AnaroAMutant ofEdwardsiella ictaluriIs Safe and Efficacious as a Live, Attenuated Vaccine

Abstract

The aroA gene of Edwardsiella ictaluri was cloned and sequenced, and the sequence data were used to construct a deletion–insertion mutation in the aroA gene. The mutated gene was transferred into a virulent, wild‐type E. ictaluri strain by conjugation and allelic exchange. Putative aroA mutants were confirmed phenotypically by demonstrating a need for supplementation with aromatic metabolites to support growth in minimal media. The genetic construction was evaluated by using the polymerase chain reaction to amplify appropriate regions of the aroA deletion–insertion, and DNA sequencing of the amplified products confirmed the predicted construction. A selected mutant, LSU‐E1, was passed 30 times in nonselective media with no reversion to the wild‐type following screening of 1.6 × 1011 colony‐forming units. The mutant was demonstrated via injection to be attenuated more than 5 logs10 compared with the wild‐type E. ictaluri strain, and it was avirulent by immersion and oral routes. Tissue persistence studies indicated that the mutant maintained the ability to invade following immersion exposure, but no viable cells were detected after 48–72 h. Significant levels of protection from disease were demonstrated following immersion vaccination of channel catfish Ictalurus punctatus.

Fur-Regulated Iron Uptake System of Edwardsiella ictaluri and Its Influence on Pathogenesis and Immunogenicity in the Catfish Host

“…5). Our results correlate with previous observations (67) and with the absence of know siderophore biosynthesis genes in the E. ictaluri chromosome (72). This observation reinforces the hypothesis of genome degradation during the process of host specialization of E. ictaluri to catfish, where siderophore genes might be lost.…”

“…5B). Our results correlate with previous observations (67) and with the fact that siderophore biosynthesis genes are not present in the E. ictaluri chromosome (72).…”

Construction of a safe, stable, efficacious vaccine againstPhotobacterium damselaessp.piscicida

“…Because 91.65 ± 6.9** LSU-P2 **13.3 ± 11.0** 85.5 Compared to the aroA strains of Edwardsiella ictaluri previously evaluated for immersion vaccination of fish, the level of protection seems to correlate to the relative virulence of the wild type. In a previous study, Thune et al (1999) determined that the injection LD 50 for wild type E. ictaluri was ca. 40 CFU g fish -1 , and that immersion in 10 7 CFU ml H 2 O -1 resulted in 73.3% mortality.…”

“…piscicida that retains its ability to invade the host and temporarily persist in the tissues should stimulate an effective CMI response, and provide protection against subsequent challenge with the wildtype strain. Other bacterial fish pathogens with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, are attenuated and confer varying degrees of protection against subsequent challenge by virulent pathogens (Vaughan et al 1993, Thune et al 1999. Bacterial pathogens with stable defects in the aro genes of the shikimate pathway require supplementation with aromatic metabolites for growth; because the metabolites required are essentially unavailable in vertebrate tissues, they are incapable of sustained growth in those tissues and are, thus, nutritionally attenuated.…”

A comparison of invasive capabilities between Edwardsiella ictaluri and Aeromonas hydrophila on Pangasius hypophthalmus

“…Sau đó, 1 ml dịch nuôi cấy được cấy chuyền vào 100 ml BHI lỏng và nuôi cấy 200 vòng/phút ở 28 o C, lắc qua đêm đối với E. ictaluri đến khi đạt OD 600 0,8-1,0 (~10 9 CFU/ml), với A. hydrophila nuôi cấy lắc khoảng 5-6 giờ đến OD 600 đạt 1,4-1,5 (~10 9 CFU/ml). Sinh khối vi khuẩn được ly tâm ở 2200 g/15 phút ở 4 o C, được huyền phù và pha loãng trong NaCl 0,65% theo Ronald (1999) [7].…”