Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, in the People’s Republic of China and in the United States, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish. Multiple lines of evidence indicate that US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila and 26 were epidemic strains. Phylogenetic analyses indicate that all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci were identified that are unique within A. hydrophila as well as genes that are present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates supports that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than with other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes revealing that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Background: Hypervirulent Aeromonas hydrophila (vAh) is an emerging pathogen in freshwater aquaculture that results in the loss of over 3 million pounds of marketable channel catfish, Ictalurus punctatus, and channel catfish hybrids (I. punctatus, ♀ x blue catfish, I. furcatus, ♂) each year from freshwater catfish production systems in Alabama, U.S.A. vAh isolates are clonal in nature and are genetically unique from, and significantly more virulent than, traditional A. hydrophila isolates from fish. Even with the increased virulence, natural infections cannot be reproduced in aquaria challenges making it difficult to determine modes of infection and the pathophysiology behind the devastating mortalities that are commonly observed. Despite the intimate connection between environmental adaptation and plastic response, the role of environmental adaption on vAh pathogenicity and virulence has not been previously explored. In this study, secreted proteins of vAh cultured as free-living planktonic cells and within a biofilm were compared to elucidate the role of biofilm growth on virulence. Results: Functional proteolytic assays found significantly increased degradative activity in biofilm secretomes; in contrast, planktonic secretomes had significantly increased hemolytic activity, suggesting higher toxigenic potential. Intramuscular injection challenges in a channel catfish model showed that in vitro degradative activity translated into in vivo tissue destruction. Identification of secreted proteins by HPLC-MS/MS revealed the presence of many putative virulence proteins under both growth conditions. Biofilm grown vAh produced higher levels of proteolytic enzymes and adhesins, whereas planktonically grown cells secreted higher levels of toxins, porins, and fimbrial proteins. Conclusions: This study is the first comparison of the secreted proteomes of vAh when grown in two distinct ecological niches. These data on the adaptive physiological response of vAh based on growth condition increase our understanding of how environmental niche partitioning could affect vAh pathogenicity and virulence. Increased secretion of colonization factors and degradative enzymes during biofilm growth and residency may increase bacterial attachment and host invasiveness, while increased secretion of hemolysins, porins, and other potential toxins under planktonic growth (or after host invasion) could result in increased host mortality. The results of this research underscore the need to use culture methods that more closely mimic natural ecological habitat growth to improve our understanding of vAh pathogenesis.

Hypervirulent Aeromonas hydrophila (vAh) is an emerging pathogen in freshwater aquaculture systems. In the U.S.A., outbreaks of motile aeromonad septicemia associated with vAh result in the loss of over 3 million pounds of channel catfish from Southeastern production systems each year. A. hydrophila is a well-known opportunistic pathogen that secretes degradative and potentially toxigenic proteins, and the rapid mortality that occurs when catfish are challenged with vAh by intraperitoneal injection suggests that vAh-induced motile aeromonad septicemia may be, in part, a toxin-mediated disease. While vAh isolates from carp isolated in China possess complete Type I, Type II, and Type VI secretion systems, many of the US catfish isolates only possess complete Type I and Type II secretions systems. In order to determine the role of secreted proteins in vAh-induced disease, and to determine the extent of protein secretion by the Type II secretion pathway, an exeD secretin mutant was constructed using a recombineering method in the well-characterized US vAh strain, ML09-119. Wild-type and mutant secretomes were analyzed for protein content by SDS-PAGE and by assays for specific enzymes and toxins. Type II secretion-deficient mutants had a near complete loss of secreted proteins and enzyme/toxin activity, including hemolytic and proteolytic activity. The intact Type II secretion system was cloned and used to complement the deletion mutant, ML09-119 exeD, which restored protein secretion and the degradative and toxigenic potential. In vivo challenges in channel catfish resulted in complete attenuation of virulence in ML09-119 exeD, while the complemented mutant was observed to have restored virulence. These results indicate that secreted proteins are critical to vAh virulence, and that the Type II secretion system is the primary secretory pathway utilized for multiple effectors of vAh pathogenesis.

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An adult female osprey (Pandion haliaetus) was found weak and unable to fly in Auburn, Alabama in August 2019. The bird was captured and submitted to the Southeastern Raptor Center of the Auburn University College of Veterinary Medicine for evaluation. On presentation, the bird was thin with a body condition score of approximately 1.5 out of 5. The bird died during the examination and was submitted for necropsy. At the necropsy, there was a severe loss of muscle mass over the body, and the keel was prominent. The liver and spleen were moderately enlarged with pale tan to red foci randomly scattered throughout the parenchyma. A histopathologic observation revealed multifocal to coalescing areas of necrosis and hemorrhage with intralesional protozoans in the liver, spleen, lungs, kidney, sciatic nerve, esophagus, cerebrum, heart, and proventriculus. Immunohistochemistry using anti-Toxoplasma gondii-specific antibodies showed a strong positive labeling of the parasite. Semi-nested PCR, specific for the B1 gene of T. gondii, successfully identified T. gondii. This is the first confirmed case of T. gondii infection in an osprey.