Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium

Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium.     

Decreased colonization of chicks by Salmonella enterica serovar Gallinarum expressing mannose-sensitive FimH adhesin from Salmonella enterica serovar Enteritidis

To investigate the role of non-hemagglutinating type 1 fimbriae in the pathogenesis of Salmonella Gallinarum, the isogenic mutant elaborating type 1 fimbriae with mannose-sensitive (MS) variant of the FimH adhesin from Salmonella Enteritidis and the mutant strain with no FimH expression were constructed. Their binding to chicken leukocytes in vitro and invasiveness in 1-day-old chicks were studied. Our results demonstrated that S. Gallinarum type 1 fimbriae with an endogenous variant of the FimH adhesin mediated mannose-resistant (MR) binding to avian leukocytes and did not bind to human epithelial cells. However, after allelic replacement of the FimH, mutated fimbriae with S. Enteritidis variant of the FimH adhesin bound to both cell types in a mannose-dependent manner. In chick model, S. Gallinarum expressing wild-type FimH variant colonized cecal tonsils and bursa of Fabricius more effectively and invaded the spleen and liver in greater numbers than S. Gallinarum fimH knockout strain or mutant expressing MS FimH variant from S. Enteritidis. The invasive potential of the latter was greatly reduced in chicks since no viable bacteria expressing MS variant of the adhesin could be recovered from intestinal lymphoid tissues or liver over a 6 days course of infection. Together, these results demonstrate that the S. Gallinarum type 1 fimbriae with the endogenous MR variant of the FimH protein increase systemic dissemination of S. Gallinarum and colonization of internal organs in chicks indicating the importance of these adhesive structures in the virulence of S. Gallinarum.     

Sequence analysis demonstrates the conservation of fimH and variability of fimA throughout avian pathogenic Escherichia coli (APEC)

In this study we sequenced and analysed the fimH and fimA genes of 24 avian pathogenic Escherichia coli (APEC) isolates, in order to investigate their possible conserved nature. Additional parameters (serotype, presence of aerobactin receptor, expression of F1 pili and virulence for chickens) were investigated to look for correlations with the obtained sequences. The sequence analysis demonstrated that FimH is highly conserved among all investigated APEC strains (>99% homology), whereas the major subunit FimA is less conserved, presenting 6 variable regions distributed along the protein. A hydrophilicity analysis suggested several variable domains of FimA to be potential epitopes. We were able to classify the investigated strains into three main groups, on the basis of the amino-acid sequences of the variable regions. This grouping was consistent throughout all variable regions and was independent of serotype, leading to an improved classification of the F1 pili. No correlation was found between the fimH and fimA sequences and the following parameters: avian species, organ of isolation, serotype, presence of aerobactin receptor and virulence for chickens. This study elucidated the molecular structure and the degree of conservation of FimH and FimA among various avian pathogenic E. coli strains.     

Escherichia coli colonization of the trachea in poultry: comparison of virulent and avirulent strains in gnotoxenic chickens

In chickens, virulent Escherichia coli strains express their pathogenicity in the respiratory tract. A quantitative comparison of tracheal colonization by virulent and avirulent E. coli was carried out in gnotoxenic chickens after intestinal implantation. Two-week-old axenic chicks reared in isolators were inoculated per os with various associations of identified E. coli strains. No clinical sign of disease was observed in any of the chicks, despite the presence of virulent strains in all the intestines and most of the tracheas. The virulent organism reached greater population sizes in the trachea and feces of monocontaminated chicks and of chicks contaminated simultaneously with a virulent and an avirulent strain. In holoxenic chicks, identified virulent and avirulent strains were outnumbered by the E. coli population of the intestinal flora previously established and could not be recovered from the tracheas of most chicks.     

