Ultrasound-enhanced latex agglutination for the detection of bacterial antigens in urine.

An ultrasound-enhanced latex agglutination technique has been applied to the detection of bacteria in urine. The approach combines the use of ultrasound, the dilution of latex to allow agglutination with low levels of antigen, and microscopy. Using commercially available latex coated with antibody to Esch. coli O157 or K1, ultrasound enhanced the detection of Esch. coli strains carrying these antigens by x512 and x2048 respectively, compared with the standard test card procedure. The latex particles in the commercial kits were 0.4-1.0 micron in diameter. As larger particles are more effectively manipulated in a sound field, particles of 2.8 microns diameter were coated with antiserum against a urinary tract isolate of Esch. coli (SP3112). The application of ultrasound with these particles facilitated the detection of 6 x 10(3) cells/mL of Esch. coli SP3112 within 2 min, a > 10,000-fold increase in sensitivity compared with the normal agglutination procedure. The possible exploitation of this technique in the clinical laboratory for the rapid, sensitive detection of bacterial antigens in urine is discussed.

Incidence of the aerobactin iron uptake system among Escherichia coli isolates from infections of farm animals.

To assess the importance of aerobactin-mediated iron uptake as a bacterial virulence determinant in animal infections, a total of 576 strains of Escherichia coli isolated from cattle, chickens, sheep and pigs were screened by colony hybridization to determine the presence of the aerobactin genetic determinants, and by a bioassay to detect aerobactin secretion in iron-limited conditions. Results obtained by the two complementary methods correlated well. The incidence of the aerobactin system was very high among septicaemia isolates, particularly those from cattle and chickens, an observation that strongly suggests an important role for this mechanism of iron assimilation in pathogenesis. On the other hand, the incidence of the aerobactin system among mastitis strains was not significantly higher than among faecal isolates from healthy animals. No classical enterotoxigenic E. coli strains tested carried the aerobactin genetic determinants. Although most strains that produced aerobactin were also able to make colicin V, the fact that the two characteristics existed separately in a significant minority of isolates suggested that colicin testing alone could not be reliably used to determine the presence of the aerobactin system.

Aerobactin-mediated iron uptake by Escherichia coli isolates from human extraintestinal infections.

A total of 516 strains of Escherichia coli were screened for the presence and expression of the aerobactin iron uptake system. The incidence was markedly higher among clinical isolates from patients with septicemia (68.8%), pyelonephritis (74.6%), and symptomatic (59.8%) and asymptomatic (63.2%) lower urinary tract infections than among normal human fecal isolates (34.3%).

A comparative study of the mannose-resistant and mannose-sensitive haemagglutinins of Escherichia coli isolated from urinary tract infections.

The distribution of mannose-resistant (MRHA) and mannose-sensitive (MSHA) fimbrial haemagglutinins was examined in 482 strains of Escherichia coli isolated from 390 adult women and 45 pregnant mothers with a variety of urinary tract infections (UTI), and from 47 healthy controls. The proportion of MRHA strains was significantly higher in patients with symptomatic UTI (75%) than in women with non-significant bacteriuria (30%, p less than 0.001), pregnant women with asymptomatic UTI (34%, p less than 0.0001) and healthy controls (0%). The proportion of MSHA strains was significantly lower in patients with symptomatic UTI (22%) than in women with non-significant bacteriuria (46%, p less than 0.001) and pregnant women with asymptomatic UTI (52%, p less than 0.01). Only 17% of the strains from healthy controls had MSHA activity. In pregnant women with UTI, whether this was symptomatic or asymptomatic, there was a significant association between infection with MRHA strains of E. coli and a past history of UTI. Thus, in a pregnant woman with an infection and a past history of UTI there is a seven-fold greater chance that this infection is due to an MRHA-bearing organism than in pregnant women without such a history. There was also a significant association between MRHA organisms and symptomatic infection. The risk of symptomatic patients having an infection with an MRHA strain is six times greater than that for a patient with a covert infection.

The role of secretory IgA in anti-coccidial immunity in the chicken.

