The diversity of Escherichia coli serotypes and biotypes in cattle faeces

AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.     

Prevalence and diversity of class 1 integrons and resistance genes in antimicrobial-resistant Escherichia coli originating from beef cattle administered subtherapeutic antimicrobials

Aims: To characterize class 1 integrons and resistance genes in tetracycline‐resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control). Methods and Results: Tetracycline‐resistant E. coli (control, n = 111; AS700, n = 53; A44, n = 40) were studied. Class 1 integrons, inserted gene cassettes and the presence of other antimicrobial resistance genes, as well as phylogenetic analysis, were performed by PCR, restriction enzyme analysis and sequencing. Susceptibilities to 11 antimicrobials were conducted on all isolates. Prevalence of class 1 integrase was higher (P < 0·001) in isolates from AS700 (33%) and A44 (28%) steers as compared to control (7%). Most integron gene cassettes belonged to the aad or dfr families. Correlations were found between the tet(A) gene and the genetic elements sul1 (r = 0·44), aadA1 (r = 0·61), cat (r = 0·58) and intI1(r = 0·37). Both closely and distantly related isolates harboured integrons with identical gene cassette arrays. Conclusions: Subtherapeutic administration of chlorotetracycline alone or in combination with sulfamethazine may select for class 1 integrons in bovine tetracycline‐resistant E. coli isolates. Vertical spread and horizontal transfer are responsible for the dissemination of a particular type of class 1 integron, but this study could not differentiate if this phenomenon occurred within or outside of the feedlot. Tetracycline‐resistant E. coli strains with sul1 and tet(A) genes were more likely to harbour class 1 integrons. Significance and Impact of the Study: Subtherapeutic use of chlortetracycline and sulfamethazine may promote the presence of class 1 integrons in tetracycline‐resistant E. coli isolated from feedlot cattle.     

Genetic diversity measures of local European beef cattle breeds for conservation purposes

This study was undertaken to determine the genetic structure, evolutionary relationships, and the genetic diversity among 18 local cattle breeds from Spain, Portugal, and France using 16 microsatellites. Heterozygosities, estimates of Fst, genetic distances, multivariate and diversity analyses, and assignment tests were performed. Heterozygosities ranged from 0.54 in the Pirenaica breed to 0.72 in the Barrosã breed. Seven percent of the total genetic variability can be attributed to differences among breeds (mean F_st = 0.07; P < 0.01). Five different genetic distances were computed and compared with no correlation found to be significantly different from 0 between distances based on the effective size of the population and those which use the size of the alleles. The Weitzman recursive approach and a multivariate analysis were used to measure the contribution of the breeds diversity. The Weitzman approach suggests that the most important breeds to be preserved are those grouped into two clusters: the cluster formed by the Mirandesa and Alistana breeds and that of the Sayaguesa and Tudanca breeds. The hypothetical extinction of one of those clusters represents a 17% loss of diversity. A correspondence analysis not only distinguished four breed groups but also confirmed results of previous studies classifying the important breeds contributing to diversity. In addition, the variation between breeds was sufficiently high so as to allow individuals to be assigned to their breed of origin with a probability of 99% for simulated samples.     

Effect of finishing diets on Escherichia coli populations and prevalence of enterohaemorrhagic E. coli virulence genes in cattle faeces

AIM: To determine the effect of different carbohydrate-based finishing diets on fermentation characteristics and the shedding of Escherichia coli and enterohaemorrhagic E. coli (EHEC) virulence genes in cattle faeces. METHODS AND RESULTS: The size of faecal E. coli populations and fermentation characteristics were ascertained in three experiments where cattle were maintained on a range of finishing diets including high grain, roughage, and roughage + molasses (50%) diets. Increased E. coli numbers, decreased pH and enhanced butyrate and lactate fermentation pathways were associated with grain diets, whereas roughage and roughage + molasses diets resulted in decreased concentrations of ehxA, eaeA and stx(1) genes, this trend remaining at lairage. In one experiment, faecal E. coli numbers were significantly lower in animals fed roughage and roughage + molasses, than animals fed grain (4.5, 5.2 and 6.3 mean log10 g(-1) digesta respectively). In a second experiment, faecal E. coli numbers were 2 log lower in the roughage and roughage + molasses diets compared with grain-fed animals prior to lairage (5.6, 5.5 and 7.9 mean log10 g(-1) digesta respectively) this difference increasing to 2.5 log at lairage. CONCLUSIONS: The type of dietary carbohydrate has a significant effect on E. coli numbers and concentration of EHEC virulence genes in faeces of cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a better understanding of the impact finishing diet and commercial lairage management practices may have on the shedding of E. coli and EHEC virulence factors, thus reducing the risk of carcass contamination by EHEC.     

