Virulent Aeromonas hydrophila (vAh) is a causative agent of motile Aeromonas septicemia (MAS) in catfish. There are limitations in the current therapeutic and preventative strategies against vAh. The pathogenesis of MAS as well as the immune response of catfish to vAh infection are poorly understood. The aim of this study is to: 1) develop a dual live attenuated vaccine against MAS and enteric septicemia of catfish caused by Edwardsiella ictaluri; and 2) evaluate the vAh bacterial load and gene expression patterns in catfish tissues following vAh infection. Previously, six recombinant vAh proteins (outer membrane protein, TonB-dependent receptor, three fimbrial proteins, and an ATPase) were identified to have vaccine efficacy against MAS, and live attenuated E. ictaluri vaccine strain ESC-NDKL1 was identified as an effective vector for expressing vAh antigens. A total of 29 recombinant ESC-NDKL1 strains have been constructed with the integration of one, two, or three genes encoding vAh antigens into the ESC-NDKL1 chromosome. Vaccine efficacy of the constructed strains was evaluated in channel catfish fingerlings. Four recombinant ESC-NDKL1 strains expressing two vAh antigens (ESC-NDKL1::atpase::fimMrfG, ESC-NDKL1::fim::fimMrfG, ESC-NDKL1::tdr::fimMrfG, and ESC-NDKL1::fim::ompA) showed significant protection against MAS with relative percent of survival (RPS) values of 55.72%, 60.18%, 61.74%, and 54.81%. Four triple recombinant ESC-NDKL1 strains (ESC-NDKL1::fimMrfG::ompA::fimA, ESC-NDKL1::atpase::fimMrfG::ompA, ESC-NDKL1::fim::fimMrfG::ompA and ESC-NDKL1::atpase::tdr::fim) showed the best protection with RPS values of 77.93%, 63.18%, 67.74%, and 82.35%. To gain a better understanding of vAh pathogenesis, catfish fingerlings were intraperitoneally injected with vAh strain ML09-119. The anterior kidney, liver, and spleen were collected for determination of vAh distribution and expression of thirteen pro-inflammatory, innate, and adaptive immune-related genes using real-time PCR. Results revealed that vAh spread into catfish tissues within 2 hours and peaked at 12 hours post-infection. vAh infection initiated a strong inflammatory response in catfish tissues. Additionally, our research revealed that surviving catfish were able to develop a primary immune response and possibly generation of memory B cells against MAS. Such information will facilitate the development of vaccines and therapeutic drugs for preventing and treating MAS outbreaks in catfish aquaculture.

Channel catfish farming is the largest aquaculture industry in the U.S., and virulent Aeromonas hydrophila is an important re-emerging bacterial pathogen of farmed catfish. A. hydrophila is ubiquitous in the aquatic environment and causes motile Aeromonas septicemia (MAS) in farmed catfish. A. hydrophila infection is challenging to treat due to rapidly progressive mass mortalities. Although florfenicol has been used in managing outbreaks, acute onset of anorexia, drug cost, and emergence of antimicrobial-resistant strains are significant concerns. Prevention of MAS by vaccination could be a promising approach, but a commercial vaccine is not available. Thus, it is critical to develop effective vaccines against A. hydrophila to prevent losses in the catfish industry. The overall objective of this study is to develop effective live attenuated A. hydrophila vaccines and understand macrophage-A. hydrophila interactions. To achieve this, potential virulence-related genes from T6SS, secretion pathway, tat pathway, and flagellar system were deleted by in-frame, and mutants’ virulence and protection were evaluated in channel catfish. Also, macrophage-A. hydrophila interactions were explored by studying global gene expression to understand macrophage responses to A. hydrophila and A. hydrophila virulence mechanisms

Yes / Aeromonas hydrophila has emerged worldwide as a human pathogen. Here, we report the draft whole-genome sequence of a freshwater isolate from Malaysia, A. hydrophila strain M062, and its N-acylhomoserine lactone genes are also reported here. / University of Malaya via High-Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01, no. A-000001- 50001), and aUM-MOHEHIR grant (UM C/625/1/HIR/MOHE/CHAN/ 14/1, no. H-50001-A000027)

