Isolation of citrate-positive variants of Escherichia coli from domestic pigeons, pigs, cattle, and horses.

Twenty-seven isolates of citrate-positive variants of Escherichia coli were obtained from domestic pigeons, pigs, cattle, and horses. With the exception of citrate utilization, all isolates closely resembled typical E. coli in their biochemical reactions. These isolates were multiply resistant to antibiotics in in vitro susceptibility tests. Transfer experiments of multiple-drug resistance to the E. coli K-12 strain showed that all citrate-positive isolates from domestic pigeons, pigs, and cattle, resistant to three or more drugs, carried R plasmids showing temperature-sensitive transfer.     

Isolation and characteristics of sorbitol-fermenting Escherichia coli O157 strains from cattle

Cattle can be a reservoir of sorbitol-fermenting Escherichia coli O157 (SF E. coli O157) and a source of human diseases. In this study, six strains of SF E. coli O157 were isolated and characterized from cattle using an immunomagnetic separation procedure. PCR analysis of the SF E. coli O157 virulence markers showed that all six isolates tested positive for sfpA, rfbE and eaeA, and negative for terA, ureA, katP and espP. Two of the isolates contained the stx genes. Four isolates tested positive for enterohemorrhagic E. coli hlyA (EhlyA) by PCR but were nonhemolytic on the blood agar. Five isolates tested positive for the cdtA gene. The possession of these virulence factors was an indication of their pathogenic potential. The random amplified polymorphic DNA patterns, which were generated by the arbitrarily primed PCR of the SF E. coli O157 isolates from the cattle, were significantly different from those of the non-sorbitol-fermenting E. coli O157 (NSF E. coli O157) strains originating from cattle or humans. GelCompar analysis showed that the SF E. coli O157 isolates had only a 57% genetic similarity with the NSF E. coli strains. The minimal inhibitory concentration assay showed that imipenem inhibited the growth of the six isolates at a concentration of <4 microg/ml.     

Trimethoprim resistance plasmids in Escherichia coli isolated from cases of diarrhoea in cattle, pigs and sheep

A total of 1572 isolates of Escherichia coli obtained from the faeces of young farm animals with diarrhoea over the period 1980–1983 were screened for resistance to trimethoprim (Tp). Resistance to Tp was detected in263/954 (28%) of bovine isolates,59/441 (13%) of porcine isolates and15/177 (9%) of ovine isolates. Seventy-five resistant isolates from separate outbreaks of infection on farms within a 25 mile radius of Nottingham were examined in detail. Sixty-eight (91%) of the 75 isolates were resistant to > 1024 mg Tp/1 and 34 (50%) of these 'highly resistant' isolates (45% of total resistant isolates) transferred their Tp resistance to E. coli K12. A further 13 (17%) isolates were demonstrated to carry non-self-transferable plasmids which were capable of being mobilized to E. coli K12 by the broad host range plasmid RP4. Thirty-one self-transferable Tp R plasmids were divided between the following incompatibility groups: IncB (14 plasmids), IncF_***H (4 plasmids), IncH₂ (1 plasmid), IncIaP (10 plasmids), IncIdT (1 plasmid) and IncP (1 plasmid). In terms of antibiotic resistance patterns and incompatibility properties, many of these plasmids closely resembled those isolated from human patients in the same area, suggesting that there may be a common pool of Tp R plasmids.     

Isolation of plasmid-protein complexes from Escherichia coli.

A procedure is described for the isolation of complexes between pMB9 plasmids and protein from Escherichia coli which are stable during centrifugation on sucrose gradients and are not destroyed in the presence of competitor DNA. The proteins in these complexes have been analysed by dodecyl sulphate/polyacrylamide gel electrophoresis. Only 10 polypeptide species are found in significant quantities, many of which are bound to both the plasmid and host DNA. We have also detected the presence of one protein which binds to a specific DNA sequence inserted in the plasmid.     

Sperm morphology of cattle and domestic pigs

Sperm morphology was evaluated (using Blom classification) in 44 ejaculates of 11 bulls and 44 ejaculates of 11 boars. Significant differences in sperm morphology were found between bulls and boars. In addition, the correlations between frequency of morphological changes and morphometrical traits of boar spermatozoa were demonstrated. More morphological anomalies of spermatozoa were recorded in ejaculates containing longer spermatozoa.     