interactions between escherichia coli and Newcastle disease virus in chickens

We investigated the interaction between Newcastle disease virus (NDV) and Escherichia coli in cell cultures, embryonated eggs, and 8-wk-old chickens. We measured the interactions on the basis of bacterial adherence and NDV hemagglutination titer in chickens, chicken embryos, and chicken embryo cell culture. Depending on the inoculation order of E. coli, a significant alteration of the growth of NDV was observed in both chickens and chicken embryos. When certain strains of E. coli were given before NDV exposure, the virus titers were lowered. In chickens, the mean virus titer was significantly (P < 0.05) lowered in the crop, the proventriculus, the gizzard, and the jejunum. However, there were no significant differences (P < 0.05) between the two groups for NDV titers in the duodenum, ileum, and cecum. In chicken embryos, when E. coli serotypes O78 and O119:B14 were inoculated before NDV exposure, the mean NDV titers were significantly (P < 0.5) lowered. However, there were no significant differences (P < 0.05) in NDV titer between the two groups when E. coli serotypes O78:K80:NM and O1ab:K NM were inoculated 24 hr before NDV exposure. When NDV was given prior to E. coli exposure, NDV titer was higher in both chickens and chicken embryos. In chickens, when NDV was given 48 hr before E. coli inoculation, NDV was detected in the proventriculus, gizzard, jejunum, ileum, and cecum, whereas no virus was detected in the control groups (NDV only). In the crop, NDV was detected at a significantly (P < 0.05) higher titer in the E. coli-inoculated group when compared with the control group that received NDV alone. In chicken embryos, virus titer was significantly (P < 0.05) higher when NDV was given 24 hr before E. coli inoculation for all three NDV strains used (Ulster and V4 strains). Adherence of E. coli to chicken embryo kidney (CEK) cells was significantly higher (P < 0.05) when the CEK cells were infected first with NDV and then by E. coli. The mean bacterial count per microscopic field in NDV-uninfected monolayers was eight compared with 112 for the NDV-infected monolayers. In approximately 10% of the fields in NDV-infected monolayers, the bacteria were too numerous to count.     

Role of Campylobacter jejuni potential virulence genes in cecal colonization

Campylobacter jejuni, a common commensal in chickens, is one of the leading causes of bacterial gastroenteritis in humans worldwide. The aims of this investigation were twofold. First, we sought to determine whether mutations in the C. jejuni ciaB and pldA virulence-associated genes impaired the organism's ability to colonize chickens. Second, we sought to determine if inoculation of chicks with C. jejuni mutants could confer protection from subsequent challenge with the C. jejuni wild-type strain. The C. jejuni ciaB gene encodes a secreted protein necessary for the maximal invasion of C. jejuni into cultured epithelial cells, and the pldA gene encodes a protein with phospholipase activity. Also included in this study were two additional C. jejuni mutants, one harboring a mutation in cadF and the other in dnaJ, with which we have previously performed colonization studies. In contrast to results with the parental C. jejuni strain, viable organisms were not recovered from any of the chicks inoculated with the C. jejuni mutants. To determine if chicks inoculated with the C. jejuni mutants become resistant to colonization by the C. jejuni parental strain upon subsequent challenge, chicks were inoculated either intraperitoneally (i.p.) or both orally and i.p. with the C. jejuni mutants. Inoculated birds were then orally challenged with the parental strain. Inoculation with the C. jejuni mutants did not provide protection from subsequent challenge with the wild-type strain. In addition, neither the C. jejuni parental nor the mutant strains caused any apparent morbidity or mortality of the chicks. We conclude that mutations in genes cadF, dnaJ, pldA, and ciaB impair the ability of C. jejuni to colonize the cecum, that chicks tolerate massive inoculation with these mutant strains, and that such inoculations do not provide biologically significant protection against colonization by the parental strain.     

Effect of antibodies to type 1 fimbriae on clearance of fimbriated Escherichia coli from the bloodstream of turkeys

Young turkeys (n = 20) were inoculated IV with fimbriated, virulent Escherichia coli ECl (O78:K80: H9:F1). Blood samples were collected for bacterial quantitation at postinoculation minutes (PIM) 10, 20, 30, 40, 50, and 60. Immediately after the PIM 30 sampling, the turkeys were allotted into 4 groups (5 turkeys/group) and were injected IV with 1 of the following antisera: group 1, antibodies to F1 fimbriae (AF); group 2, antibodies to E coli O78 antigen (AO); group 3, antibodies to live, fimbriated (F1+) homologous E coli (ALEC); or group 4, normal turkey serum (NTS) collected from a healthy turkey. Compared with NTS, ALEC and AO caused a significant reduction in blood-borne E coli, whereas AF did not reduce bacterial numbers. In addition, 2 groups of 10 turkeys were inoculated IV with live, F1+ or nonfimbriated (F1-) E coli ECl. Numbers of viable bacteria were determined in blood samples and liver specimens collected 2 minutes after inoculation. Compared with F1- bacteria, significantly more F1+ bacteria were found in liver specimens and significantly fewer F1+ bacteria were found in blood samples. Results indicated that antibodies to F1 fimbriae do not enhance clearance of F1+ E coli from the bloodstream of turkeys probably because F1+ bacteria are selectively cleared by the liver, even without antibody.     