Aeromonas hydrophila is an opportunistic pathogen that causes a wide range of symptoms and diseases in fish. Development of a commercial vaccine has been problematic due to the heterogenicity between isolates of A. hydrophila. A new approach using immunoproteomics was used in this study to try to develop a vaccine that would protect against a wide range of A. hydrophila strains. The virulence of 14 isolates of A. hydrophila from different geographical regions was determined in common carp (Cyprinus carpio) indicating that 6 isolates were virulent, while 8 isolates were avirulent. Expression of cellular and extracellular products (ECP) of six of these isolates (4 virulent and 2 avirulent isolates) were examined following culture of the bacterium in vitro, in tryptic soy broth, and in vivo, in dialysis tubing placed within the peritoneal cavity of carp. Two types of molecular weight cut off tubes (25 and 100 kDa) were used for the implants. Whole cell (WC), outer membrane protein (OMP) and ECPs of the bacteria grown in vitro and in vivo were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations of A. hydrophila grown in vitro and in vivo. The production of unique proteins and up and down-regulation of protein expression were observed in all the preparations of bacteria grown in vitro and in vivo. Unique bands were seen in the 1D SDS-PAGE at 58 and 55 kDa for WC and OMP preparations, respectively, for all the isolates cultured in vivo. Bands of increased intensity were observed at 70, 55, 50 and 25 kDa with WC preparations for the virulent isolates cultured in vivo. Analysis of WC preparations by 2D SDS-PAGE indicated differences in the expression of spots between bacteria cultured in vitro and in vivo. A number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0-6.0 were observed in the bacteria grown in vivo. The protein profiles of different preparations (WC, OMP, ECP) of bacteria cultured in vitro and in vivo were screened by 1D Western blot using antibodies from carp artificially infected with different isolates of A. hydrophila to identify potential vaccine candidates. The WC preparations of A. hydrophila (T4 isolate) grown in vitro were also analysed by 2D Western blot. A 50 kDa protein of A. hydrophila was found to be the most immunogenic molecule in both WC and OMP of bacteria grown both in vitro and in vivo. The protection efficacy of this protein was determined in goldfish by vaccinating fish with electro-eluted 50 kDa protein then challenging the fish with A. hydrophila. Fish were also passively immunised with fish sera raised to the 50 kDa protein and then challenged. The relative percentage survival (RPS) was 67 % in the vaccination trial, while the results were inconclusive for the passive immunisation trial. The 50 kDa protein was confirmed to be the S-layer protein of A. hydrophila following identification using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Recombinant S-layer protein was then produced and the cross-protection efficacy of this protein against six virulent isolates of A. hydrophila was confirmed in a large scale vaccination trial using carp. The RPS value for the 6 isolates of A. hydrophila ranged from between 56 and 87 %. The results of this project suggest that the immunogenic S-layer protein of A. hydrophila could be used as a common antigen to protect fish against infection by different isolates of this pathogenic bacterium.

Microcystin-LR is a hepatotoxin produced by cyanobacteria. Aeromonas hydrophila and Edwardsiella piscicida infections are leading causes of losses in market-sized channel catfish (Ictalurus punctatus). These older fish should have natural immunity in place and a predisposing factor is likely a prerequisite for these disease outbreaks. While microcystin-LR rarely causes mortality in warm-water aquaculture, we believe it may be a predisposing factor that leads to bacterial disease outbreaks during the summer months due to its ability to damage the liver. Our study investigated microcystin-LR’s effects on channel catfish susceptibility to these pathogens. We found that a sublethal dose of microcystin-LR induced substantial damage to multiple immune organs. In our challenges with both the toxin and bacteria, we saw a significant increase in mortality of fish. Our findings suggest that microcystin-LR increases channel catfish susceptibility to Aeromonas hydrophila and Edwardsiella piscicida infections.

No description available.

Pike-perch (Sander lucioperca) is a species of fish whose breeding in recirculation systems has a great prospect in the years to come. Due to the unfavorable conditions of intensive rearing of these fish in recirculation systems, they are often exposed to stress resulting in reduced fish defenses. Thus, fish are exposed to attack of a wide range of bacteria. Although there are preventive measures to protect fish against pathogen attack, knowledge of the immune system and the immune response of the fish is crucial for further evolution of vaccination. Even though we know the mechanisms of immune response of many fish species,but the knowledge of the pike-perch's immune response is very limited.

