F41 Fimbriae Research Articles

Isolation and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains From Industrial Dairy Farms of Hamedan, Iran

Background: Enterotoxigenic Escherichia coli (ETEC) is considered as one of the most common causes of infectious diarrhea in calves, infecting animals during the first week of age. The secretory diarrhea is attributed to the virulence factors of ETEC strains mainly including heat stable toxin (STa), as well as F5 (K99) and F41 fimbriae. Objectives: The present study was undertaken to investigate ETEC infection in neonatal calves of industrial dairy farms of Hamedan, Iran. Additionally, it was undertaken to investigate the genotypic screening of virulence genes in enterotoxigenic E. coli isolated from from dairy farms calves of Hamedan county. Materials and Methods: A total of 120 rectal swab samples were collected from healthy and diarrheic calves at one week of age belonging to eight farms. Conventional bacteriological methods, multiplex PCR, and antibiotic susceptibility test of the ETEC isolates were performed. Results: Nine E. coli isolates were found to be ETEC strains, carrying STa enterotoxin along with F5 and/or F41 fimbriae as the indicators of ETEC cells. Additionally, antibiotic susceptibility test of the ETEC isolates revealed that all of them were sensitive to trimethoprim-sulfamethoxazole, ciprofloxacin, and enrofloxacin, whereas complete resistance was observed against amoxicillin-clavulanic acid (100%) and polymyxin B (100%). The present study, conducted for the first time in Hamedan, indicated a prevalence of 7.5% for Enterotoxigenic Escherichia coli in the examined animals. Conclusion: Regarding economic losses of the infection in claves as well as the zoonotic nature of ETEC cells, it is recommended that measures should be taken, such as immunization of pregnant cows prior to the delivery, feeding of adequate colostrum to newborn calves at the right time, and adherence to hygiene practices on the farms to prevent and/or reduce the incidence of diarrhea cases caused by infection with these bacteria.

A systematic review and meta-analysis of the epidemiology of pathogenic Escherichia coli of calves and the role of calves as reservoirs for human pathogenic E. coli.

Escherichia coli bacteria are the most common causes of diarrhea and septicemia in calves. Moreover, calves form a major reservoir for transmission of pathogenic E. coli to humans. Systematic reviews and meta-analyses of publications on E. coli as calf pathogens and the role of calves as reservoir have not been done so far. We reviewed studies between 1951 and 2013 reporting the presence of virulence associated factors (VAFs) in calf E. coli and extracted the following information: year(s) and country of sampling, animal number, health status, isolate number, VAF prevalence, serotypes, diagnostic methods, and biological assays. The prevalence of VAFs or E. coli pathotypes was compared between healthy and diarrheic animals and was analyzed for time courses. Together, 106 papers with 25,982 E. coli isolates from 27 countries tested for VAFs were included. F5, F17, and F41 fimbriae and heat-stable enterotoxin (ST) – VAFs of enterotoxigenic E. coli (ETEC) were significantly associated with calf diarrhea. On the contrary, ETEC VAF F4 fimbriae and heat-labile enterotoxin as well as enteropathogenic (EPEC), Shiga toxin-producing (STEC), and enterohemorrhagic E. coli (EHEC) were not associated with diarrhea. The prevalence increased overtime for ST-positive isolates, but decreased for F5- and STEC-positive isolates. Our study provides useful information about the history of scientific investigations performed in this domain so far, and helps to define etiological agents of calf disease, and to evaluate calves as reservoir hosts for human pathogenic E. coli.

Colibacillosis in lambs is associated to type I heat-stable enterotoxin in a farm in São Paulo State, Brazil

Twenty seven (48.2%) culture supernatants of 56 Escherichia coli isolated from diarrheic lamb feces (7 to 10 days old) in São Paulo State, Brazil, presented positive results to suckling mice assay (fluid accumulation) but none caused cytopathic effects on Vero and CHO cells, indicating that these strains did not produced LT or VT toxins. PCR assays showed that these 27 E. coli strains harbored estA, that codifies for STa, but not for stx1, stx2 or cnf genes. The positive STa strains were checked for genes that codify for F41, F17 and K99 fimbriae, wich are considered colonization factors in ETEC. Only F17 was detect in two samples (7.4%). Twelve of 27 STa positive carried hlyA gene and presented hemolytic activity in blood Agar. Presence of rotavirus was not detected among the diarrheic feces. These data suggests that STa must be an important diarrheagenic factor to small ruminants in São Paulo State.

