of small intestines

The small intestine is an important digestive organ and plays a vital role in the life of a pig. We tested the hypothesis that the length of the small intestine is related to growth performance and intestinal functions of piglets. A total of 60 piglets (Duroc × Landrace × Yorkshire), weaned at day 21, were fed an identical diet during a 28-day trial. At the end of the study, all piglets were sacrificed, dissected and grouped according to small intestine lengths (SILs), either short small intestine (SSI), middle small intestine (MSI) or long small intestine (LSI), respectively. Positive relationships between SIL and BW, average daily gain (ADG), average daily feed intake (ADFI) and gain-to-feed ratios (G : F) were observed. Final BW, ADG, ADFI and G : F significantly increased (P < 0.05) in MSI and LSI piglets compared with SSI piglets. Short small intestine and MSI had greater jejunal mucosa sucrase and alkaline phosphatase activities (P < 0.05) than LSI piglets. The mRNA level of solute carrier family 2 member 2 (Slc2a2) in the jejunal mucosa of SSI piglets was the greatest. The MSI piglets had a greater (P < 0.05) ileal villus height than other piglets and greater (P < 0.05) villus height-to-crypt depth ratios than LSI piglets. However, the LSI piglets had a greater (P < 0.05) ileal crypt depth than SSI piglets. No significant differences in duodenal, jejunal, caecal and colonic morphologies were detected among the groups. Moreover, luminal acetate, propionate, butyrate and total short-chain fatty acid contents were greater (P < 0.05) in SSI and MSI piglets than those in LSI piglets. In addition, there was greater serum glucose concentration in MSI piglets than other piglets. Serum albumin concentration in SSI piglets was the lowest. In conclusion, these results indicate that SIL was significantly positively associated with growth performance, and in terms of intestinal morphology and mucosal digestive enzyme activity, the piglets with a medium length of small intestine have better digestion and absorption properties.     

pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis

Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless beta-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. beta-Galactosidase was expressed in E. coli and E. faecalis at levels of 10(3) and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.     

Dietary N-Carbamylglutamate Supplementation Boosts Intestinal Mucosal Immunity in Escherichia coli Challenged Piglets

N-carbamylglutamate (NCG) has been shown to enhance performance in neonatal piglets. However, few studies have demonstrated the effect of NCG on the intestinal mucosal barrier. This study was conducted to determine the effects of dietary NCG supplementation on intestinal mucosal immunity in neonatal piglets after an Escherichia coli (E. coli) challenge. New-born piglets (4 d old) were assigned randomly to one of four treatments (n = 7), including (I) sham challenge, (II) sham challenge +50 mg/kg NCG, (III) E. coli challenge, and (IV) E. coli challenge +50 mg/kg NCG. On d 8, pigs in the E. coli challenge groups (III and IV) were orally challenged with 5 mL of E. coli K88 (10⁸ CFU/mL), whereas pigs in the sham challenge groups (I and II) were orally dosed with an equal volume of water. On d 13, all piglets were sacrificed, and samples were collected and examined. The results show that average daily gain in the E. coli challenged piglets (III and IV) was decreased (P_E.coli<0.05). However, it tended to be higher in the NCG treated piglets (II and IV). Ileum secretory IgA, as well as IFN-γ, IL-2, IL-4 and IL-10 in ileal homogenates, were increased in E. coli challenged piglets (III and IV). Similarly, ileum SIgA and IL-10 levels, and CD4⁺ percentage in NCG treated piglets (II and IV) were higher than no-NCG treated piglets (P_NCG<0.05). However, the IL-2 level was only decreased in the piglets of E. coli challenge + NCG group (IV) compared with E. coli challenge group (III) (P<0.05). No change in the IL-2 level of the sham challenged piglets (III) was observed. In conclusion, dietary NCG supplementation has some beneficial effects on intestinal mucosal immunity in E. coli challenged piglets, which might be associated with stimulated lymphocyte proliferation and cytokine synthesis. Our findings have an important implication that NCG may be used to reduce diarrhea in neonatal piglets.     

Conjugal transfer of plasmid pWBG637 from Staphylococcus aureus to Staphylococcus epidermidis and Streptococcus faecalis

Plasmids pWBG636 (GmR) and pWBG642 (EmR) derived from the conjugative plasmid pWBG637 were tested for ability to transfer conjugatively to Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis and Escherichia coli. Both plasmids transferred to S. epidermidis and Strept. faecalis but not to B. subtilis and E. coli. Once in S. epidermis and Strept. faecalis they were transferred back to S. aureus. Restriction endonuclease analysis showed that the plasmids were stably maintained in S epidermidis and Strept. faecalis.     

Intestinal development and growth performance of early-weaned piglets fed a low-threonine diet

High dietary threonine extraction by the digestive tract suggests that threonine contributes to maintain gut integrity. The aims of this study were to investigate the intestine development and the growth performance of early-weaned piglets pair-fed either a control well-balanced (C: 9.3 g threonine/kg diet) or a low-threonine diet (LT: 6.5 g threonine/kg diet) for 2 weeks. As expected, LT piglets presented lower plasma free threonine compared with C piglets (118 v. 356 ± 12 μmol/l, P < 0.001). Dietary threonine supply altered neither growth performance nor growth of the intestine and of the other portal-drained viscera (stomach, spleen and pancreas). Nevertheless, villus height was reduced in the ileum of the LT piglets compared with C piglets (446 v. 714 ± 74 μm, P < 0.05). This was also associated with a decrease in crypt width (P < 0.05) and villus height-to-crypt depth ratio (P < 0.05). Whereas maltase and lactase activities did not change between the two groups, aminopeptidase nitrogen activity was decreased in the ileum of LT piglets (269 v. 374 ± 27 IU/mg protein, P < 0.05). The number of mucin-containing goblet cells was not modified in the ileum and in the proximal part of the large intestine of the LT piglets compared with the C piglets. In conclusion, despite no alteration of intestinal growth, villus hypotrophy associated with a reduction of aminopeptidase nitrogen activity suggest an alteration of the structure of the ileum in early-weaned piglets fed a diet supplying inadequate dietary threonine.     

