Simple and direct detection of Aeromonas hydrophila infection in the goldfish, Carassius auratus (L.), by dot blotting using specific monoclonal antibodies

Abstract

A combination of eight isolates of Aeromonas hydrophila was used to produce monoclonal antibodies (MAbs). Ten different groups of MAbs specific to Aeromonas were selected. The first five groups of MAbs demonstrated high specificity and bound to only one or two isolates of A. hydrophila. The sixth and the seventh groups of MAbs were A. hydrophila specific. They recognized seven of eight A. hydrophila isolates (AH1, 2, 3, 4, 5, 6, 8); however, the MAb in the seventh group also showed cross-reactivity to one isolate of Aeromonas caviae (AC3). The eighth MAb group recognized two isolates of A. hydrophila (AH2 and AH5) and demonstrated cross-reactivity to one isolate of Aeromonas sobria (AS1) and one isolate of A. caviae (AC3). The tenth group of MAbs bound to all isolates of Aeromonas spp. tested (AH1-8, AS1-6, AC1-5, Aeromonas veronii and Aeromonas jandaei) without cross-reactivity to any of the other bacteria tested. MAbs in the ninth group showed similar specificity to those in the tenth group but did not recognize two isolates of A. sobria (AS4 and AS6) or A. jandaei. All the MAbs could be used to identify Aeromonas by dot blotting with a sensitivity ranging from 10⁵ to 10⁷ CFU mL⁻¹. However, the sensitivity of detection was increased to 10²-10³ CFU mL⁻¹ after inoculation of the sample in tryptic soy broth for 3-6 h before performing the dot blotting. The dot blot method can be used for the direct detection of A. hydrophila infection in symptomatic and asymptomatic goldfish. This study demonstrated a convenient immunological tool that can be used for the direct detection of A. hydrophila and Aeromonas infections in a complex sample without the requirement for separation of the bacteria or isolation and biochemical tests.

Production and use of immunoglobulin Y in the diagnosis of haemorrhagic septicemia caused by Aeromonas hydrophila in Piaractus mesopotamicus

“…Also, they showed specificity to three inner membrane proteins (10, 25 and 37 kDa) and two cytoplasm proteins (15 and 20 kDa) (Figure 4). The study showed a molecular mass of 10 kDa which was also described by Longyant et al (2010) and is compatible with a lipopolysaccharide. Poobalane et al (2008) described two bands of 25 and 37 kDa as components of the bacterial membrane, which were purified from virulent isolates of A .…”

Aeromonas veronii, Associated with Skin Ulcerative Syndrome, Isolated from the Goldfish (Carassius auratus) in China

“…The virulence of the isolate A. veronii CAV-134 could be associated with the virulence gene Aha1 (major adhesion). A. veronii infection is a growing problem in cultured goldfish causing major economic losses in China (Longyant et al, 2010;Yang, 2013;Song et al, 2009;Hu et al, 2008;Xu et al, 2006).…”

Development of a PCR Assay Based on a Single–Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene

“…Various biochemical tests to identify the bacteria were performed in a conventional format. The origins and sources of the 41 Aeromonas isolates tested are shown in Table 1 Verification of Aeromonas spp.-A monoclonal antibody (MAb) developed in our laboratory and designated AH-396 was used to verify the bacteria in the genus Aeromonas (Longyant et al 2010), and an MAb designated AC-4A3 (unpublished data) was used to verify A. caviae. Briefly, bacterial cultures grown on tryptic soy agar (TSA; Table 1) were harvested and resuspended in phosphate-buffered saline (PBS) at pH 7.2, heat-killed at 60 • C for 1 h, and adjusted to an optical density at 600 nm equal to 1 (approximately 10 9 CFU/mL); finally, this suspension was diluted 10 times (10 8 CFU/mL).…”