实验性鸡大肠杆菌病病理学动态变化

用致病性大肠杆菌O18分离株和／或低致病性禽流感病毒（Mildly pathogenic avian influenza virus ,MPAIV)接种10－12日龄SPF鸡。在接种后1－96h进行临床症状与大体病理变化、组织学观察发现：大肠杆菌接种组、MPAIV接种组和健康接种组除扑杀鸡外未见鸡死亡，MPAIV与大肠杆菌混合接种组除扑杀鸡外死亡率为24％。混合接种组的病变比大肠杆菌接种组出现的时间早，恢复也慢，各脏器的病理变化更严重。MPAIV主要引起各实质器官的坏死，结果表明，大肠杆菌经气管内接种后试验鸡主要表现为呼吸道的炎症反应；MPAVI可使鸡大肠杆菌病严重化。     

Conservation of deduced amino acid sequence of FimH among Escherichia coli of bovine, porcine and avian disease origin

The FimH subunit of type 1 pili mediates adhesion of Escherichia coli to epithelium in different animal hosts. In this study, we sequenced and analyzed the fimH genes of 24 E. coli strains from bovine and porcine clinical cases. The obtained sequences were compared among each other and also with 24 known fimH sequences from avian E. coli strains. This comparison revealed a substantial homology (>99%) among strains from the different animal species origins. Moreover, specific mutations were found, some of which were present more frequently in avian strains or in bovine and porcine strains.     

Escherichia coli multiplication and lesions in the respiratory tract of chickens inoculated with infectious bronchitis virus and/or E. coli.

Escherichia coli numbers and histopathological changes were studied in the respiratory tract of line 151 chickens intranasally inoculated with infectious bronchitis virus (IBV) and/or virulent E. coli; this line is highly susceptible to IBV. Chickens inoculated with IBV alone showed increased numbers of E. coli in the trachea and had tracheitis, airsacculitis, and bronchiolitis. One of 17 chickens inoculated with IBV alone died with fibrinopurulent serositis. Chickens inoculated with IBV and E. coli had more severe and persistent respiratory lesions than those inoculated with IBV alone. E. coli was isolated from tracheas of chickens inoculated with IBV and E. coli more frequently than from chickens inoculated with IBV alone. In this group, 14 of 27 chickens died with tracheal plugs or with fibrinopurulent serositis. There was neither increased numbers of E. coli nor significant lesions in the respiratory tract of the group inoculated with E. coli alone. These results suggest that IBV may facilitate E. coli invasion into the lower respiratory tract of the chicken.     

Construction, characterization, and evaluation of the vaccine potential of three genetically defined mutants of avian pathogenic Escherichia coli

The delta galE, delta purA, and delta aroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis. The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively. The delta purA and delta aroA mutants did not grow on minimal medium, whereas the delta galE mutant grew on minimal medium but was sensitive to galactose-induced lysis. The reversion frequencies of all three mutants were <10(-12). The mutants were highly attenuated for virulence as determined by administration of approximately 10(7) colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route. Chickens were vaccinated with the mutants by spray (droplet size approximately 20 microm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy. The mutants were found to be safe. Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E. coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay. In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline-treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain. In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain. The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E. coli 4 days later. Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge. This study indicates that the delta galE, delta purA, and delta aroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.     

鸡致病性大肠杆菌I型菌毛交叉保护性研究及其结构基因 FimA 的序列分析

从山东省各地分离到107株鸡致病性大肠杆菌，选择了其中2株高致病性大肠杆菌，血清型分别为O78和OM，经增菌培养后提取菌毛制备为单价菌毛油乳苗，分别接种于1日龄和14日龄雏鸡，于4周龄攻毒。结果二者之间有一定的交叉保护作用。根据Genbank收录的人源大肠杆菌I型菌毛FimA基因和P型菌毛papA基因序列，分别设计了2对引物。通过PCR对上述2株大肠杆菌进行扩增，结果只有FimA的一对引物扩增出相应的条带，经测序证明为FimA基因。papA基因的引物未扩出任何条带，证明这2株大肠杆菌表达I型菌毛。通过对2株大肠杆菌结构基因FimA进行分析，发现二者具有高度的同源性。本研究的目的是探讨菌毛亚单位之间的交叉保护性与其菌毛的结构基因之间是否存在相关性。     

Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens.