Virulence genes in Escherichia coli isolated from calves with diarrhoea in Iran

This study was conducted to determine the prevalence and characteristics of pathogenic Escherichia coli strains from diarrhoeic calves in Iran. A total of 156 E. coli isolates obtained from 180 diarrhoeic calves were evaluated with two multiplex PCR protocols for the detection of the K99, F41, Sta, Stx1, Stx2 and eaeA genes. Of these 156 isolates, 32 (20.5%) carried at least one virulent gene, 22 (14.1%) possessed enterotoxigenic E. coli (ETEC) virulent genes and 10 (6.4%) possessed shiga toxin genes, virulence factors of shiga toxin-producing E. coli (STEC). None of the isolates carried eaeA gene. All ETEC but one were isolated from 1- to 4-day-old calves (95.4%), and one was isolated from a 6-day-old calf. All STEC were isolated from 7- to 28-day-old calves. All ETEC carried both K99 and F41 fimbriae and possessed Sta enterotoxin gene.

Induction of Immune Responses in Mice after Oral Immunization with RecombinantLactobacillus caseiStrains Expressing EnterotoxigenicEscherichia coliF41 Fimbrial Protein

In an effort to develop a safe and effective vaccine for the prevention of enterotoxigenic Escherichia coli (ETEC) F41 infections, we have developed a surface antigen display system using poly-gamma-glutamate synthetase A (PgsA) as an anchoring matrix. The recombinant fusion proteins comprised of PgsA and fimbrial protein of F41 were stably expressed in Lactobacillus casei 525. Surface localization of the fusion protein was verified by immunoblotting, immunofluorescence microscopy, and flow cytometry. Oral inoculation of recombinant L. casei 525 into specific-pathogen-free BALB/c mice resulted in significant mucosal immunoglobulin A (IgA) titers that remained elevated for >16 weeks. High levels of IgG responses in sera specific for F41 fimbriae were also induced, with prominent IgG1 titers as well as IgG2a and IgG2b titers. The helper T-cell (Th) response was Th2-cell dominant, as evidenced by increased mucosal and systemic interleukin-4-producing T cells and a concomitant elevation of serum IgG1 antibody responses. More than 80% of the mice were protected against challenge with a 2 x 10(4)-fold 50% lethal dose of standard-type F41 (C83919). The induced antibodies were important for eliciting a protective immune response against F41 infection. These results indicated that the use of recombinant L. casei 525 could be a valuable strategy for future vaccine development for ETEC.

Prevalence of enterotoxigenic Escherichia coli virulence genes from scouring piglets in Zimbabwe

World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe. We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened in toxin positive isolates. Toxin positive strains lacking tested fimbriae genes were characterized using transmission electron microscopy, agglutination and agglutination inhibition tests. Approximately 32% of the 1,984 isolates tested positive for STa, STb, LT or Stx-2e genes. Of these, approximately 81% had F4, F5, F6, F18 or F41 fimbriae genes. The remaining toxin positive strains lacked tested fimbriae genes and appeared to either express F1-like fimbriae, or lacked fimbriae. The data constitute an important framework for implementation of prevention measures, such as using relevant fimbriae-based vaccines against ETEC induced diarrhea or VTEC-induced edema.

Virulence Factor Gene Profiles of Escherichia coli Isolates from Clinically Healthy Pigs

Nonpathogenic, intestinal Escherichia coli (commensal E. coli) supports the physiological intestinal balance of the host, whereas pathogenic E. coli with typical virulence factor gene profiles can cause severe outbreaks of diarrhea. In many reports, E. coli isolates from diarrheic animals were classified as putative pathogens. Here we describe a broad variety of virulence gene-positive E. coli isolates from swine with no clinical signs of intestinal disease. The isolation of E. coli from 34 pigs from the same population and the testing of 331 isolates for genes encoding heat-stable enterotoxins I and II, heat-labile enterotoxin I, Shiga toxin 2e, and F4, F5, F6, F18, and F41 fimbriae revealed that 68.6% of the isolates were positive for at least one virulence gene, with a total of 24 different virulence factor gene profiles, implying high rates of horizontal gene transfer in this E. coli population. Additionally, we traced the occurrence of hemolytic E. coli over a period of 1 year in this same pig population. Hemolytic isolates were differentiated into seven clones; only three were found to harbor virulence genes. Hemolytic E. coli isolates without virulence genes or with only the fedA gene were found to be nontypeable by slide agglutination tests with OK antisera intended for screening live cultures against common pathogenic E. coli serogroups. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations and that high numbers of E. coli cells harboring virulence genes and/or with hemolytic activity do not necessarily correlate with disease.

