Differential production and secretion of potentially toxigenic extracellular proteins from hypervirulent Aeromonas hydrophila under biofilm and planktonic culture.

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.

Disseminated Toxoplasma gondii Infection in an Adult Osprey (Pandion haliaetus).

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.

Type II Secretion Is Essential for Virulence of the Emerging Fish Pathogen, Hypervirulent Aeromonas hydrophila.

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.

Emphysematous gastritis in a cat.

OBJECTIVE: To describe the presentation, diagnosis, treatment, and outcome of a case of emphysematous gastritis (EG) in a cat. CASE SERIES SUMMARY: A 15-year-old female neutered domestic short-hair cat presented for a 4-month history of weight loss and intermittent vomiting. Clinicopathologic and imaging findings suggested an underlying primary gastrointestinal (GI) disease, as well as possible hepatobiliary disease. Two days following exploratory laparotomy to obtain GI and liver biopsies, the patient became septic and intracellular bacteria were present on cytology of peritoneal effusion. On radiographs, the stomach was markedly distended with fluid and contained a thin gas opacity surrounding the stomach wall. The patient was taken back to surgery to identify a source of sepsis. At surgery, the patient's stomach was firm and emphysematous on palpation but grossly appeared normal. There were no signs of dehiscence of the previous biopsy sites. Stomach biopsy confirmed the presence of intralesional Gram-positive rods, consistent with microbial EG, and a light growth of a Clostridium sp. was cultured from abdominal fluid, consistent with clostridial peritonitis. During a third surgery for suspected septic peritonitis, a jejunostomy tube was placed for postgastric enteral feeding. The patient ultimately survived to discharge and is clinically stable 10 months later. NEW/UNIQUE INFORMATION PROVIDED: EG is a rare but potentially fatal clinical entity in the human and veterinary literature with only 1 other case reported in cats. Though clostridial organisms have been reported in EG in people, this is the first implication of EG secondary to a Clostridium sp. in the cat. This is also the first report to document the use of a jejunostomy tube for postgastric enteral nutrition to treat EG in the veterinary literature.

Classification of a Hypervirulent Aeromonas hydrophila Pathotype Responsible for Epidemic Outbreaks in Warm-Water Fishes.

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, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate 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, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion 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 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. These studies revealed 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.

Detection and quantification of virulent Aeromonas hydrophila in channel catfish tissues following waterborne challenge.

The aim of this study was to understand the pathogenesis of motile aeromonas septicemia caused by an emergent, high virulent Aeromonas hydrophila (vAh) in channel catfish, Ictalurus punctatus Adipose fin clipped catfish were challenged with vAh using a waterborne challenge method, and the distribution of vAh over a time course was detected and quantified using real-time polymerase chain reaction. The results showed that 77.8% of fish died within 48 h post challenge with mean day to death of 1.5 days. At 2 h post challenge, vAh (inferred from genomic DNA copies or genome equivalents) was detected in all external and internal tissues sampled. Gill had the highest vAh cells at 1 h post challenge. Spleen harbored the most vAh cells among internal organs at 4 h post challenge. The tissues/organs with most vAh cells detected at 8 h post challenge were adipose fin, blood, intestine, kidney and skin, while liver showed the highest vAh cells at 24 h post challenge. These results suggest that vAh was able to rapidly proliferate and spread, following wound infection, through the fish blood circulation system and cause mortality within 8-24 h.

Oxidative stress responses of gulf killifish exposed to hydrocarbons from the Deepwater Horizon oil spill: Potential implications for aquatic food resources.

Ecosystem effects of polycyclic aromatic hydrocarbons (PAHs) remain under investigation following the Gulf of Mexico Deepwater Horizon oil spill. Fundulus grandis, an established indicator of aquatic ecosystem health, was investigated because this species shares genes and biochemical pathways with higher trophic-level fish and plays an important role in the gulf food chain. Oxidative stress responses including hepatic cytochrome P4501A (CYP1A) and serum antioxidant capacity were evaluated in fish exposed to PAHs. Fish were exposed to water-accommodated fractions (WAFs) of crude oil (7.0 ± 0.10 mg/L C6-C28) after which solutions were diluted below the level of detection over 8 h using 15 ppt aerated artificial seawater. Before euthanasia, fish remained in aquaria for 12 h, 24 h, or 48 h. Three replicate experiments were conducted at each time point using unexposed fish as experimental controls. Significant differences (p < 0.05) in CYP1A induction were observed in exposed versus control fish at 24 h. Expression of CYP1A increased by 25%, 66%, and 23% in exposed fish at 12 h, 24 h, and 48 h, respectively. Significant increases were observed in antioxidant capacity of nonenzymatic antioxidants in exposed versus control fish at each time point. Given the activity of CYP1A, radicals formed during PAH detoxification likely resulted in increased oxidant load requiring elevated antioxidant defenses. Research is needed to determine the duration of oxidative stress responses considering the potential for lipid oxidation in exposed fish or species feeding on exposed fish.

Implication of lateral genetic transfer in the emergence of Aeromonas hydrophila isolates of epidemic outbreaks in channel catfish.

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.

Identification of Bacillus strains for biological control of catfish pathogens.

Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7) CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.

Multifocal cutaneous histiocytic sarcoma in a young dog and review of histiocytic cell immunophenotyping.