Avian pathogenic strains of Escherichia coli cause a number of extraintestinal diseases in poultry, including airsacculitis and colisepticemia. Expression of O78 lipopolysaccharide (LPS) is frequently associated with pathogenic isolates. Salmonella, a common poultry contaminant, is a major public health concern. The purpose of this work was to develop an E. coli vaccine for poultry with the use of an attenuated Salmonella typhimurium carrier that would benefit both the bird and the consumer. Orally administered attenuated S. typhimurium delta cya delta crp strains have been shown to provide excellent protection against wild-type Salmonella challenge in chickens. This work describes the construction of a delta cya delta crp derivative of an avian pathogenic S. typhimurium that expresses both the homologous group B determinants (O1,4,5,12) and the heterologous E. coli O78 LPS O antigens. This was accomplished by inserting the E. coli rfb region, which encodes the genes required for O78 expression, into the chromosomal cya gene of S. typhimurium, creating a defined deletion/insertion mutation. A delta crp mutation was introduced in a subsequent step. Expression of both O antigens was stable in vitro and in vivo. Vaccination of white leghorn chicks at day of hatch and 14 days with the recombinant vaccine strain induced serum immune responses against both S. typhimurium and E. coli LPS and protected the birds against subsequent challenge with an avian pathogenic E. coli O78 strain. Introduction of a mutation in rfc, which encodes the O antigen polymerase, reduced the chain length of the S. typhimurium LPS without affecting the expression of O78. The rfc mutation further enhanced the ability of the vaccine strain to protect chickens against E. coli challenge.     

Avian pathogenic Escherichia coli (APEC).

Avian pathogenic Escherichia coli (APEC) cause aerosacculitis, polyserositis, septicemia and other mainly extraintestinal diseases in chickens, turkeys and other avian species. APEC are found in the intestinal microflora of healthy birds and most of the diseases associated with them are secondary to environmental and host predisposing factors. APEC isolates commonly belong to certain serogroups, O1, O2 and O78, and to a restricted number of clones. Several experimental models have been developed, permitting a more reliable evaluation of the pathogenicity of E. coli for chickens and turkeys. Hence, virulence factors identified on APEC are adhesins such as the F1 and P fimbriae, and curli, the aerobactin iron sequestering system, K1 capsule, temperature-sensitive hemagglutinin (Tsh), resistance to the bactericidal effects of serum and cytotoxic effects. Experimental infection studies have shown that the air-exchange regions of the lung and the airsacs are important sites of entry of E. coli into the bloodstream of birds during the initial stages of infection and that resistance to phagocytosis may be an important mechanism in the development of the disease. They have also demonstrated that F1 fimbriae are expressed in the respiratory tract, whereas P fimbriae are expressed in the internal organs of infected chickens. The role of these fimbrial adhesins in the development of disease is not yet, however, fully understood. The more recent use of genetic approaches for the identification of new virulence factors will greatly enhance our knowledge of APEC pathogenic mechanisms. Diagnosis of APEC infections is based on the clinical picture, lesions and isolation of E. coli. This may be strengthened by serotyping and identification of virulence factors using immunological or molecular methods such as DNA probes and PCR. Approaches for the prevention and control of APEC infections include the control of environmental contamination and environmental parameters such as humidity and ventilation. Antibiotherapy is widely used, although APEC are frequently resistant to a wide range of antibiotics. Vaccines containing killed or attenuated virulent bacteria protect against infection with the homologous strain but are less efficient against heterologous strains. Hence, vaccination for colibacillosis is not widely practised because of the large variety of serogroups involved in field outbreaks.     

Resistance of broiler chickens to Escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies

Egg-yolk antibodies induced by immunizing hens with selected Escherichia coli antigens were evaluated for their ability to protect broiler chickens against respiratory/septicemic disease caused by avian pathogenic E. coli (APEC). Seven groups of broiler breeder hens were vaccinated three times, 1 week apart with live E. coli, killed E. coli, E. coli antigens [lipopolysaccharide (LPS), type 1 pilus adhesin (FimH), P pilus adhesin (PapG), aerobactin outer membrane receptor (IutA)] or phosphate buffered saline (PBS). An O78 APEC strain was used for preparation of all the antigens. Egg yolk immunoglobulins (IgY) were purified from eggs of each group and antibody activity in serum and purified IgY was determined by enzyme-linked immunosorbent assay (ELISA). IgY (100mg) was injected intramuscularly into 11-day-old broiler chickens, which were challenged 3 days later with homologous (O78) or heterologous (O1 or O2) E. coli by the intra-air sac route. Mortality was recorded and surviving chickens were euthanized 1 week after the challenge and examined for macroscopic lesions. Passive antibodies against all antigens except FimH were protective (90-100%) against the homologous challenge, but only anti-PapG and anti-IutA were effective against heterologous challenge. Anti-PapG IgY provided the greatest protection against the three serogroups of E. coli used for challenge. Hence vaccination of broiler breeders to induce anti-PapG and anti-IutA antibodies may provide passive protection of progeny chicks against respiratory/septicemic disease caused by APEC.     