Survival of Escherichia coli O157 in faeces of experimentally infected rats and domestic pigeons

In order to evaluate the role of some synanthropic animals in the spreading of Escherichia coli O157, laboratory rats and domestic pigeons were experimentally infected per os with E. coli O157. Rats infected with 10(5) colony forming units (cfu) (n = 5) and 10(9) cfu (n = 5) shed E. coli O157 for 2 +/- 1.7 d and 9.8 +/- 1.3 d, respectively. In the faeces of infected rats stored at 4 degrees C in a moist environment, at 4 degrees C in a dry environment or at 20 degrees C in a moist environment, E. coli O157 survived for 34 weeks. When stored at 20 degrees C or - 20 degrees C in a dry environment, E. coli O157 survived for greater than or = 36 weeks. Pigeons infected with 10(5) cfu (n = 5) and 10(9) cfu (n = 5) shed the pathogen for 14.8 +/- 3.4 d and 20.2 +/- 5.2 d, respectively. Both species, rats and pigeons, can be important in spreading of the E. coli O157 infection in cattle.     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle.

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

The enzyme histochemistry of developing odontoblasts in cattle, pigs and horses

Synopsis The histochemical distribution of some hydrolytic and oxidative enzymes in developing odontoblasts and subodontoblasts in cattle, pigs and horses has been observed in cryostat sections of teeth that have been decalcified with neutral EDTA.Undifferentiated dental epithelium and immature odontoblasts of the bell stage tooth germ showed lower levels of enzymatic activity as compared with the well-developed tooth germ.When the dentine matrix began to form, the young odontoblasts appeared to have a significantly positive reaction for acid phosphatase, and gradually other enzymes developed an activity at the top of the cusp.Odontoblasts as well as subodontoblastic-rich cells showed strong enzymatic activities for hydrolytic and oxidative enzymes, that is, they were strongly reactive for alkaline and acid phosphatase and lactate and malate dehydrogenases, and moderately reactive for other oxidative enzyme systems.It is suggested that the subodontoblastic layer is concerned with the biosynthesis of dentinal matrix as well as with the odontoblasts themselves.     

Spontaneous emergence of an Hfr strain with a cit plasmid from natural isolates of citrate-positive Escherichia coli bovine origin.

From citrate-utilizing (Cit+) Escherichia coli strain C53 of bovine origin, strains C53A and C53B were obtained. Upon mating with recA+ but not with recA mutant recipients of K-12, C53A produced chromosomal recombinants at quite high frequencies, leading to the following conclusions: (i) C53A is an Hfr strain; (ii) the site of integration of the Cit plasmid (IncH1) is between metA (89 min) and ara (1 min); (iii) the direction of chromosome transfer is clockwise; and (iv) the plasmid-associated determinants are transferred as the terminal markers. A transductant of a dnaA(Ts) strain, CRT46, which acquired Cit determinants from a recombinant, SG13, was also an Hfr strain similar to SG13, and thermoresistant due to suppressive integration. On the other hand, unstable C53B did not produce recombinants, but the frequency of RecA-independent transfer of the Cit plasmid was high, indicating that the Cit plasmid (IncH1) exists autonomously in C53B. Attempts to isolate an Hfr strain from C53B failed.     

Isolation of shiga toxin-producing Escherichia coli strains from healthy cattle of the region of Lower Silesia

The aim of the study was to determine if cattle from the region of Lower Silesia is the reservoir of shiga toxin-producing E. coli strains (STEC) and the analysis of virulence factors of isolated STEC strains. The ability of tested animal strains to shiga toxin synthesis was analysed in cytotoxicity assay in vitro on Vero cell line and then confirmed by detection of shiga toxin-encoding genes by PCR. STEC strains were isolated from 12 (15,2%) of animals examined, 21,4% of these strains were obtained from 9 of 42 calves, and 8,1% from 3 of 37 cows. Most of STEC isolated (75%) was enterohemolysin-producing. The cattle from the region of Lower Silesia is the reservoir of pathogenic for humans sorbitol-fermenting non-O157 STEC strains.     

Proximity-dependent inhibition in Escherichia coli isolates from cattle

We describe a novel proximity-dependent inhibition phenotype of Escherichia coli that is expressed when strains are cocultured in defined minimal media. When cocultures of "inhibitor" and "target" strains approached a transition between logarithmic and stationary growth, target strain populations rapidly declined >4 log CFU per ml over a 2-h period. Inhibited strains were not affected by exposure to conditioned media from inhibitor and target strain cocultures or when the inhibitor and target strains were incubated in shared media but physically separated by a 0.4-μm-pore-size membrane. There was no evidence of lytic phage or extracellular bacteriocin involvement, unless the latter was only present at effective concentrations within immediate proximity of the inhibited cells. The inhibitory activity observed in this study was effective against a diversity of E. coli strains, including enterohemorrhagic E. coli serotype O157:H7, enterotoxigenic E. coli expressing F5 (K99) and F4 (K88) fimbriae, multidrug-resistant E. coli, and commensal E. coli. The decline in counts of target strains in coculture averaged 4.8 log CFU/ml (95% confidence interval, 4.0 to 5.5) compared to their monoculture counts. Coculture of two inhibitor strains showed mutual immunity to inhibition. These results suggest that proximity-dependent inhibition can be used by bacteria to gain a numerical advantage when populations are entering stationary phase, thus setting the stage for a competitive advantage when growth conditions improve.     

