USDA-ARS - Aquatic Animal Health Research Unit
USDA-ARS - Aquatic Animal Health Research Unit
Program overview
Emily M. Churchman1, Miles D. Lange2, Nithin Muliya Sankappa2,3, Megan Justice2, Victoria L. Quiroz1, Jason Abernathy2, Mark R. Liles1
1Department of Biological Sciences, Auburn University, Auburn, AL, USA; 2United States Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, Auburn, AL, USA; 3Oak Ridge Institute for Science and Education (ORISE), ARS Research Participation Program,
Oak Ridge, TN, USA
Columnaris disease is one of the leading causes of mortality in channel catfish production. Flavobacterium covae, an etiological agent of columnaris disease, has shown to be highly virulent in channel catfish and is a major problem for the US aquaculture industry. Here, we test the immunogenicity and efficacy of several biofilm-associated F. covae proteins to be used as recombinant subunit vaccines. The genes encoding these predicted proteins were cloned into expression vector pET-28a(+). After expression in Escherichia coli strain BL21 (DE3), the antigens were purified under native conditions. Channel catfish were injected intraperitoneally with purified protein (20 ug/mL), and peripheral blood was collected 30 days post-vaccination. Preliminary data shows vaccinated fish exhibited sera IgM antibody specificity to the respective antigens when blotted to the reduced proteins and had an increase of sera IgM antibodies. In our next trial, groups of fish (n=540) were immunized by bath immersion with the recombinant protein(s) (1 ug/ml) or sham immunized. There was no significant mucosal IgM antibody production among the vaccinated groups. However, each vaccinated group showed significant survival when challenged with F. covae (>30% compared to the control group) at nine weeks post-immunization. Significant upregulation of innate and adaptive and immune genes was seen in vaccine groups compared to baseline gene regulation in the sham immunized group. The efficacy of the vaccine(s) delivered orally via top-coating feed will be discussed as well. These results lay the groundwork for potential vaccine candidates to protect farmed fish against columnaris disease during the production cycle.
Yesutor K. Soku1, Miles Lange2, Craig Shoemaker2, Jason Abernathy2, Nithin M. Sankappa2, Megan Justice2, Emily M. Churchman3, Victoria L. Quiroz3, Abdelrahman Mohamed1
1 College of Veterinary Medicine, Tuskegee, University, Tuskegee, AL, 36088; 2 Aquatic Animal Health Research Unit, USDA-ARS, Auburn, AL, 36832; 3 School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849
Virulent Aeromonas hydrophila (vAh) is documented as the main pathogen in recent motile Aeromonas septicemia (MAS) outbreaks in commercial-sized channel catfish (Ictalurus punctatus) and hybrid catfish (Ictalurus punctatus X I. furcatus). In 2021, over 2 million pounds of catfish were lost to vAh in West Alabama alone, and at current live fish prices, the estimated monetary loss is roughly US $2.6 million annually. Quantifying bacterial cells within MAS-infected channel catfish reveals that one of the tissues with the highest recovery of vAh is the intestines, inferring that the gut may play a vital role in the pathogenesis of MAS. Additionally, field reports suggest that mortality is often observed after feeding catfish. Therefore, using an existing bath immersion challenge model with fin clipping, we investigated the dynamics of MAS infection by investigating the role of breaking skin integrity (fin-clipping) and dietary status (feeding) in the disease process. 425 channel catfish (mean weight = 30g) were challenged with ALG-15-097 strain of vAh at a concentration of 1.5×107 CFU/mL in 4 treatment groups. Group 1 was fin clipped and fed (FCF, n=100), group 2 was not fin clipped but fed (NCF, n=100), group 3 was fin clipped but not fed (FCN, n=100), and group 4 was not fin clipped and not fed (NCN, n=100), and the control (FCF and NCF, n=25) was challenged with sterile tryptic soy broth. Groups 1 and 2 were fed on a commercial diet two hours before the challenge at 3% of the mean weight. The catfish were anesthetized with MS-222 at 150mg/L. Fin-clipped treatments had their adipose fin cut off with scissors before being placed in flow-through tanks. “No fin-clipped fish” were placed in the tanks after anesthesia. The mortalities were recorded at 0, 1, 2, 4, 6, 8, 12, 20, 24, 36, 48 and 72h. The intestines and stomach were sampled at times points 2, 4, and 8 hours for histopathology using a similar setup. After 72 h post-challenge, we observed a 30%, 38%, 23%, and 55% survival rate in groups 1 (FCF), 2 (NCF), 3 (FCN) and 4 (NCN) respectively. Overall, statistically significant differences in survival were observed between treatments, except between groups 1 (FCF) and 3 (FCN), where both groups were fin-clipped, suggesting that a break in skin integrity is detrimental to the survival of channel catfish in MAS infections. The major histological lesions revealed include ulceration, inflammatory infiltration, hemorrhage, epithelial necrosis, and edema. A semiquantitative grading system of the histopathological lesions showed statistically significant differences between the control and the fed groups (FCF and NCF) only after 4 hours. This study reveals that significant pathological lesions become apparent at 4 hours leading up to mortalities within 12 hours post-infection, probably due to blood rush to the gut region to aid in feed digestion in catfish that were fed. However, the absence of feed in injured catfish before infection delays mortalities but becomes marked after 12 hours post-infection.
Nithin Muliya Sankappa
Oak Ridge Institute for Science and Education (ORISE), ARS Research Participation Program, Oak Ridge, TN, 37830, USA; USDA-ARS Aquatic Animal Health Research Unit (AAHRU), 990 Wire Road, Auburn, AL 36832, USA
Numerous farmed fish species, such as carps, tilapia, salmon, and catfish, have faced considerable economic losses in aquaculture due to motile Aeromonas septicemia caused by Aeromonas hydrophila. Phages infecting hypervirulent Aeromonas hydrophila (vAh) had not been isolated anywhere in the world; however, phages have been isolated against motile Aeromonas septicemia (MAS) with only few of them having been characterized. This study aimed at developing therapeutic phage against hypervirulent Aeromonas hydrophila infection in aquaculture systems. A total of 110 water samples were collected aseptically. A novel phage AhFM11 specific to hypervirulent A. hydrophila was isolated and shows lytic activity against reference A. hydrophila (ATCC 35654). Soft agar overlay method was used to determine titer and found to be 1.58 x 1010 pfu/mL. Host range of the AhFM11 phage was performed for 131 Aeromonas spp. and 10 non Aeromonas. The results indicated that AhFM11 had a broad host range, infecting 65 Aeromonas species. Also, it was found that this phage did not harbor any antibiotic resistance genes. We isolated and characterized a novel lytic bacteriophage, AhFM11, which infects hypervirulent Aeromonas hydrophila (vAh). This is the first documented instance of a phage against vAh. Phage AhFM11 showed lytic activity against both vAh strains and the A. hydrophila reference strain ATCC 35654. The genome of AhFM11, consisting of 168,243 bp with an average G+C content of 41.5%, was sequenced and assembled, revealing no antibiotic resistance genes. Genomic data and transmission electron microscopy classified AhFM11 as belonging to the Straboviridae family. Therapeutic application of AhFM11 in fish resulted in 100% survival following injection, 95% survival via immersion, and 93% survival through oral administration with phage-coated feed. These results suggest that phage AhFM11 could serve as an effective biocontrol agent against vAh, offering an alternative to antibiotics in aquaculture.