Expression of Escherichia coli antigens in Salmonella typhimurium as a vaccine to prevent airsacculitis in chickens

Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis. A number of E. coli serotypes are associated with these diseases, although the most prevalent serotype is O78. Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli. We have been working to develop an effective vaccine to protect chickens against these diseases. We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E. coli strain. In this work, we have constructed an attenuated S. typhimurium that expresses both the O78 lipopolysaccharide and E. coli-derived type 1 fimbriae. In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age. Birds were challenged at 4 wk of age. We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S. typhimurium that did not express any E. coli antigens. In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates. We were not able to demonstrate protection against E. coli O1 or O2 serotype challenge, nor against challenge with wild-type S. typhimurium.     

SPF鸡人工感染禽源大肠杆菌、H9亚型禽流感病毒后的体液免疫应答和死亡率分析

SPF鸡经不同顺序、不同时间间隔人工感染MPAIV和E．coli 173株（04）后，对试验鸡的病死率和针对大肠杆菌不同抗原体液免疫应答水平等进行研究。结果表明：各混合感染组试验鸡病死率明显高于单独接种组，且以先接种MPAIV再接种E．coli组死亡率最高；各混合感染组试验鸡针对E．coli OMPs和LPS的抗体水平低于单独感染E．coli组，其中又以先接种MPAIV再接种E．coli组为最低，提示MPAIV和E．coli间存在协同致病作用，这种协同机理可能与因MPAIV的感染导致一定程度的免疫抑制并进而促进了E．coli在体内的定居与繁殖有关。     

Pathogenicity of various isolates of Alcaligenes faecalis for broilers

Day-old broilers or specific-pathogen-free chickens were inoculated intranasally with approximately 1 X 10(8) organisms of eight different field isolates of Alcaligenes faecalis. Major differences in the pathogenicity of isolates and their ability to colonize the trachea were found. Only two isolates (Wilson and Lockamy) produced mild clinical signs of respiratory disease ("snicking," dyspnea). The same two also colonized the respiratory tract, especially the trachea, in large numbers; they persisted for 31 days. Of the remaining six isolates, five were also able to colonize the respiratory tract but did so to a lesser degree and less persistently, without causing clinical signs. Only one isolate (CS) was incapable of becoming established in the respiratory tract of chicks after intranasal inoculation.     

Inactivated Salmonella expressing the receptor-binding domain of bacterial adhesins elicit antibodies inhibiting hemagglutination

We examined the potential of inactivated Salmonella strains to induce protective antibodies against two adhesins of pathogenic Escherichia coli. The receptor-binding domains of the F17a-G adhesin of F17a fimbriae and of the FimH adhesin of type 1 fimbriae were fused to the translocator domain of the autotransporter AIDA-I. An IgG response against F17a-G or FimH was induced after immunization of mice with acetone-inactivated Salmonella displaying the corresponding fimbrial receptor-binding domain. These sera inhibit in vitro agglutination of erythrocytes by E. coli carrying these fimbriae. Our results demonstrate that induced and subsequently acetone-inactivated Salmonella are useful delivery vehicles for the stimulation of an IgG antibody response against heterologous antigens.     

Effect of a commercial competitive exclusion culture on reduction of colonization of an antibiotic-resistant pathogenic Escherichia coli in day-old broiler chickens

One-day-of-age broiler chickens were administered a commercial competitive exclusion (CE) product and then challenged by three different methods with an Escherichia coli O78:K80 that was pathogenic for poultry and resistant to six antibiotics. Three challenge methods were used on 2-day-old broilers: direct challenge, precolonized seeder, and instant seeder. Direct challenge was accomplished by administering the challenge E. coli per os. The precolonized seeder challenge had two chicks that had received the challenge E. coli 24 hr previously, whereas the instant seeder challenge had two chicks given the challenge E. coli per os with immediate placement with the experimental birds. One oral dose of the commercial CE product significantly reduced the colonization of the small intestine, large intestine, and ceca by the highly antimicrobial resistant poultry pathogenic E. coli O78:K80 at 7 and 14 days postchallenge by all three challenge methods. The overall mean reductions in colonization were 3.0 log10 for the large intestine, 3.0 log10 for the small intestine, and 4.0 log10 for the cecum. The most severe challenge method, on the basis of the least amount of reduction of colonization of the challenge E. coli by the CE, was by the direct oral gavage at 2 days of age.