Validation of Fermentation and Processing Procedures for the Commercial-Scale Production of a Live, Attenuated Edwardsiella ictaluri Vaccine for Use in Channel Catfish Aquaculture.

Mortality associated with Edwardsiella ictaluri infection is a serious impediment to the commercial production of fingerling Channel Catfish Ictalurus punctatus. A patented, live, attenuated, orally delivered vaccine has been developed that offers exceptional protection against E. ictaluri infection in both laboratory and small-scale pond trials. Further vaccine development is contingent on the successful completion of large-scale field trials that accurately reflect industry conditions. This current work focuses on the validation of fermentation protocols and the optimization of downstream processing procedures to produce sufficient quantities of vaccine to conduct commercial-scale field trials. Eight vaccine serials were produced from a master seed stock (S97-773-340X2) in a 50-L floor model fermenter over two consecutive years. Following fermentation, cells were harvested, concentrated 10-fold, and cryogenically stored (-74°C). To assess processing protocols and determine shelf life of cryogenically stored vaccine, serials were tested for cell viability and vaccine potency at various intervals over 24 months. There were no significant differences in cell viability between the fresh vaccine and the stored frozen product. All serials provided a high level of protection (77-100% relative percent survival) against E. ictaluri infection in juvenile Channel Catfish and exhibited excellent poststorage viability. This data demonstrates that the live, attenuated, orally delivered vaccine can be stored at -74°C for at least 2 years with no reduction in cell viability or vaccine potency. Received May 17, 2016; accepted January 19, 2017.

Efficacy of water-dispersible formulations of biological control strains of Aspergillus flavus for aflatoxin management in corn.

Field experiments were conducted in 2011 and 2012 to evaluate the efficacy of water-dispersible granule (WDG) formulations of biocontrol strains of Aspergillus flavus in controlling aflatoxin contamination of corn. In 2011, when aflatoxin was present at very high levels, there was no WDG treatment that could provide significant protection against aflatoxin contamination. The following year a new WDG formulation was tested that resulted in 100% reduction in aflatoxin in one field experiment and ≥ 49% reduction in all five WDG treatments with biocontrol strain 21882. Large sampling error, however, limited the resolution of various treatment effects. Corn samples were also subjected to microbial analysis to understand better the mechanisms of successful biocontrol. In the samples examined here, the size of the A. flavus population on the grain was associated with the amount of aflatoxin, but the toxigenic status of that population was a poor predictor of aflatoxin concentration.

Antimicrobial properties of nest volatiles in red imported fire ants, Solenopsis invicta (Hymenoptera: Formicidae).

The antimicrobial property of volatiles produced by red imported fire ants, Solenopsis invicta, against Beauveria bassiana, a common entomopathogenic fungus, was demonstrated. The germination rate of B. bassiana spores was significantly reduced after they were exposed to volatiles within an artificial ant nest. Since the air that contained the same level of O2 and CO2 as that in artificial fire ant nests did not suppress the germination rate of B. bassiana, the observed reduction of germination rate must be caused by the toxicity of nest volatiles. Nest fumigation may be an important component of the social immune system in S. invicta.

Effects of piperidine and piperideine alkaloids from the venom of red imported fire ants, Solenopsis invicta Buren, on Pythium ultimum Trow growth in vitro and the application of piperideine alkaloids to control cucumber damping-off in the greenhouse.

