Academic journal article Journal of Health Population and Nutrition

Patterns and Properties of Haemagglutinins Expressed by Shigella Serogroups in Lagos, Nigeria

Academic journal article Journal of Health Population and Nutrition

Patterns and Properties of Haemagglutinins Expressed by Shigella Serogroups in Lagos, Nigeria

Article excerpt

INTRODUCTION

The high pathogenicity of enteric bacteria, including Shigella, to cause life-threatening intestinal and extra-intestinal infections has been linked to elaborate expression of virulence factors, such as toxins and haemagglutinins (1-3). Haemagglutinins, which are ubiquitous proteinaceous adhensins with cell-agglutinating and adherence properties, have also been implicated to play a crucial role in the initiation and development of clinical symptoms and complications in shigellosis (4,5). Stathopoulos et al. demonstrated the transferability of these haemagglutinating factors among Enterobacteriaceae in a manner that familiarize them with serine proteases of the Enterobacteriaceae family (SPATEs) found in Shigella and Escherichia coli (6).

As in many developing countries, Shigella strains in circulation in Nigeria have been found to show multiple resistance to antibiotics (7) and express proteases (8), endotoxins (9), and enterotoxins (Iwalokun BA et al. Personal communication, 2003) as virulence factors. However, information gathered so far on the virulence behaviour of this organism is not substantial since there is still a paucity of information on whether they produce haemagglutinins or not. This could be a constraint in the future design of effective vaccines against shigellosis.

Presently, use of Shigella vaccines as an alternative or adjunct to chemotherapy of this dreadful disease is a leading priority in many endemic communities (10), and the fact that haemagglutinins possess immunoprotective epitopes (11,12) has made their inclusion in multivalent vaccine construct unquestionable. It was on this basis that the immunoprotective efficacy of a live oral Shigella flexneri vaccine in guinea pig was revealed (13).

Therefore, as a prelude to probable design of antiShigella vaccines for the Nigerian environment, 45 local Shigella strains were screened for haemagglutinin expression. The pattern of expression of these adhesions and their broadness of activity and effects of sugars other than mannose and trypsin on the haemagglutination reaction were also investigated.

MATERIALS AND METHODS

Shigella strains

Stock trypticase soy broth cultures of Shigella isolated from stool samples of patients with diarrhoea, attending six medical centres in Lagos, Nigeria, were studied for patterns of haemagglutinin expression. Stools were collected during February-November 2001 on Cary-Blair medium (Difco, USA) and Selenite F broth (Oxoid, UK). Suspected isolates were eventually identified on Salmonella-Shigella agar (Oxoid, UK) and speciated biochemically according to Cowan (14). The isolates were stocked under 16% glycerol at -70[degrees]C. Prior to experimentation, the isolates were re-tested for viability in Mueller-Hinton broth (Oxoid, UK), Salmonella-Shigella agar, and then in trypticase soy broth ([A.sub.600nm]=0.5-0.8 at mid-log phase). The selected strains were: S. flexneri (n=18), S. dysenteriae (n=13), S. sonnei (n=8), and S. boydii (n=6).

Haemagglutination assay

For optimal production of haemagglutinins, each Shigella strain was statically grown in brain-heart infusion (BHI) broth (Oxoid, UK) at 37[degrees]C for 48 hours. Cells were then harvested by centrifugation at 12,000 x g for one minute at 25[degrees]C and adjusted to an inoculum size of 1 x [10.sup.9] cfu/mL with phosphate-buffered saline (PBS, pH 7.2). To test for haemagglutination and mannose susceptibility, citrated guinea pig erythrocytes, washed twice with PBS and re-suspended to a final concentration of 3% (v/v) with or without 50 mM mannose, were used (15,16). Two-fold serial dilutions of bacterial suspension (1:2-1:64 per row) were first dispensed in a 96-well microtitre plate, followed by the addition of equal volumes of the erythrocyte suspension. The plates were finally incubated at room temperature for 30 minutes. A mannose-sensitive haemagglutinin (MSHA) was inferred if the red blood cells agglutinated only in the absence of mannose. …

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