Phenotypic characterization of intestinal Escherichia coli of pigs during suckling, postweaning, and fattening periods.

A highly discriminatory and standardized biochemical fingerprinting method was used to monitor the persistence and colonization of intestinal Escherichia coli isolated from the feces of four sows and their litters (four piglets from each) during the suckling, postweaning, and fattening periods. Altogether, 195 fecal samples were collected and 1,827 E. coli strains were tested (mean number of isolates tested per fecal sample per pig, 9.5). Strains were divided into similarity groups on the basis of their biochemical phenotypes (BPTs). The diversity of E. coli strains in each sample was measured with Simpson's index of diversity, and similarity between E. coli floras of piglets was calculated with a population similarity index. Each fecal sample contained several BPTs of E. coli, some of which dominated that population. The intestinal colonization of piglets consisted of successive waves of different E. coli BPTs, the tenure of which varied from a few days to 2 weeks. Most of these BPTs disappeared in the succeeding samples and were not recovered again from the same piglets. On the other hand, some E. coli strains which colonized piglets early during the suckling period persisted for a long period and were referred to as resident BPTs. Each piglet carried more than one resident BPT (mean of 2.4 BPTs per pig), some of which were also found in other piglets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of medicated feed on the development of antimicrobial resistance in bacteria at integrated pig-fish farms in Vietnam

Integrated livestock-fish aquaculture utilizes animal excreta, urine, and feed leftovers as pond fertilizers to enhance the growth of plankton and other microorganisms eaten by the fish. However, antimicrobial-resistant bacteria may be transferred and develop in the pond due to selective pressure from antimicrobials present in animal feed, urine, and feces. In an experimental pig-fish farm located in periurban Hanoi, Vietnam, nine piglets were provided feed containing 5 μg of tetracycline (TET)/kg pig weight/day and 0.45 μg of enrofloxacin (ENR)/kg pig weight/day during the second and fourth (last) months of the experiment. The aim of this study was to determine the association between the provision of pig feed with antimicrobials and the development of antimicrobial resistance, as measured in a total of 520 Escherichia coli and 634 Enterococcus strains isolated from pig manure and water-sediment pond samples. MIC values for nalidixic acid (NAL) and ENR showed that E. coli and Enterococcus spp. overall exhibited significant higher frequencies of resistance toward NAL and ENR during the 2 months when pigs were administered feed with antimicrobials, with frequencies reaching 60 to 80% in both water-sediment and manure samples. TET resistance for both indicators was high (>80%) throughout the study period, which indicates that TET-resistant E. coli and Enterococcus spp. were present in the piglets before the initiation of the experiment. PCR-based identification showed similar relative occurrences of Enterococcus faecium, Enterococcus faecalis, and other Enterococcus spp. in the water-sediment and manure samples, suggesting that Enterococcus spp. isolated in the ponds originated mainly from the pig manure. The development of antimicrobial resistance in integrated animal husbandry-fish farms and possible transfers and the impact of such resistance on food safety and human health should be further assessed.     

酶解乳蛋白对初生仔猪小肠组织学结构及隐窝细胞增殖的影响(英文)

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Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs

A mixture of two phages, B44/1 and B44/2, protected calves against a potentially lethal oral infection with an O9:K30,99 enteropathogenic strain of Escherichia coli, called B44, when given before, but not after, the onset of diarrhoea; a mixture in which phage B44/3 was replaced by phage B44/3 was effective after the onset of diarrhoea. Calves that responded to phage treatment had much lower numbers of E. coli B44 in their alimentary tract than untreated calves. Usually, high numbers of phage B44/1 and rather lower numbers of phage B44/2 or B44/3 were present in the alimentary tract of these animals. At death, most calves that had not responded to treatment with phages B44/1 and B44/2 had high numbers of mutants of E. coli B44 resistant to phage B44/1 in their small intestine. Phage-treated calves that survived E. coli infection continued to excrete phage in their faeces, at least until the numbers of E. coli B44 also excreted were low. The phages survived longer than E. coli B44 in faecal samples taken from phage-treated calves and exposed to the atmosphere in an unheated animal house. Calves inoculated orally with faecal samples from phage-treated calves that contained sufficient E. coli B44 to cause a lethal infection remained healthy. A mixture of two phages, P433/1 and P433/2, and phage P433/1 alone cured diarrhoea in piglets caused by an O20:K101,987P strain of E. coli called P433. The numbers of the infecting bacteria and phages in the alimentary tract of the piglets resembled those in the calves. Another phage given to lambs 8 h after they were infected with an O8:K85,99 enteropathogenic strain of E. coli, called S13, reduced the numbers of these organisms in the alimentary tract and had an ameliorating effect on the course of the disease. No phage-resistant mutants of E. coli S13 were isolated from the lambs. The only mutants of E. coli B44 and P433 that emerged in the calves and piglets were K30- or K101- and resistant to phage B44/1 or P433/1 respectively; those tested were much less virulent than their parent strains.