Passive immunity in cattle against enterotoxigenic Escherichia coli: serologic evaluation of a bacterin containing K99 and F41 fimbriae in colostrum of vaccinated females and calf serum

A bacterin from enterotoxigenic Escherichia coli (ETEC), containing fimbriae K99 and F41, was produced and its capacity to induce anti-K99 and anti-F41 antibodies in colostrum of vaccinated cows and in calf serum, and the persistence of these antibodies in neonates were determined. Three experiments were performed on two commercial farms. In all experiments animals were allotted randomly to the blocks, each block consisting of two pregnant females (a vaccinated one and a control one) and their respective calves. In experiment A (farm 1), comprised of 18 blocks, the animals received a vaccine dose 30 days before delivery. In experiment B (farm 1), consisted of 26 blocks, the animals received two vaccine doses (60 and 30 days before delivery). In experiment C (farm 2), consisted of 22 blocks, the animals received two vaccine doses (60 and 30 days before delivery). In experiments A and B pregnant cows and heifers were used and colostrum and serum from 24- to 36-hour-old calves were collected. In experiment C, pregnant embryo-recipient heifers were used and colostrum and sera from calves at 7, 14, 28 and 42 days of age were collected. Anti-K99 and anti-F41 antibodies were detected by ELISA using purified K99 and F41 fimbrial antigens. In experiment A no difference between treated and control groups was observed for the concentration of anti-K99 and anti-F41 antibodies in colostrum and calf serum. In experiment B a difference (P<0.001) was observed for colostrum of vaccinated females and for serum of their calves. In experiment C, difference between vaccinated and control animals was observed for colostrum and calf serum at 7, 14, 28 (P<0.001 in all cases) and 42 days of age (P= 0.003). The results showed the efficiency of the bacterin to induce detectable humoral immune response.

Enterotoxigenic Escherichia coli strains bind bovine milk gangliosides in a ceramide-dependent process.

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is the main infectious disease of newborn calves. The first step of infection involves bacterial attachment to the intestinal mucosa. This adhesion is mediated by fimbriae that recognize some glycoconjugates on the host cell surface, in particular, several gangliosides. Because milk also contains gangliosides, these have been suggested to serve as ligands for bacterial fimbriae and thus prevent the bacterial attachment to mucosa. The most relevant ETEC strains in calves, including those with K99 and F41 fimbriae, were assayed to determine whether they are able to bind gangliosides isolated from several stages of bovine lactation. Both GM3 and GD3, the main gangliosides of milk, were recognized by ETEC strains, although the different fimbriae showed diverse levels of affinity. Unexpectedly, the adhesion to colostral gangliosides was considerably weaker than that to gangliosides from the other stages of lactation. Because the carbohydrate moiety did not change and because differences in the percentages of unsaturated FA and sphingosine between colostrum and other stages were observed, we conclude that the differences in adhesion could be due to a different composition of the ganglioside ceramide.

Isolation and association of Escherichia coli AIDA-I/STb, rather than EAST1 pathotype, with diarrhea in piglets and antibiotic sensitivity of isolates.

To identify emerging Escherichia coli that have the potential to cause diarrhea in pigs, the prevalence of E. coli pathotypes was determined among 170 and 120 isolates from diarrheic and nondiarrheic piglets, respectively. The isolates were tested for F4, F5, F6, F18, and F41 fimbriae, for E. coli attaching and effacing (EAE), porcine attaching and effacing-associated (Paa), and adhesin involved in diffuse adherence (AIDA-I) factors, for LT, STa, STb, and enteroaggregative heat-stable (EAST1) enterotoxins, and for Shiga toxins (Stxl, Stx2, and Stx2e), using DNA hybridization and polymerase chain reaction. All isolates were O-serotyped and tested for antibiotic resistance against 10 drugs. Seventeen different pathotypes, accounting for 40.0% of the isolates, were recovered from diarrheic piglets. The main pathotypes included EAST1 (13.5%), F4/LT/STb/EAST1 (6.5%), AIDA-I/STb/EAST1 (4.1%), F5/STa (2.9%), EAE/EAST1 (2.9%), and AIDA-I/F18 (2.3%). Only 3 pathotypes, EAE (11.7%), EAST1 (10.8%), and EAE/EAST1 (3.3%), were recovered from nondiarrheic piglets. Paa factor was detected in 8.8% and 7.5% of isolates from diarrheic and nondiarrheic piglets, respectively, and always was associated with other virulence determinants. Overall, 22.9% of isolates from diarrheic piglets appeared to be enteropathogens: enterotoxigenic E. coli (11.7%), enteropathogenic E. coli (3.5%), and E. coli isolates (3.0%) for which none of the above adherence factors was detected. Pathotypes AIDA-I/STb/EAST1 and AIDA-I/STb were isolated only from diarrheic piglets and accounted for 4.7% of isolates. Strains of these pathotypes induced diarrhea when inoculated into newborn colostrum-deprived pigs, in contrast to an isolate positive only for EAST1, which did not induce diarrhea. Antibiotic sensitivity test showed that isolates of the AIDA-I/STb/EAST1 and AIDA-I/STb pathotypes were the only strains sensitive to enrofloxacin, gentamicin, neomycin, and trimethoprim-sulfamethoxazole. This study showed that at least 20.5% of isolates from diarrheic piglets appeared to be associated with AIDA-I/STb pathotype and that EAST1 pathotype is probably not an important marker for diarrhea in piglets.