The serological and secretory immune responses of the chicken to infection with Eimeria tenella were evaluated in terms of various anti-coccidial activities. Serological responses were detected in the forms of precipitating, sporozoite neutralizing, anti-merozoite and anti-schizont antibodies. Similarly, anti-schizont and sporozoite neutralizing activities were found in caecal contents (containing mainly IgA) from infected birds and these also had the capacity to damage second generation merozoites. Moreover, the functional importance of IgA could be implied from the substantial predominance of IgA synthesizing cells in the intestinal immunocyte response as revealed by immunohistology. This was reflected in the immunoglobulin profile of caecal contents, for primary and secondary infection resulted in elevated levels of IgA whilst IgG and IgM generally remained extremely low or were usually undetectable. Taken with the well established lack of correlation between serum antibody and protection, these results suggest that the intestinal secretory IgA system plays an essential role in the protective immune response to E. tenella.

Characterization and localization of secretory component in the chicken.

A component found free in intestinal contents and caecal contents of conventional and germ-free chickens (lacking IgA producing cells) was found to have similar characteristics to mammalian secretory component (SC). Free secretory component (FSC) showed a classic reaction of partial identity with secretory IgA (SIgA) from bile, intestinal contents and cystic oviduct fluid. Furthermore, there was demonstrable cross-reactivity between FSC and a low molecular weight component released from SIgA by mild reductive dissociation, confirming the presence of a disulphide-linked accessory polypeptide chain. Fractionation of serum IgA revealed two molecular classes of IgA, a high molecular weight 15S IgA which possessed SC and could not be differentiated antigenically from SIgA and a low molecular weight 7S IgA which showed a reaction of partial identity with 15S IgA and non-identity with FSC. Fluorescent localization of SC in young germ-free chicks demonstrated its presence in the supranuclear golgi zone, apical cytoplasm and basement membrane of crypt epithelial cells. It is concluded that the characteristics of chicken SIgA are closely aligned with those of its mammalian counterpart and are consistent with a system in which SIgA is the wynthetic product of two distinct cells, final assembly occurring in the crypt epithelium.

Intestinal immune response to E. coli antigens in the germ-free chicken.

The secretory intestinal immune response to live and heat-inactivated E. coli 02 has been studied in young germ-free chicks. A response to live organisms was evident from an infiltration of the intestinal mucosa with IgA and IgM immunoglobulin-producing cells (IPC). Antibody associated with both immunoglobulin classes which was specific for E. coli 02 was demonstrated in saline extracts of contents from the small intestine. Repeated oral immunization with heat-killed E. coli 02 failed to stimulate serum or intestinal antibody. This finding reflected the complete absence of IPC within the intestinal mucosa of these birds. The IPC profile of unimmunized germ-free chicks was identical to that seen in chicks orally immunized with inactivated E. coli. An interesting feature of all gnotobiotic birds was a considerable elevation of serum IgM levels, compared to those of conventional birds, which was unassociated with immunization procedures. Serum IgG and IgA levels in gnotobiotes were much lower than those in the serum of conventional birds of comparable age. Studies on the fate of orally administered antigen using radiolabelled E. coli endotoxin indicated that a proportion remained intact as far down the intestinal tract as the caecum. However, unaccountably high levels of low mol. wt antigen in the faeces suggested degradation, intestinal absorption and subsequent excretion of endotoxin fragments in the urine. The differences between the avian response and that observed in mammals are discussed in relation to the comparative roles of Peyer's patches and the bursa of Fabricius in initiating intestinal immununity.

Significance of immune mechanisms in relation to enteric infections of the gastrointestinal tract in animals.

