Experience of fish mortalities caused by epidemic clonal strains of Aeromonas hydrophila carrying a lysogenic bacteriophage Wes Baumgartner*, Mark Liles, Mark L. Lawrence* *Mississippi State University College of Veterinary Medicine
Motile aeromonads • A group of loosely associated Aeromonas species with similar biochemistry, genetics, and serology. • Optimal growth temperature of 35 o C to 37 o C. • Commonly associated with disease in warmwater fish. • A. hydrophila was the first motile aeromonad species named.
Aeromonas hydrophila • A. hydrophila is found in almost all freshwater environments; sediment, domestic tap water, sewage, and as normal flora on the skin and intestines of fish. • Therefore, the presence of A. hydrophila by itself is not indicative of disease in fish. • Diseases attributable to A. hydrophila have been reported in channel catfish (Ictalurus punctatus), minnows and baitfish, carp ( Cyprinus carpio ), gizzard shad ( Dorosoma cepedianum ), striped bass ( Morone saxatilis ), largemouth bass ( Micropterus salmoides ), and tilapia.
Motile aeromonad septicemia • Disease caused by A. hydrophila and other motile aeromonads -Motile Aeromonad Septicemia (MAS)- characterized by a widespread systemic infection. • Fish typically exhibit epidermal hemorrhage, ulceration, and soft tissue necrosis, severe periophthalmic cellulitis with exophthalmia, iridial hemorrhages, and swollen abdomens. • Skin lesions start as irregularly-shaped depigmented areas that eventually ulceate, exposing muscle and bone. Internal signs include generalized hyperemia and • petechiation, renomegaly, splenomegaly, mottled livers, cloudy and bloody ascites, and flaccid intestines with intramural hemorrhage
A. hydrophila is an opportunistic pathogen • Disease potential is based on complex interactions between multiple biotic (host and bacterium) and abiotic (climate, water chemistry, etc.) factors. • The strains of A. hydrophila commonly found on the skin and intestines of healthy fish are considered opportunistic. • Disease occurs secondarily to preexisting diseases, weakened immune systems, injury, or following periods of low oxygen, high ammonia, extreme temperatures, or other forms of stress (crowding). • Diseased fish are often infected with other bacteria such as Flavobacterium columnare and Edwardsiella ictaluri .
Epidemic A. hydrophila (EAh) • Outbreaks of EAh started in West Alabama in April 2009, and they continued through September. • Disease was characterized by acute onset of anorexia followed by high mortality rates. • Affected fish had clinical signs typical of a systemic bacterial infection, but distinct from E. ictaluri infection. • Pure isolates of A. hydrophila were grown-BBL crystal and API testing found differences from typical A. hydrophila • Mortality in ponds ranged up to 50-60%, and predominantly marketable size fish were affected.
Epidemic A. hydrophila (EAh) • Histopathology- consistent lesions in clinically diseased fish were limited to the gastrointestinal tract – The entire length affected, varying severity – Acute epithelial necrosis with mucosal sloughing – Small numbers of inflammatory cells in the lamina propria – Sub-epithelial, large macrophages with abundant proteinaceous and karyorhectic ingested material – Few bacteria seen-morphology consistent with A.hydrophila – Suspect bacterial enterotoxin
Impact on catfish aquaculture • In 2009, EAh was documented on at least 48 farms with an estimated loss of 3,184,000 lbs. of catfish. • In spring and summer of 2010, the disease reemerged and spread to at least 60 farms (including the 48 affected in 2009) with an estimated loss of 2,400,000 lbs. of catfish. • Relative to 2009, there was a 25% increase in the number of infected farms in 2010 over 2009, but 20% of the farms infected in 2009 did not have a reoccurrence in 2010. • Data from 2011 and 2012 are also similar to 2010, with a loss of over 2 million lbs. of catfish per year
Importance of biosecurity • Cases of EAh have occurred in east Mississippi, but the disease is currently not active in the delta of Mississippi and Arkansas. • Contaminated fish, water, seining and hauling equipment are good sources for spreading this disease. • Implementation of biosecurity measures to stop the spread of EAh, which requires a reliable detection method, is critical to prevent further spread of the disease in the west Alabama/east Mississippi region and keep it from entering the Mississippi delta and Arkansas
Formation of a multistate research group • Due to the severity of outbreaks in 2009, fish health diagnosticians and researchers began communicating and sharing information on the disease in summer 2009. • Participants in the Aeromonas working group were located at Auburn University, Mississippi State University (College of Veterinary Medicine in east Mississippi and Thad Cochran National Warmwater Aquaculture Center in the Mississippi delta), University of Arkansas-Pine Bluff, Louisiana State University, and the USDA ARS.
