Repeated vaginal administration of trimeric HIV-1 clade C gp140 induces serum and mucosal antibody responses.

Vaccine-mediated prevention of primary infection with human immunodeficiency virus (HIV) may require the sustained production of antibody at mucosal portals of entry. Here, we describe a novel approach of repeated mucosal immunization by delivering an HIV-1 envelope glycoprotein (gp) in a gel formulated for intravaginal delivery. Rabbits were immunized over one to three 19-day cycles of intravaginal dosing with soluble recombinant trimeric HIV-1 clade C gp140 administered in Carbopol gel. The formulation was well tolerated. A single immunization cycle induced immunoglobulin G (IgG) antibody detected in the serum and female genital tract, and titers were boosted on further immunization. Vaccine-induced serum antibodies neutralized the infectivity of a pseudovirus carrying a heterologous clade C envelope. Our data prove the concept that repeated exposure of the female genital tract to HIV envelope can induce mucosally detectable antibody.

Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep.

Definitive diagnosis of Johne's disease in ruminants depends on confirming the presence of the causative bacterium, Mycobacterium avium subsp. paratuberculosis, in tissues of the host. This is readily achieved in most ruminant species by culture. However, culture of clinical specimens from sheep in many countries has been unrewarding. Such a culture from sheep was achieved recently in Australia by using a radiometric culture medium. The aims of the present study were to evaluate the culture of M. avium subsp. paratuberculosis from sheep by using modified BACTEC 12B radiometric medium, to determine the sensitivity of culture in relation to histopathology, and to evaluate a range of solid media. Culture of M. avium subsp. paratuberculosis from sheep with Johne's disease is a sensitive method of diagnosis: intestinal tissues from all 43 animals with multibacillary disease and all 22 animals with paucibacillary disease were culture positive, while 98% of feces from 53 animals with multibacillary disease and 48% of feces from 31 animals with paucibacillary disease were culture positive. Of sheep without histological evidence of Johne's disease from infected flocks, intestinal tissue from 32% of 41 were culture positive, while feces from 17% of 41 were culture positive. Consequently, culture is recommended as the "gold standard" test for detection of ovine Johne's disease. Of the wide range of solid media that were evaluated, only modified Middlebrook 7H10 and 7H11 agars, which were very similar in composition to modified BACTEC 12B medium, yielded growth of ovine strains of M. avium subsp. paratuberculosis. The sensitivity of detection of M. avium subsp. paratuberculosis on solid media was slightly lower than that in modified BACTEC 12B radiometric medium. Both egg yolk and mycobactin J were essential additives for growth of ovine strains of M. avium subsp. paratuberculosis in both liquid and solid media.

Studies on primary cell cultures derived from ovarian tissue of Penaeus monodon.

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2x L-15 media. Various supplements including growth factors, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with large nuclei) were maintained for up to 17 months. Epithelioid cells grew best in modified Grace's medium but were generally short-lived (less than two months). Fibroblast-like cells formed confluent monolayers in modified 2x L-15 medium, were passaged three times and survived for 17 months. In other cultures, millions of rounded cells migrated from tissue. They survived for prolonged periods (up to ten months), either loosely attached to the flask or suspended in the medium. A change in dominant cell type from fibroblastic to epithelioid was observed in some cultures after three or nine months incubation. These epithelioid cells which had very large nuclei, grew to confluence but could not be sub-cultured. It is noteworthy that the rounded cells and the epithelioid cells with the large nuclei both produced vitellogenin in protein-free media.

Application of the theory of adaptive polymorphism to the ecology and epidemiology of pathogenic yeasts.

The theory of adaptive polymorphism predicts that species occupying broad ecological niches will be phenotypically and genotypically more varied than those occupying narrow niches. It is suggested that this theory has direct relevance to the epidemiology of microbial pathogens in that environmental pathogens inhabit a broader niche and should be expected to exhibit greater variation than pathogens that are obligate commensals. This proved to be the case when one obligate commensal, the pathogenic yeast Candida albicans, was compared with other Candida spp. and an environmental pathogen, Cryptococcus neoformans. Further evidence of this relationship is derived from the literature. This observation adds further support to the theory of adaptive polymorphism, although the mechanisms of maintenance of polymorphism is asexually reproducing populations must be different from those in sexually reproducing populations. This observation may give important clues to the epidemiology of those infections for which it is not already known.

Cross-infection and diversity of Candida albicans strain carriage in patients and nursing staff on an intensive care unit.

Colonization of patients and nursing staff on an intensive care unit by Candida albicans was studied over a 4-month period. Multiple swabs and samples were taken from patients and their nurses on 12 occasions during the study period. During this study there were no obvious clinically relevant candidal infections. Patients yielded C. albicans from at least one body site on 68%, and nurses on 57% of occasions. All isolates of C. albicans were further characterized by both morphotyping and resistotyping. All patients but one were colonized by a single strain throughout their stay on the unit, whereas nurses were often colonized by more than one strain type. Strains isolated from nurses' hands were all indistinguishable from strains colonizing the patient under their care, whilst strains isolated from nurses' mouths were usually distinct from their patients'. The probability that a C. albicans culture positive nurse carried an indistinguishable strain from that of her positive patient was p = 0.632, compared with p = 0.325 that a randomly chosen positive nurse would carry a strain indistinguishable from that of a randomly chosen positive patient. Diversity in patients' strains was low (D = 0.667) compared with nurses' strains (D = 0.778). These results suggest that cross-infection by C. albicans is a common occurrence on intensive care units. The reason outbreaks of candidal disease are not more common may be related to differences in virulence of the strains.

