Immunological quantification of the nematode parasitic bacterium Pasteuria penetrans in soil

Abstract

Currently, the abundance of Pasteuria penetrans in soils, an unculturable bacterial parasite of root-knot nematodes (Meloidogyne spp,). is estimated by the percentage of nematode juveniles infected with bacteria and the number of spores attached to their cuticle. Indirect immunofluorescence led to detection of free spores directly in soil suspensions using UV light and polyclonal antibodies raised against two P. penetrans populations (ORS-21414-Sen and PPI). Three extraction methods were compared in order to improve spore recovery. A gentle shaking/sieving method recovered more than 90% of the spores inoculated in soils and was more efficient and simple than aqueous two-phase partitioning and polyethylene glycol extractions. All the spores inoculated in sandy or sandy-clay soils were detected with immunofluorescence microscopy. The quantification of the spores was improved using an ELISA technique that showed a good correlation between optical density and spore concentration in inoculated soils. Specific antibodies provide a suitable method to quantify A penetrans and may be used to follow the evolution of the real pool of bacteria either in native suppressive soils or in inoculated ones. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved

Exploitation of immunofluorescence for the quantification and characterization of small numbers of Pasteuria endospores

“…This was due to higher than expected concentrations being detected for low initial concentrations of endospores in the standard curve, and therefore correspondingly lower than expected concentrations of endospores for higher initial concentrations. The same effect has been reported before (Fould et al , 2001), however in this case we believe it to be due to and inherent counting error, as Bayesian estimates within the same normal distribution of observed values provided a corrected and workable curve. The observed number of endospores in the three different substrates used to optimise the quantification method did not differ significantly.…”

“…The immunofluorescence method described here permitted the detection of endospores in suspensions, even when these contained organic matter and other debris; it also facilitated the identification of Pasteuria endospores in natural populations. The threshold of detection for this method was lower than for other methods, which varied from a minimum of an estimated 100 endospores g −1 in soil using PCR with specific primers (Duan et al , 2003), to about 300 endospores g −1 for antibody‐based ELISA techniques (Schmidt et al , 2003), and up to 1000 endospores g −1 (Fould et al , 2001) and above. Most authors do not present an overall efficiency estimate, which for our work was key as it enables quantification; using a similar method but at very large spore densities (Fould et al , 2001), 94% of all spores were extracted from soil, but the immunodetection method is comparable with the 59% estimate obtained after a complex differential centrifugation extraction (Davies et al , 1990).…”

“…A polyclonal antibody,∞PPORS pAB, previously raised in rabbit against Pasteuria penetrans endospores was used (Davies et al , 1992; Fould et al , 2001). The immunodetection technique was optimized through a series of preliminary experiments using different endospore suspensions, sand substrates, exposure‐periods and incubation temperatures, from an existing method (Duponnois et al , 2000).…”

Environmental quantification of Pasteuria penetrans endospores using in situ antigen extraction and immunodetection with a monoclonal antibody

“…Methods employing DNA primers and PCR‐based detection are widely used for the detection of microorganisms in the environment, but suffer limitations for detection of spore DNA [16–18]. The detection of a signature gene in an environmental sample does not assure the presence of a viable or virulent form of an endospore‐forming bacterium.…”

“…A series of monoclonal antibodies (MAbs) to P. penetrans biotype Pp1 endospores were developed to study spore coat epitopes in relationship to host specificity [23]. Recently, immunofluorescent and enzyme‐linked immunosorbent assay (ELISA) detection by a polyclonal antibody raised against P. penetrans biotype ORS‐21414‐Sen and Pp1 in rabbits has been used to detect whole spores in soil following spore recovery by differential sieving [16].…”