Abstract Medicinal plants have been for long remedies for human diseases because they contain components of therapeutic value. The growing problem of antibiotic resistance by organisms demands the search for novel compounds from plant based sources. The present study was aimed at evaluating the bioactivity and phytochemical analysis of Hydnora africana on clinical and standard strains of Helicobacter pylori (PE 252C and ATCC 43526), Aeromonas hydrophila ATCC 35654, and Staphylococcus aureus NCT 6571 in an effort to identify potential sources of cheap starting materials for the synthesis of new drugs against these strains. Ethyl acetate, acetone, ethanol, methanol, and water crude extracts of H. africana were screened for activity against the test organisms using the agar well diffusion assay. The Minimum Inhibitory Concentration (MIC50) and Minimum Bactericidal Concentration (MBC) of the most potent extracts were determined by the microdilution method, followed by qualitative phytochemical analysis. Results were analyzed statistically by ANOVA one - way test. Different concentrations (200,100, 50mg/mL) of the methanol, acetone, ethanol and ethyl acetate extracts showed activity against S. aureus and A. hydrophila while for H. pylori, only methanol and ethyl acetate extracts were active; water showed no activity for all studied bacterial pathogens. Mean zone diameter of inhibition which ranged from 0-22mm were observed for all test bacterial pathogens and 14-17mm for ciprofloxacin. The activity of methanol and ethyl acetate extracts were statistically significant (P< 0.05) compared to all the other extracts. MIC50 and MBC ranged from 0.078 – 2.5mg/mL, 0.78-25mg/mL respectively for all tested bacterial pathogens. For ciprofloxacin, the MIC50 and MBC ranged from 0.00976 – 0.078mg/mL and 0.098– 0.78mg/mL respectively. There was no statistically significant difference between extracts (methanol, acetone, ethanol, ethyl acetate) and the control antibiotic (ciprofloxacin) (P> 0.05). Qualitative phytochemical analysis confirmed the presence of alkaloids, saponins, steroids, tannins and flavonoids in the methanol, acetone,ethanol and ethyl acetate extracts. The results demonstrate that H. africana may contain compounds with therapeutic potentials which can be lead molecules for semi-synthesis of new drugs.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

MSc (Microbiology) / Department of Microbiology / Background: The occurrence of microorganisms and endocrine disrupting chemicals (EDCs) in water poses a serious concern due to their effects on humans, animals and environment. In recent years, EDCs have been increasingly reported in rivers that receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic hormones are among those that are recognized for their potential to mimic or interfere with normal hormonal functions of humans and animals. The present study aimed at assessing the occurrence of these hormones in relation to the molecular diversity of Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to assess anti-bacterial activity of two selected traditional medicinal plants. Methods: Wastewater, water and fish samples were collected from various sources (rivers, wastewater treatment plants, taps, and dams) for the detection of hormones and isolation of Aeromonas species. The analysis of hormones from various organs of the fish and from water samples was conducted, after extraction using enzymelinked immunosorbent assays (ELISA). Different types of hormones including Estriol, Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations determined. Aeromonas spp were isolated rom the samples using microbiological methods and Conventional PCR was used for genotyping as well as for detection of the beta-lactamase genes. Kirby-bauer method was used to determine the susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia milneana) extracts against Aeromonas species. Results: A total of 144 samples were collected from 23 different locations in two countries: South Africa and Zambia. These included wastewater and treated wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65 ng/ml. The hormones were also detected from drinking water, with testosterone detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment plants were not able to remove the EE2 from the wastewater as the concentration of this hormone in the final effluent was almost always higher than that in the influent. These homones were also detected in drinking water at high concentrations of up to 53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were completely resistant to cefuroxime accounting for 100% resistance. Aeromonas isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila), cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A. veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic resistance was also observed with the Aeromonas isolates being resistant to up to 11 antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were the most predominant species in the presence of total estriol, A. veronii had a prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila and Aeromonas caviae had the lower prevalence in the presence of hormones with the percentages of 26.1% and 27.8% respectively. The methanol extracts of both Zornia milneana and Annona species showed good activity against the Aeromonas spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective. Conclusion: This study has shown high occurrence of steroid hormones in all types of environmental samples tested. These included tap water, river water, wastewater and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and important health problem in the Southern African Sub-Region. The incapacity of the wastewater treatment plants to remove EE2 is an important problem that needs to be tackled immediately. The prevalence of Aeromonas species is very high in our environmental water as well as in drinking water, with the highest prevalence observed in fish and wastewater. It was also revealed that there is relationship between steroid hormones and Aeromonas species, with the hormones supporting the growth of Aeromonas species. The presence of beta-lactamase genes which causes Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia milneana and Annona spp were the most effective against Aeromonas spp and could serve as primary sources for the isolation of lead compounds. / NRF