Presence and enterotoxigenicity of F5 and F41 Escherichia coli strains isolated from diarrhoeic small ruminants in Spain

Five hundred and eighty four strains of Escherichia coli isolated from 146 diarrhoeic lambs and goat kids in Spain were analysed for the presence of F5 and F41 fimbriae by slide agglutination. Only two strains positive for both F5 and F41 fimbriae from one lamb were isolated. These two strains F5 and F41 positive for slide agglutination were also heat-stable enterotoxin a, F5 and F41 positive by PCR. Thus, our results show that F5 and/or F41 enterotoxigenic E. coli are a very infrequent cause of small ruminant diarrhoea in Spain.

Prevalence ofeaeand Shiga Toxin Genes amongEscherichia coliStrains Isolated from Healthy Calves

Strains ofEscherichia coli(n = 390) isolated from 132 healthy, 4–8‐week‐old calves, were tested by polymerase chain reaction (PCR) for theeae(intimin) gene and shiga toxin genes (stx1andstx2). All strains were also analysed for F5, F17 and F41 fimbriae and for the heat‐labile (LT) and heat‐stable (STI and STII) genetic markers. Overall, theeaegene was detected in 84 (21.5 %) of the strains tested. Only 21 (5.4 %) isolates were positive forstx1(18 strains) orstx2(three strains); nine of thestx1‐positive isolates also possessed theeaegene. A high percentage (29.2 %) of the isolates tested expressed F17 but no enterotoxin genes were detected. None of theeae‐ orstx‐positive strains belonged to the O157 serogroup.

Prevalence of eae and shiga toxin genes among Escherichia coli strains isolated from healthy calves.

Strains of Escherichia coli (n = 390) isolated from 132 healthy, 4-8-week old calves, were tested by polymerase chain reaction (PCR) for the eae (intimin) gene and shiga toxin genes (stx1 and stx2). All strains were also analysed for F5, F17 and F41 fimbriae and for the heat-labile (LT) and heat-stable (STI and STII) genetic markers. Overall, the eae gene was detected in 84 (21.5%) of the strains tested. Only 21 (5.4%) isolates were positive for stx1 (18 strains) or stx2 (three strains); nine of the stx1-positive isolates also possessed the eae gene. A high percentage (29.2%) of the isolates tested expressed F17 but no enterotoxin genes were detected. None of the eae- or stx-positive strains belonged to the O157 serogroup.

Prevalence of virulence factors of Escherichia coli strains isolated from diarrheic and healthy piglets after weaning

This study determined the prevalence of F4, F5, F6, F17 and F41 fimbriae and the genes for FedA (F18 fimbriae), LT and ST enterotoxins, and Shiga toxins Stx1, Stx2 and Stx2e among E. coli isolated from 372 weaned pigs with diarrhea and 46 healthy pigs of the same age. Agglutination tests showed that most isolates were negative for all five fimbrial antigens. The F4 antigen was found in 71 (19.1%) and the F5, F6, or F41 antigen was detected in 6.4% of isolates from diseased pigs. Genes for the F18 fimbriae were detected in 10 (2.7%) strains from diarrheic pigs and in 1 of 46 isolates from healthy pigs. Most isolates (213, 57.3%) from pigs with diarrhea were positive for LTI only or for LTI and STI or Stx2e toxin genes. Fifteen strains (13.7%) possessed only the STI or STII toxin genes. All F4-positive bacteria had genes for LTI or LTI and STI, whereas F18-positive isolates had genes for LTI, LTI/STI, or LTI/Stx2e. Of the strains isolated from diseased pigs, 264 (71.0%) were negative for the fimbrial antigens (genes) examined in this study. The fimbria-negative isolates frequently possessed genetic determinants for LTI (118, 31.7%) or for STII (16, 4.3%) enterotoxins.

