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 35o C to 37o 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 (Moronesaxatilis), 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 Edwardsiellaictaluri.

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. ictaluriinfection.

• 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. ictaluriand 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 gyraseB-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 1x105 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 7966T).

• 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 7966T

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. hydrophilagenomes.

• This assembly revealed that all of the other six EAhstrains shared this genomic region(s) in common with ML09-119, whereas the reference A. hydrophilagenomes 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.

Phenotypic differentiation of EAh

• EAh isolates were capable of good growth in a M9 minimal medium containing only inositol as a sole carbon source.

• The differential growth of EAh and RAh strains on a M9 minimal medium with inositol was consistently observed for all sequenced A. hydrophila strains, as well as for A. hydrophila strains isolated from diseased fish in ponds experiencing a MAS epidemic (n=129).

• The West Alabama Fish Farming Center (W. Hemstreet, director) has used this low-cost assay since the summer of 2012 for diagnostic differentiation of EAhversus environmental A. hydrophila isolates.

Unique O antigen

• A. hydrophila ML09-119 along with all other sequenced EAh strains contain a unique 33 kb O-antigen biosynthesis gene cluster.

• There are 29 total predicted genes within this cluster, all organized in the same transcriptional orientation, suggesting that this is an O-antigen biosynthesis operon.

• Genes required for biosynthesis of the building blocks of surface polysaccharides are often found clustered in bacteria.

• 24 of the 29 genes do not have any similarity to known A. hydrophila genes.

Unique lysogenic prophage

• More than 20 prophage-linked open reading frames (ORFs) were identified.

• Only three of these ORFs had any known affiliation with any previously described Aeromonas phage, suggesting that this prophage may be a novel Aeromonas phage.

• Twenty-eight of the genes encoded in these regions were similar to known virulence factors in other species, and several putative virulence loci (including the myo-inositol utilization pathway) appear to be linked to prophage elements within the EAh genome.

Genetic differentiation of EAh

• The set of consensus sequences present in all of the sequenced EAh strains was used to develop primer sets that were specific to these strains and that would be useful for diagnostic and epidemiological studies.

• Eah-specific sequences met the following criteria:

– 1) present in each sequenced EAh strain

– 2) did not have significant homology to known genes in GenBank

– 3) did not have an overly high or low %G+C content

– 4) were sufficiently long enough (>500 bp) for development of primer sets.

Diagnostic PCR for EAh

• Selected primer sets had an approximate Tm of 60oC and an expected PCR product length of 300 bp.

• The primer sequences were evaluated by diagnostic laboratories in MS, AL, and AR.

• Three primer sets were selected as providing the best specificity and robust PCR for known EAh strains and not giving any significant amplification for environmental or reference strains.

• The most promising primers have been refined to provide even better results in real-time PCR assays

Summary

• 1) a unique prophage is ubiquitous among EAhstrains

• 2) this prophage is not present within reference A. hydrophila strains

• 3) there is physical linkage between virulence associated-ORFs and this prophage region.

• In conclusion, lateral gene transfer has contributed to the pathogenicity of epidemic A. hydrophila strains, mediated by lysogenic conversion by a temperate phage.

Summary

• In addition to prophage, EAh has unique O

antigen biosynthetic genes.

• EAh has unique inositol metabolism genes and

can be phenotypicallly differentiated based on

inositol utilization.

• Using comparative genomics, a diagnostic PCR

assay was developed to differentiate EAh from

environmental or reference A. hydrophila.