BACKGROUND: Pythium ultimum is a plant pathogen that causes significant yield losses on many economically important crops. Chemical treatment has been used for disease control. In searching for alternatives, venom piperidine and piperideine alkaloids from red imported fire ants were tested against P. ultimum in vitro, and piperideines were employed to control cucumber damping-off in the greenhouse as drench treatments. Results Piperidine and piperideine alkaloids of the red imported fire ant significantly inhibited mycelium growth of P. ultimum. Piperidine alkaloids were stable at both room and elevated temperatures. The inhibitory activity positively correlated with the concentrations of piperidine alkaloids in the medium, and the EC(50) = 17.0 µg ml(-1). Germination of sporangia of P. ultimum was negatively correlated with the concentrations of piperidine alkaloids in the medium, and the EC(50) = 12.3 µg ml(-1). The piperideine alkaloid drenching treatment significantly improved seedling emergence and seedling height of cucumber. CONCLUSION: This is the first report describing the use of venom alkaloids from the red imported fire ant to inhibit P. ultimum in the laboratory and the application of piperideine alkaloids to control damping-off disease caused by P. ultimum in the greenhouse. These findings may lead to the development of a new group of fungicides.

Identification and expression profile of multiple genes in response to magnesium exposure in Culex quinquefasciatus larvae.

Magnesium is crucial for baculovirus transmission in Culex nigripalpus (Theobald) and Culex quinquefasciatus (Say) larvae. However, the mechanistic role of magnesium in baculovirus transmission is unknown. To investigate the possible role of host response factors in baculovirus transmission, suppression subtractive hybridization was used to identify genes differentially transcribed after magnesium exposure in Cx. quinquefasciatus larvae. Suppression subtractive hybridization was performed in both directions enriching for cDNAs differentially transcribed between a nonmagnesium larval control and magnesium (15 mM MgSO4) treatment of Cx. quinquefasciatus larvae held for 1 h at 27 degrees C. Clones from differentially transcribed genes were evaluated by sequencing, and relative gene transcription levels were analyzed using quantitative real-time polymerase chain reaction quantitative real-time polymerase chain reaction. Target transcripts up/downregulated by magnesium included Cx. quinquefasciatus troponin C, isocitrate dehydrogenase, allergen, cytochrome b5, chymotrypsinogen, apolipophorins, tryptase gamma, carboxylesterase, prolylcarboxypeptidase, imaginal disc growth factor, aldehyde dehydrogenase, tropomyosin-1, chitotriosidase, heat shock protein 70 B2, inorganic phosphate cotransporter, and many other hypothetical protein genes. Magnesium can alter gene transcription in a vector mosquito population, and understanding this process can provide insight into the mechanistic role of magnesium in baculovirus transmission.

Interspecific variation of Delta1,6-piperideines in imported fire ants.

Delta(1,6)-piperideines have been recently reported in the venom of the red imported fire ants, Solenopsis invicta Buren and the black imported fire ants, Solenopsis richteri Forel. However, they have never been quantified in either species. Furthermore, there is no information available about those piperideines in the hybrid imported fire ants (S. invicta x S. richteri). The abundance of six Delta(1,6)-piperideines was investigated in both species and their hybrid using gas chromatography-mass spectrometer (GC-MS). They include 2-methyl-6-tridecenyl-6-piperideine, 2-methyl-6-pentadecenyl-6-piperideine, 2-methyl-6-heptadecenyl-6-piperideine, 2-methyl-6-tridecyl-6-piperideine, 2-methyl-6-pentadecyl-6-piperideine, and 2-methyl-6-heptadecyl-6-piperideine. S. invicta produced all six Delta(1,6)-piperideines, whereas, S. richteri did not produce 2-methyl-6-heptadecenyl-6-piperideine and 2-methyl-6-heptadecyl-6-piperideine. The Delta(1,6)-piperideine profiles of the hybrid was similar to that of S. richteri, except trace amounts of 2-methyl-6-heptadecenyl-6-piperideine and 2-methyl-6-heptadecyl-6-piperideine were found in some of the samples. The ratio of 2-methyl-6-pentadecenyl-6-piperideine to 2-methyl-6-pentadecyl-6-piperideine (C(15:1)/C(15:0)) was significantly different among two species and their hybrid. In addition to Delta(1,6)-piperideines, hybrid workers also contained significantly more piperidines than their parent species. This is the first evidence of heterosis of imported fire ants in venom production.