A 9-month-old male Great Dane had progressive generalized nodular dermatopathy for several months. There were > 100 raised, alopecic, firm, painful nodules throughout the skin. Aspirates from several lesions yielded moderate numbers of irregularly round or polygonal to spindle-shaped cells with mild to moderate anisocytosis and few inflammatory cells, and the cytologic interpretation was proliferation of mesenchymal or histiocytic cells. On histopathologic examination, nodules were composed of densely packed sheets of round to spindle-shaped cells with mild anisokaryosis and low mitotic activity. Multifocal histiocytic sarcoma with a spindle-cell pattern was diagnosed based on morphologic features and intense expression of CD18. Additional immunophenotypic analysis on frozen sections of tissue confirmed the diagnosis of histiocytic sarcoma; expression of CD18, CD45, CD1a, CD11b, and CD11c, limited expression of Thy-1 (CD90) and CD80, and lack of expression of CD4, CD11d, and CD86 indicated that the cells were likely interstitial dendritic cells; a review of reactive and neoplastic dendritic cells is provided. Based on staging, internal organs were not affected. Sequential treatment with lomustine and doxorubicin failed to prevent progression of the cutaneous lesions, and the dog died 3 months after initial diagnosis. At necropsy, a focus of neoplastic cells was present in one lymph node, but except for skin other organs were not involved. The clinical presentation of histiocytic sarcoma may be unusual, and neoplastic cells may lack overt features of malignancy on cytologic and histopathologic examination. In some instances, immunophenotyping is required to differentiate histiocytic sarcoma from other histiocytic disorders.

Suspected systemic calcinosis and calciphylaxis in 5 horses.

Five horses were presented with signs of myopathy along with systemic malaise, hyperfibrinogenemia, hyperphosphatemia, and an elevated calcium phosphorus product (Ca*P). Postmortem findings were consistent with systemic calcinosis, a syndrome of calcium deposition in the tissue of organs including lungs, kidneys, muscle, and heart that has not been previously described in horses.

Experimental infection of cats (Felis catus) with Tritrichomonas foetus isolated from cattle.

Tritrichomonas foetus is recognized as the causative agent of venereal trichomoniasis in cattle. It is characterized by embryonic and early fetal death and post-coital pyometra, and feline trichomoniasis, manifest as chronic, large bowel diarrhea. Many of the infected cats are less than 2 years old and specific routes of transmission remain unknown. We recently demonstrated that feline isolates of T. foetus can successfully infect heifers, resulting in pathologic changes similar, but not identical to those previously reported as representative of bovine trichomoniasis. In this study, we experimentally infected six cats less than 1 year of age with a bovine (D-1) isolate of T. foetus and one cat with a feline (AUTf-1) isolate of T. foetus. Within 2 weeks, the cat infected with the feline (AUTf-1) isolate was culture positive for trichomonads in weekly fecal samples. At the end of 5 weeks, only one cat infected with the bovine (D-1) isolate was fecal culture positive for trichomonads. At necropsy, the intestine of each cat was removed and divided into five sections (ileum, cecum, anterior, medial and posterior colon). Contents from each section were collected and cultured. The cat infected with the feline (AUTf-1) isolate was culture positive in the ileum, cecum, medial and posterior colon. Two cats infected with the bovine (D-1) isolate were culture positive in the cecum only. Additionally, each intestinal section was submitted to a pathologist for histopathological examination. The combined results indicate that there are demonstrable differences between the feline (AUTf-1) and bovine (D-1) isolates regarding their infectivity in cats.

Immunology and immunotherapy of the infections caused by Pythium insidiosum.

Although infections caused by the straminipilan pathogen Pythium insidiosum were described in 19th century, it has been only recently that its epidemiology, immunology, treatment and other important traits were extensively studied. These studies were of paramount importance to theorize about the ecological niche for this pathogen, its host-parasite relationships, the antigens used for diagnosis, and the management of the infection using immunotherapy. P. insidiosum triggers in the infected host a T helper 2 [Th2] subset with an inflammatory reaction composed mainly of eosinophils and mast cells. These cells degranulate around the hyphal elements of P. insidiosum where a Splendore-Hoeppli-like reaction develops. In horses this reaction is so intensive that firm concretions called 'kunkers' develop. These data indicated that this pathogen might have developed an evolutionary strategy to conceal important antigens from the host immune system. Immunotherapy, a treatment approach that relies on the injection of antigens of P. insidiosum from in vitro cultures, has been successfully used in humans and horses to manage this disease. A switch from a Th2 to Th1 response is postulated as the most likely explanation of the curative properties of this approach. This review provides details on the serological, immunological, and immunotherapeutic methodologies used to diagnose and treat the infections caused by this pathogen.

Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae.

Species-specific polymerase chain reaction (PCR) primers have been designed to identify the causative agent of columnaris disease, Flavobacterium columnare. The 16S rRNA gene sequences of F. columnare (eight sequences representing the different genotypes of the species) and related species (18 sequences) were aligned and compared to choose specific regions that are unique to F. columnare and do not have significant intraspecies variability. The species-specific regions in the 16S rRNA gene were used to design a pair of species-specific PCR primers, ColF and ColR. The PCR technique produced a specific amplicon of about 675 base pairs (bp) in 27 isolates of F. columnare and there was no amplification in the closely related species. The specificity of the amplified product was confirmed by digesting with HhaI. The PCR primers did not produce a 675 bp product with F. columnare ATCC43622 strain. This ATCC43622 strain was characterized by biochemical and ribotyping methods and renamed Flavobacterium johnsoniae. The American Type Culture Collection has confirmed these findings and made the change.