Isolation and characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production.

Type 1 pili of Escherichia coli are the prototype of the somatic class of pili found on many strains of bacteria. As a first step in the genetic analysis of type 1 piliation, an extensive series of nonpiliated derivatives of E. coli K-12 strain AW405, was characterized to produce attached or free pili when examined in the antiserum or appeared to produce attached or free pili when examined in the electron microscope. The derivatives fell into two classes; phase variants and mutants. Phase variants that formed colonies of two distinctive types, one associated with a predominantly piliated (P+), and the other associated with a nonpiliated (P-) phase, were obtained. Each phase could give rise to the other at a relatively high rate, which was greater in the P- to P+ direction during culture in unshaken liquid medium. In addition, 77 Pil- mutants were selected on the basis of a subtle difference in colonial morphology. The mutants reverted, if at all, at a much lower rate than that of the P- to P+ change. The stability of Pil- derivatives grown in unshaken liquid medium was used as a criterion for distinguishing between phase variants and mutants, Phase variation also effected colonial morphology and chemotactic swarming. These properties did not directly depend upon piliation since Pil- mutants were only slightly altered in colonial form and unaltered in chemotactic swarming. Piliation of Pil+ bacteria was quantitatively affected by growth conditions.     

Effective population sizes in cattle,sheep, horses,pigs and goats estimated from census and herdbook data

Accurate measures of effective population sizes (N_e) in livestock require good quality data and specialized skills for their computation and analysis. N_e can be estimated by Wright’s equation N_e=4MF/(M+ F) (M, F being sires and dams, respectively), but this requires assumptions which are often not met. Total census sizes N_c of livestock breeds are collated globally. This paper investigates whether estimates of N_e can be made from N_c; this would facilitate conservation monitoring. Some N_e methodologies avoid the assumptions of Wright’s equation and permit measurement, rather than estimation, of N_e. Those considered here employ, respectively, linkage disequilibrium (LD) of single-nucleotide polymorphisms (yielding N_e(LD)), and genealogical analysis (rate of increase of inbreeding, DF), yielding N_e(DF). Considering breeds of cattle, sheep, horses, pigs and goats for which N_c and either N_e(LD) or N_e(DF) are known (totals of 203 breeds and 321 breeds, respectively), proportionality has been investigated between N_c and these measures of N_e. N_e(LD) was found to increase with N_c, significantly in sheep and horses, less so in cattle, but not at all in pigs. N_e(DF) was correlated with log₁₀(N_c) in cattle, sheep and horses (53, 56, 43 breeds, respectively). N_e(LD) was correlated in cattle (73 breeds) and pigs (31 breeds) with the log₁₀ transformation of N_e as calculated by Wright’s equation. Further verification and refinement are needed, particularly of census data, but credible predictions of N_e are obtainable by applying the following multipliers to log₁₀(N_c): cattle 17.61, sheep 97.72, horse 70.78. For cattle and pigs, multiplying log₁₀(N_e(Wright)) by, respectively, 40.69 and 60.09, also gives credible predictions. Such census-based estimates of N_e could in principle be generated by non-specialists and are likely to be suited to audits of conservation activity when financial resources or availability of data are limiting. The ratio N_e/N_c varied among species with an overall median value of 0.03, less than a tenth of that typically observed in wild mammals. Characteristics were also investigated of a distinct herdbook-based methodology, namely the development of Wright’s equation to take into account variances of progeny numbers to yield what has been termed here N_e (Hill). Comparison of these values with N_e (Wright) could help to identify breeds with breeding structures conducive or inimical to genetic conservation. However, N_e(Hill) requires breed-specific values for these variances, and this restricts its applicability.     

Isolation and immunochemical characteristics of the immunodepressive substance from Escherichia coli.

A substance, inhibiting the production of haemolysins against sheep erythrocytes in mice was isolated from two non-pathogenic strains of E. coli, 020:K4 and M-17, by the methods of differential centrifugation and gel filtration. Spectrophotometric studies and chemical analysis have shown the isolated substance to be glycolipid. The immunodepressive substance is localized in the cytoplasm of the microbial cell. The isolated and partly purified immunodepressive substance did not contain any admixture of O-antigen (endotoxin) of the cell wall or of antigens giving cross reactions with sheep erythrocytes. The isolated substance exhibited weak antigenic properties and was not toxic for mice when administered in a dose of 2 mg (dry weight).