Application of a numerical index of discriminatory power to a comparison of four physiochemical typing methods for Candida albicans.

Four typing methods for the strain differentiation of Candida albicans (morphotyping, resistotyping, extracellular enzyme production, and carbon source assimilation reactions) were compared on a single population of 100 strains. An index of discriminatory power was used to facilitate this comparison. Extracellular enzyme production had poor discriminatory power, and carbon source assimilation reactions were poorly reproducible. Morphotyping and resistotyping had acceptable reproducibility and discrimination. The use of resistotyping and morphotyping in parallel enhanced discrimination without an unacceptable decrease in reproducibility, although other combinations did not enhance discrimination because reproductibility was impaired.

Morphotype markers of virulence in human candidal infections.

A study of the morphotypes of 446 strains of Candida albicans, isolated from a variety of clinical specimens, is reported. The method was based on a morphotyping scheme that has recently been described, but not all of the potential characters were used in this analysis. By this limited code, 50 different morphotypes were distinguished, the largest group comprising 23% of the population. The simplicity and good discrimination of the method make it a useful typing scheme for C. albicans. Discontinuous colonial fringes were associated with strains from oral sites and deep infections. Significantly, 67% of strains from fatal infections were of the discontinuous fringe type, compared to only 11% of strains from other infections. Further associations between morphotype and anatomical source included narrow-coarse fringes in genitourinary isolates.

Preparation of specific antisera to the opacity factors of group-A streptococci.

Type-specific antibody to the opacity factor (OF) of group-A streptococci can be demonstrated in human sera but the multiplicity of antibodies to different serotypes limits their usefulness in anti-OF typing. The antibody response in rabbits is inconsistent; only 61 of 138 (44%) of rabbit anti-M sera tested contained OF antibody. Of these only about half had titres of greater than 16, and usable sera to only 11 of 23 OF-positive serotypes were obtained. On the other hand good anti-OF sera (titres greater than 16) to 27 of 28 serotypes resulted from the interperitoneal and subcutaneous inoculation of heat-killed whole-cell vaccines in guinea-pigs and the frequency of response in groups of animals given injection of the same vaccine was 100% for all but three serotypes. Antibody response was not obtained with M-type 13. A comparison of routes of inoculation for M-type 25 showed that the subcutaneous route alone could probably be used in the routine production of anti-OF typing sera. Use of the set of 27 sera in OF-inhibition tests confirmed the remarkable specificity of OF antigens and their parallelism with M-antigen specificity, with the exception fo a reciprocal cross reaction between M-type 61 and provisional type PT3875.

A shortened scheme for the identification of indifferent streptococci.

A shortened biochemical scheme was devised for the reliable identification of the 'viridans' streptococci. It compared favourably with the classical identification system of Colman and could be recommended for use in the busy routine laboratory to investigate streptococcal isolates from clinical sources.

Use of the API-ZYM system in rapid identification of alpha and non-haemolytic streptococci.

The API-ZYM method of detecting enzymes was tested using 99 streptococci isolated from clinical material and 14 type species obtained from the National Collection of Type Cultures. We found the method easy and reliable. The results obtained indicate that this method could be a useful identification system in busy routine clinical laboratories.

Serological characterization of group-A streptococci associated with skin sepsis in meat handlers.

A series of outbreaks of skin sepsis among meat handlers in England during 1974 and 1975 afforded an opportunity to study the group-A streptococci commonly isolated from the lesions. Few of these streptococci could be M typed with existing antisera. Intensive study of strains from two outbreaks of sepsis in one abattoir in Shropshire led to the recognition of three new provisional M types. The streptococci were first sorted according to T-typing pattern and ability to produce opacity factor. Opacity-factor producing strains with the same T pattern were then screened for inhibition of opacity production by the sera of convalescents from the same outbreak. Finally, M antisera were made in rabbits against representative cultures. Sera against the three new provisional types were used to re-examine streptococci from 20 other outbreaks or incidents of sporadic infection among meat handlers. This increased the proportion of typable strains from 3% to 55%. Two of the new provisional types (nos. 2015 and 1658; both T25/Imp 19, opacity-factor positive) were confined to the Shropshire outbreak, but the third (no. 2681; T14, opacity-factor negative) was found among strains from meat workers in eight other geographically distinct areas. In all, 31% of 131 distinct strains from meat workers, but less than 1% of 2816 strains from other British sources, belonged to provisional type 2681. Thus, in Britain, one M-type of group-A streptococcus appears at present to be almost exclusively associated with sepsis in meat workers.

Antibody to streptococcal opacity factor in human sera.

Two tests are described for detecting antibody to the type-specific opacity factor (OF) of group A streptococci. This antibody was detected among patients convalescent from streptococcal sore throat in two communities in which outbreaks due to opacity factor-producing strains of group A streptococci occurred.In an outbreak due to streptococci of M-type 22 there was a close correspondence between the distribution of anti-OF and of bactericidal M-antibody for the type. In a smaller outbreak due to M-type 58 streptococci, however, M-antibody was detected more often than antibody to OF.