The impact of bacterial colonization on the alimentary tract in early life is reflected in gross changes in morphology. Subsequent health, if not survival, may largely be determined by a continuum of local intestinal immune mechanisms and it is essential for antibody development during the neonatal period to compensate adequately for declining passive maternal antibody. Consequent upon the development of the gut microflora the lamina becomes infiltrated with immunocytes in which the dominant immunoglobulins produced are IgM and IgA. Both immunoglobulins are transported across the epithelium by a process involving membrane-bound vesicles. Germ-free and fistulated pigs and calves are shown to be able to respond to oral immunization with Escherichia coli O somatic antigens during the first week of life. Resistance to infection with enteropathogenic E. coli was significantly enhanced, along with other parameters of nutrition and performance. However, in the young chick, although the intestinal response to infection with E. coli was similar to that in the mammal, no response to E. coli O antigens could be determined on oral administration in germ-free or local intestinal applications in fistulated birds. In the mammalian intestine secretory antibodies participate in the control of pathogenic E. coli by blocking adhesion to the mucosal epithelium, interfering with the elaboration of surface antigens, inhibiting toxins, and facilitating rapid elimination from the alimentary tract by agglutination and bacteriostasis. In consequence fewer enteropathogens are excreted into the environment, an important feature in modern intensive systems of animal production.

Further characterization of IgA in chicken serum and secretions with evidence of a possible analogue of mammalian secretory component.

Immunochemical studies of the intestinal secretory immune system of the chicken have led to further characterization of IgA in bile, intestinal contents and serum. A component was detected in late Sephadex G-200 fractions of caecal and intestinal contents which showed partial identity with bile, intestinal and a high molecular weight fraction of serum IgA. This component showed similar sedimentation characteristics to bovine serum albumin in sucrose density gradients, a fast electrophoretic mobility on polyacrylamide gel and is a possible analogue of mammalian secretory component (SC). Fractionation of serum from birds affected with infectious synovitis revealed two moleculare classes of IgA. Comparative double diffusion studies produced a reaction of complete identity between bile IgA and high molecular weight serum IgA (15S) and partial identity with low molecular weight serum IgA (7S), suggesting a lack of an SC determinant on the latter. A spur of partial identity between 15S and 7S serum IgA was also observed. Although no direct structural homology with mammalian or human IgA could be demonstrated by immunological cross-reactivity, the similarities of molecular characteristics, particularly emphasized by the presence of a secretory component, favour a functional analogy between the secretory immune system of the fowl and mammalian species.

Local immunity in the respiratory tract of the chicken. I. Transudation of circulating antibody in normal and virus-infected birds.

Three-week-old chickens were given sheep erythrocytes or bovine serum albumin intravenously. Seven days later their tears and saliva possessed low levels of antibody to those antigens. Concurrent infection with lentogenic Newcastle disease virus (NDV) caused a significant increase in transuded antibody in those fluids. In chickens with circulating antibody to NDV, induced by parenterally administered inactivated vaccine, respiratory infection with heterologous infectious bronchitis virus resulted in limited transudation of anti-NDV. In contrast, the tears, saliva and tracheal fluid of non-vaccinated chickens undergoing primary infection with NDV acquired considerable levels of specific anti-NDV. The difference between the two groups is attributed to locally synthesized antibody.

Local immunity in Newcastle disease: some recent experiments.

Following primary exposure by the ocular route to lentogenic Newcastle disease virus (NDV) the lacrymal fluid, saliva and tracheal washes of three-week old specific pathogen free chickens acquired specific virus-neutralizing activity which considerably exceeded transudation of circulating antibody. All three secretions contain IgA which, at least in saliva, accounted for 85% of its activity, the remanider being due to IgG. Antibody in secretions limited, but did not prevent, reinfection of the trachea when birds were challenged two weeks later. In contrast to an elevation of circulating antibody titre, challenge induced only a repeated primary response in secreted antibody. Ocular infection induced marked lymphoid and plasma cell activity in the Harderian gland which is a major source of specific antibody in lachrymal and possibly other fluids. Functional ablation of the gland can be effected by occlusion of its draining duct, providing a means for evaluation of its immunological significance.

Immunoglobulin A in some avian species other than the fowl.

In immunodiffusion tests rabbit and pheasant antisera monospecific for fowl IgA detected a cross-reacting homologous protein in sera and/or secretions (saliva and bile) of pheasant, Japanese quail, guinea fowl, turkey and pigeon. Negative findings with duck samples do not preclude the occurrence of a similar immunoglobulin class in that species.