The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland

E. coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively. The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR. Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea. PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61,9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease. The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea. Shiga toxin 2 variant ( stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea. The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E. coli strains.

Multiplex PCR for enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains from calves.

A multiplex PCR was developed to identify enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains by amplifying genes encoding K99 and F41 fimbriae, heat-stable enterotoxin a, intimin, and Shiga toxins 1 and 2. This multiplex PCR was specific and sensitive. It will be useful for identification of E. coli strains which cause diarrhea in calves.

豚の大腸菌症における2134P線毛保有大腸菌の分布

A total of 263 Escherichia coli strains without K88, K99, 987P and F41 fimbriae, which were isolated from pigs with neonatal diarrhea, postweaning diarrhea or edema disease, were examined for a novel adhesin factory of enterotoxigenic E. coli, 2134P, using dot-blot immunoassay. The 2134P fimbriae were found in 7 (8.2%) of 85 isolates from postweaning diarrhea cases and 3 strains (O141: H4) produced both STa and VT, while 1 (O8: H-) both STa and LT. The remaining 2 (OUT: H-) and 1 (O141: H4) produced STa alone and VT alone, respectively.

Structural studies on the Escherichia coli O101 lipopolysaccharide found in association with F41 and K99 fimbriae

In this study it was shown that the O101 lipopolysaccharide isolated from Escherichia coli B41 did not contain an O-specific polysaccharide and that its sugar moiety is probably restricted to the core oligosaccharide. It is characterized by the presence of galactose, glucose, N-acetylglucosamine, heptose and 3-deoxy- d- manno-2-octulosonic acid and the fatty acid composition is typical of an Enterobacteriaceae lipopolysaccharide. Methylation analysis indicated terminal non-reducing galactose and glucose and also 1,2-linked glucose which is a substitution pattern typical of an E. coli lipopolysaccharide core oligosaccharide. The obtained structural information is sufficient to explain the previously observed interactions between the O101 lipopolysaccharide and the K99 lectin.

Fimbriae extracts from enterotoxigenic Escherichia coli strains of bovine and porcine origin with K99 and/or F41 antigens

Fimbriae extracts obtained using the thermal shock method, from bovine and porcine enterotoxigenic Escherichia coli strains with K99 and/or F41 antigens, were analyzed by SDS-PAGE, immunoblotting and haemagglutinating activity. Three major protein bands with molecular weights 17 kDa, 29.3 kDa and 30.9 kDa were detected depending on the strain assayed. A 17 kDa band was identified as the fimbrial subunit for K99 fimbriae and was detected in strains of bovine and porcine origin. The 30.9 kDa band was identified as the fimbriae subunit for F41 fimbriae and was detected in all porcine strains with F41 antigen and only in the bovine strains of serogroups O9 and O101 that proved positive for F41 antigen. The 29.3 kDa band was shown to be antigenically related to F41 and K88, and was only detected in bovine strains of serogroups O8 (5 strains) and O20 (a single strain). We speculate that the 29.3 kDa band may be related to the CS31A antigen.

Clonal analysis of Escherichia coli of serogroups O9, O20, and O101 isolated from Australian pigs with neonatal diarrhea

The genetic diversity of 87 isolates of Escherichia coli recovered from Australian pigs with neonatal diarrhea was examined by multilocus enzyme electrophoresis. The isolates were of serogroups O9, O20, and O101, and although most isolates lacked K88(F4), K99(F5), 987P(F6), and F41 fimbriae, they were considered to be involved in the etiology of the diarrhea. The isolates were extremely diverse, considering their origin from a single pathological condition in one country. There were estimated to be 18, 16, and 12 clones of the three respective serogroups in the collection, with serogroup diversities of 0.387, 0.448, and 0.275, respectively. Comparison with the results previously obtained for isolates from piglets with postweaning diarrhea suggested that bacteria from piglets with these two conditions did not come from any particular common genetic background. The overall genetic diversity for the combined collection was the same as that reported by others for representative isolates selected from throughout the species (0.47). The current results indicate that if isolates of these O groups are involved in porcine diarrhea, their pathogenicity is directly linked to their O somatic antigen type and is not simply due to the wide distribution of a small number of virulent clones.