Genetic diversity and differentiation in Portuguese cattle breeds using microsatellites

Genotype data from 30 microsatellites were used to assess genetic diversity and relationships among 10 native Portuguese cattle breeds, American Charolais and the Brazilian Caracú. Hardy–Weinberg equilibrium was observed for all loci/population combinations except for five loci in Brava de Lide and one locus in Alentejana that exhibited heterozygote deficiency. Estimates of average observed and expected heterozygosities, total number of alleles (TNA) per breed and mean number of alleles (MNA) per locus/population were obtained. A total of 390 alleles were detected. TNA among Iberian cattle ranged from 170 to 237 and MNA ranged from 5.67 to 8.07. The highest observed heterozygosities were found in the Caracú, Maronesa, Garvonesa and Arouquesa and the lowest in Brava de Lide and Mirandesa. Estimation of population subdivision using Wright's F_ST index showed that the average proportion of genetic variation explained by breed differences was 9%. Neighbour‐joining phylogenetic trees based on D_A distances showed that the genetic relationships of present‐day Portuguese native breeds are consistent with historical origins in the Brown Concave (Arouquesa, Mirandesa, Marinhoa) and Red Convex (Mertolenga, Alentejana, Garvonesa, Minhota) evolutionary groups. The Iberian Black Orthoide group, represented by Brava de Lide and Maronesa, and the Barrosã breed appeared to be more closely related to the Brown Concave group but may represent a separate lineage. The Caracú breed was not found to be closely associated with any of the native Portuguese breeds.     

基因工程半成品中大肠杆菌蛋白残余量测定方法研究—双抗体夹心ELISA法

用大肠杆菌ＤＨ５α菌体蛋白免疫家兔制备抗血清,建立了双抗体夹心ＥＬＩＳＡ方法。经初步测定,该方法的灵敏度为１．５ｎｇ／ｍｌ,与间接ＥＬＩＳＡ法的灵敏度相似,比聚丙烯酰胺凝胶电泳的考马斯亮兰染色方法的灵敏度高将近７００倍,比银染色方法的灵敏度高将近３０倍。在２０～１００００ｎｇ／ｍｌ范围内呈直线关系,直线相关系数为０．９９６。经八次重复测定,显示很好的重复性。该方法可用于测定基因工程产品中ＤＨ５α菌体蛋白的残余量。     

The incidence of Shiga toxin-producing Escherichia coli in cattle with mastitis in Brazil

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Brazil. METHODS AND RESULTS: A total of 2144 milk samples from dairy cattle showing mastitis were screened for the presence of E. coli. A total of 182 E. coli isolates were selected and examined. All were subjected to dot blot analysis using the CVD419 probe for the detection of the enterohaemolysin (hly) gene, and to a multiplex PCR for the detection of stx1, stx2 and eaeA genes. STEC were isolated from 22 (12.08%) milk samples. All the STEC isolates were tested for sensibility to 10 antimicrobials; the resistances most commonly observed were to cephalothin (86.3%), tetracycline (63.6%) and doxycycline (63.6%). CONCLUSION: STEC isolates were found in bovine mastitic milk in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: STEC isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhoea and haemolytic-uraemic syndrome, some of them were stx2, eaeA and hly positive.     

Genetic diversity of north-east Asian cattle based on microsatellite data

In order to assess the genetic variability and population structure of north-east Asian cattle, 13 microsatellite loci were analysed for a total of 200 individuals including Korean, Chinese, Japanese Black and European Holstein cattle. Observed and expected heterozygosity, two estimators (F(ST) and G(ST)) of gene differentiation, and Nei's DA distance were evaluated. Based on expected mean heterozygosity, the lowest genetic diversity was exhibited in Japanese Black cattle (H(E)=0.471), and the highest in Chinese cattle (H(E)=0.744). Korean cattle revealed a relatively high degree of genetic diversity (H(E)=0.728). Average proportion of genetic variation because of interpopulation subdivision among north-east Asian cattle varied between 10.9 and 9.9%, depending on the estimator used. N-J tree based on Nei's DA genetic distance showed that Korean and Chinese cattle are closely related, whereas Japanese Black cattle are clearly distinct from the other two populations, forming a north-east Asian outgroup.     

Transfer of a rifampicin-resistant Escherichia coli strain among feedlot cattle

AIMS: To determine the incidence of transfer of a naturally occurring rifampicin-resistant strain of Escherichia coli (RREC) among cattle in a research feedlot. METHODS AND RESULTS: During three separate experiments, steers in three different pens were orally inoculated with RREC originally isolated from bovine faeces. Faecal swabs were performed on all steers in the feedlot at approximately 5 week intervals thereafter. Faecal grab samples were collected from steers in the inoculated and the immediately adjacent pens for up to 4 months. In all three experiments, the inoculated steers and penmates shed RREC within 48 h, and then shed intermittently throughout the sampling periods. Transfer of RREC to steers in an adjacent pen was confirmed only during the first experiment, but never to those in non-adjacent pens. All recovered RREC isolates were compared with the inoculated strain using multiple methods indicating that all RREC isolates were descendants of the original inoculated strain. CONCLUSIONS: Detection of the RREC strain on the pen floor and within the animal handling system, but not in the feed troughs or water bowls, suggests faecal-oral to be the primary mode of transmission among animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that in the absence of selective pressure, antibiotic-resistant bacteria may persist in cattle for a short duration but widespread transfer among cattle in a feedlot environment may be an exception rather than the norm. Modifications to feedlot management are discussed.     

Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil

AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.     

Genetic diversity,introgression and relationships among West/Central African cattle breeds

Genetic diversity, introgression and relationships were studied in 521 individuals from 9 African Bos indicus and 3 Bos taurus cattle breeds in Cameroon and Nigeria using genotype information on 28 markers (16 microsatellite, 7 milk protein and 5 blood protein markers). The genotypes of 13 of the 16 microsatellite markers studied on three European (German Angus, German Simmental and German Yellow) and two Indian (Nelore and Ongole) breeds were used to assess the relationships between them and the African breeds. Diversity levels at microsatellite loci were higher in the zebu than in the taurine breeds and were generally similar for protein loci in the breeds in each group. Microsatellite allelic distribution displayed groups of alleles specific to the Indian zebu, African taurine and European taurine. The level of the Indian zebu genetic admixture proportions in the African zebus was higher than the African taurine and European taurine admixture proportions, and ranged from 58.1% to 74.0%. The African taurine breed, Muturu was free of Indian zebu genes while its counter Namchi was highly introgressed (30.2%). Phylogenic reconstruction and principal component analysis indicate close relationships among the zebu breeds in Cameroon and Nigeria and a large genetic divergence between the main cattle groups – African taurine, European taurine and Indian zebu, and a central position for the African zebus. The study presents the first comprehensive information on the hybrid composition of the individual cattle breeds of Cameroon and Nigeria and the genetic relationships existing among them and other breeds outside of Africa. Strong evidence supporting separate domestication events for the Bos species is also provided.     

Serogroup distribution and virulence characteristics of sorbitol‐negative Escherichia coli from food and cattle stool

Aims: To (i) study the serogroup distribution and virulence characteristics of non‐sorbitol‐fermenting Escherichia coli isolates from foods of animal origin and cattle faeces and (ii) re‐examine the true sorbitol and β‐d ‐glucuronidase (GUD) reactions of sorbitol‐negative (Sor^?) strains from MacConkey sorbitol agar (SMAC) to assess their phenotypic similarity with E. coli O157. Methods and Results: One hundred and thirty Sor^?E. coli were isolated from 556 food samples and 177 cattle stool samples using cefixime tellurite–supplemented SMAC (CT‐SMAC) and chromogenic HiCrome MS.O157 agar respectively. Based on typing of somatic antigen, the isolates were classified into 38 serogroups. PCR results identified about 40% strains, belonging to O5, O8, O20, O28, O48, O60, O78, O82, O84, O101, O110, O123, O132, O156, O157, O‐rough and OUT as Shiga toxigenic. Majority of O5, O84, O101, O105, O123, O157, O‐rough and OUT strains were enterohaemolytic. Further, 39·2% and 63·1% of Sor^? isolates from CT‐SMAC fermented sorbitol in phenol red broth and hydrolysed 4‐methylumbelliferyl‐β‐d ‐glucuronide (MUG) respectively. Members of serogroups O5, O28, O32, O81, O82, O84, O101, O‐rough lacked both the sorbitol fermentation (broth test) and GUD activity and might create confusion in phenotypic identification of E. coli O157. Conclusions: Sor^?E. coli isolates from raw meat, milk, shrimp and cattle stool belonged to 38 serogroups, with E. coli O157 constituting only 14·6% of the isolates. Many of these nonclinical Sor^? strains were potentially pathogenic. Nearly 39% of these Sor^?E. coli from CT‐SMAC fermented sorbitol in broth, indicating the need for confirmation of sorbitol reaction in broth. Significance and Impacts of the Study: Classical sorbitol utilization and GUD tests are not likely definitive tests for E. coli O157. Further improvement of differential media based on these phenotypic properties is necessary for detection of pathogenic serotypes from foods and environmental samples.     

雷琼牛GH基因第五外显子遗传多样性及其分化

对16头雷琼牛GH基因第5外显子序列进行分析,发现了1个变异位点,定义了2种单倍型。引用巴州牦牛2个个体GH基因同源区序列并结合GenBank中牛属普通牛、其它瘤牛和牦牛3个种群与水牛1个远缘种GH基因同源区序列,分别采用邻接（NJ）法和最大简约（MP）法构建分子系统发育树,得到基本一致的拓扑结构,结果显示GH基因的分化早于雷琼牛（瘤牛）、其它瘤牛、普通牛、牦牛和水牛的分化,瘤牛物种内存在多型,同时证实了GH基因第5外显子区有着较高的突变率。