Primary or secondary pathogen? • Because A. hydrophila is considered a secondary pathogen, initial efforts focused on identification of an underlying primary pathogen. • Through cooperative efforts, the group ruled out underlying primary bacterial disease agents (including known pathogens such as E. ictaluri and unknown anaerobes or obligate intracellular bacteria), viruses, and parasites. • Visceral toxicosis (caused by Clostridium botulinum toxin) was ruled out. • Host genetic factors were ruled out.
Primary or secondary pathogen? • Preliminary studies using culture supernatant were unsuccessful in reproducing the disease. • Immersion and bacterial laced feed also unable to cause disease. • Intraperitoneal injections can reproduce disease-unsatisfactory method.
EAh is a clonal group • An emerging primary pathogen of catfish? • EAh strains from channel catfish had been previously typed as A. hydrophila based on biochemistry (e.g., API 20E assay) and by 16S rRNA gene sequencing (100% identity to known A. hydrophila strains). • Using phylogenetic analysis based on the DNA gyrase B-subunit gene ( gyrB ), all of the recent epidemic strains of A. hydrophila were grouped together as a single clade that could be distinguished from all other A. hydrophila . • Phenotypic characterizations by API 20E assay also indicated that the epidemic A. hydrophila had a unique biochemical profile (EAh isolates ferment inositol).
• Concatenated phylogeny based on seven evolutionarily conserved gene sequences also demonstrated the highly clonal nature of these recently emerged epidemic A. hydrophila isolates. Concatenated phylogeny of A. hydrophila based on seven gene sequences ( atpD , dnaJ , dnaX , gyrA , gyrB , recA , and rpoD ) from representative strains, based on a Maximum Parsimony analysis with 1000 iterations for bootstrap support (indicated at each node).
EAh is more virulent in channel catfish Percent survival of channel catfish or grass carp after intraperitoneal injection of 1x10 5 CFU of the EAh strain ML09-119 or RAh strain AL06-06 (n=5 tanks containing 20 fish per treatment group).
Comparative genomics • A. hydrophila isolates (n=7 epidemic A. hydrophila (EAh); n=5 historical "reference" A. hydrophila strains (RAh)) were selected for bar-coded sequencing using an Illumina Genome Analyzer IIx next-generation sequencer (Illumina, Inc., San Diego, CA). • Genome sequences from EAh strain ML09-119 were assembled against the only fully sequenced A. hydrophila genome in GenBank (ATCC 7966 T ). • All sequences that did not assemble against this reference genome were then de novo assembled to identify contiguous regions (contigs) of the ML09-119 genome that were not in common with the ATCC 7966 T strain genome.
Comparative genomics • The set of contigs that were unique to strain ML09-119 were used as the reference genome for assembly of each of the other 11 sequenced A. hydrophila genomes. • This assembly revealed that all of the other six EAh strains shared this genomic region(s) in common with ML09-119, whereas the reference A. hydrophila genomes were highly variable and lacked many of the gene sequences present in the EAh strain. • 54 ML09-119-unique contigs were found that had >99% identity among all seven EAh strains.
Comparative genomic analysis of a contig from EAh strain ML09-119 against EAh strain AL09-79 (outer ring) and two RAh strains (inner rings) along with %GC content. Graph represents BLASTn comparison using CGView (Grant & Stothard, 2008).
• ORF analysis of the 54 epidemic-associated unique genetic regions revealed 307 predicted genes that are present in EAh isolates but absent from RAh isolates. • 35% of the unique genes are located within predicted genomic islands, suggesting their acquisition through lateral gene transfer. • BLAST analysis of EAh-associated ORFs: – Numerous predicted genes involved in inositol metabolism (responsible for unique biochemical profile) – Prophage structure and regulation – Transposases – Other genes with low percent similarity to known A. hydrophila gene sequences.
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