Progress 10/01/11 to 09/30/13
Outputs
Target Audience: Researchers working with Fundulus grandis as a potential aquaculture species. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training and professional development was accomplished for graduate student Rui Wang who worked on the project. How have the results been disseminated to communities of interest? A manuscript is near completetion and will be submitted to the Journal of Aquatic Animal Health. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A. Evaluate electroporation as a method for transfer of exogenous DNA into Streptococcus agalactiae (using Gulf Coast strain LADL 97-151 or Thailand strain ST81). In hopes of developing a live attenuated vaccine we utilized a method of mutation of the sipA gene, a known virulence gene in group B streptococcus. Since US Gulf Coast strains of GBS form a homogeneous group, strain LADL 97-151 was chosen as a template to amplify sipA gene by PCR. We then constructed an integrated plasmid which contained the sipA gene with a kanamycin resistance marker which was ligated into the plasmid pJL1055 backbone by multiple digestion and ligation. This construct was prepared to serve as a shuttle/suicide vector for replacing the sipA gene with a selectable kanamycin marker. Plasmid pJL1147 contains pVE6007, a pUC19 origin of replication, a kanamycin resistance cassette, and the N- and C-terminal flanking regions from sipA,. oriTs, Ts origin of replication; oripUC, pUC19 origin of replication; Cm, chloramphenicol-resistance marker; Km, kanamycin-resistance marker; and the PCR fragment upstream from the sipA gene. We cloned the partially deleted sipA gene with kanamycin into pJL1055 the temperature-sensitive plasmid. The resulting plasmid named pJLSKE was verified by sequencing and electroporated into GBS competent cells. B. Evaluate the use of plasmid pJL1055, a thermosensitive plasmid, containing sipA or cspA with deletions replaced by the kanamycin gene. The resulting plasmid will be electroporated into S. agalactiae competent cells and double crossover mutants selected. GBS cells containing pJLSKE integrated into the genome of GBS, were plated on selective media containing kanamycin Km and chloramphenicol Cm. KmRCmR colonies were selected as the single crossovers (sco) and then by changing the temperature, the second crossover should have occurred and the integrated plasmid excised from the genome. Attempts were made on many occasions but colonies with stable double crossovers were never found. The final mutant should have been chloramphenicol sensitive and kanamycin resistant. C. Description of Streptococcus agalactiae pathogenesis in cocahoe minnow following experimental exposure by immersion, intraperitoneal and intramuscular injection of wild type strain 91-157. Mortalities of minnows challenged by IP, IC or IC with abrasion were recorded. Based on the cumulative mortalities found 14 days post-challenge, the observed median lethal dose (LD50) for the IP challenged minnows infected with S. agalactiae LADL 97-151 was 10-9.43 (<2 CFU/fish). No bacteria was recovered from the surviving fish brain at the end of the challenge (14 days post challenge). On the other hand, neither the fish challenged by IC, or IC with abrasion presented more than 40% mortalities 14 days post-challenge (Figure 6 and 7). At least 4 x 107 CFU/ml or 4 x 106 CFU/ml were necessary to cause mortalities in the IC and IC with abrasion treatments. Moreover, only in two of the 61 surviving fish in the IC with abrasion treatment presented viable S. agalactiae in the brain 14 days post-challenge. None of the surviving fish in the IC challenge presented viable bacteria in the brain. Acute mortality present in the IP infected fish was accompanied with few clinical signs of diseases. On the other hand, fish that succumb to death in a more sub-acute nature presented erratic swimming, anorexia, exophthalmia and areas in the skin with petechiae and purpura (mainly near the opercula and fins). Upon examination of the internal organs, the most consistent findings were the presence of a bloody ascites in some affected fish and the presence of petechiae in liver, intestines, and stomach. Histopathological analysis of dead and moribund fish revealed similar changes in all fish examined. Typically there were numerous free coccoid bacteria present in multiple tissues; particularly within blood vessels, in the loose connective tissues of the cranium, the falciform process of the eye, the lamina propria of the intestine, and in gill lamellar blood spaces and filament interstitium. Large numbers of bacteria were present in numerous macrophages in multiple organs, particularly in the choroid rete, spleen, pseudobranchs, pericardium (especially the bulbus arteriosus), and meninges. Although typically within and expanding vascular spaces, macrophages also formed loose aggregates associated with foci of necrosis and hemorrhage; such changes were common in the dorsal pharynx. There was moderate to severe hepatic lipidosis with mild to moderate multifocal hepatocellular necrosis and hemorrhage, particularly in heavily bacteremic specimens.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Streptococcus agalactiae (also known as Group B Streptococcus or GBS) is a gram positive coccus that has a wide host range including warm blooded tererestrial animals as well as cold blooded aquatic animals. It is well known as a serious pathogen of Fundulus grandis (cocahoe minnow) when held in aquaculture or bait shop settings. We examined Lancefield serogroup B Streptococcus isolates recovered from diseased hybrid striped bass (Morone saxatilis x M. chrysops) and cocahoe minnow (Fundulus grandis) from mariculture facilities located in the coastal waters of the U.S. Gulf of Mexico (Gulf Coast), and compared them to strains from tilapia (Oreochromis sp.) reared in Thailand, Ecuador, and Honduras. The isolates were subjected to phylogenetic, biochemical and antibiotic susceptibility analysis. Genetic analysis was performed using partial sequence comparison of the 16S rRNA gene, sipA, encoding a surface immunogenic protein, cspA, encoding a cell surface-associated protein and secY, encoding components of a general protein-secretion pathway. The gulf coast isolates belong to a unique clade which can be differentiated from the other strains from south Asia and the middle east. Challenge models utilizing both immersion and injection exposure were were developed for evaluating mutant strains of the pathogen in Fundulus grandis as potential vaccines. The challenge model may be used to evaluate virulence of different strains of the pathogen in the future. PARTICIPANTS: Dr. John Hawke and graduate student Rui Wang. Department of Pathobiological Sciences, LSU School of Veterinary Medicine. TARGET AUDIENCES: Researchers interested in pathogenesis of Streptococcus agalactiae and researchers interested in diseases of Fundulus grandis. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Attempts were made to mutate the cspA gene of Streptococcus agalactiae strain ST81. Plasmid pSTCDE was constructed as an integration plasmid for cspA gene mutation. A partially deleted cspA gene containing an erythromycin resistance cassette was carried by pSTCDE, a temperature-sensitive plasmid originated from plasmid pJL1055. This plasmid served as a shuttle/suicide vector and was electroporated into GBS strain ST81. The resulting GBS ST81cells carrying pSTCDE were screened for single and double crossovers. The double crossover event should result in a mutated cspA gene in the chromosome. Single crossover integration into GBS chromosome was achieved by making dilutions of the overnight GBS cell culture and increasing the temperature to 37˚C (nonpermissive temperature). Double crossover integration into the GBS chromosome will be done by adjusting the temperature back to 28˚C (permissive temperature) and then replicate plating at 37˚C on media contain erythromycin. The mutant strains produced will be evaluated for attenuation in a fish model of infection using cocahoe minnows.
Publications
- No publications reported this period
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