Concurrent infections of Streptococcus iniae and Aeromonas veronii in farmed Giant snakehead (Channa micropeltes).

The giant snakehead, Channa micropeltes, is an increasingly important economic freshwater fish in Thailand and other regions of Asia. Presently, giant snakehead are cultured under intensive aquaculture conditions, leading to high stress and conditions favouring disease. In this study, we reported a disease outbreak in farmed giant snakehead with a cumulative mortality of 52.5%, continuing for 2 months. The affected fish exhibited signs of lethargy, anorexia and haemorrhage of the skin and eyes. Further bacterial isolations revealed two different types of colonies on tryptic soy agar: small white, punctate colonies of gram-positive cocci and cream-coloured, round and convex colonies of rod-shaped gram-negative bacteria. Additional biochemical and species-specific PCR analysis based on 16S rRNA confirmed the isolates as Streptococcus iniae and Aeromonas veronii. Multilocus sequence analysis (MLSA) placed the S. iniae isolate into a large clade of strains from clinically infected fish worldwide. Gross necropsy findings showed liver congestion, pericarditis and white nodules in the kidney and liver. Histologically, the affected fish showed focal to multifocal granulomas with inflammatory cell infiltration in kidney and liver, enlarged blood vessels with mild congestion within the meninges of the brain and severe necrotizing and suppurative pericarditis with myocardial infarction. Antibiotic susceptibility tests revealed that S. iniae was sensitive to amoxicillin, erythromycin, enrofloxacin, oxytetracycline, doxycycline and resistant to sulfamethoxazole-trimethoprim, while the A. veronii was susceptible to erythromycin, enrofloxacin, oxytetracycline, doxycycline, sulfamethoxazole-trimethoprim and resistant to amoxicillin. Conclusively, our findings highlighted the natural concurrent bacterial infections in cultured giant snakehead, which support the implementation of appropriate treatment and control strategies.

Pathological Manifestations of Francisella orientalis in the Green Texas Cichlid (Herichthys cyanoguttatus).

Francisella orientalis (Fo) is considered to be one of the major pathogens of tilapia because of the high mortalities observed during outbreaks. Other cichlids belonging to the same family (Cichlidae) as tilapia are also quite susceptible to this pathogen. On various occasions, Fo has also been isolated from other warm water fish, including three-line grunt, hybrid striped bass, French grunt, Caesar grunt, and Indo-Pacific reef fish. However, only a few studies have reported the pathogenicity of Francisella orientalis in ornamental cichlid fish. This study fulfills Koch's postulates by showing that a strain of Fo obtained from green Texas cichlid (Herichthys cyanoguttatus) was able to produce the same pathogenicity in healthy fish. A mortality of 100% was observed after healthy green Texas cichlid were experimentally injected with Fo at a dose of 8.95 × 105 CFU/fish. DNA extracted from the organs of predilection (spleen, head kidney) gave positive results by PCR for all fish that died during the experimental period. Spleen and head kidney presented with multifocal white nodules in the affected fish, corresponding to typical vacuolated granulomas on histopathological examination of the tissues. Based on the results of this study, it is evident that Fo can indeed infect green Texas cichlid and produce a disease typical of francisellosis.

Transcriptome analysis of immune- and iron-related genes after Francisella noatunensis subsp. orientalis infection in Nile tilapia (Oreochromis niloticus).

Francisella noatunensis subsp. orientalis (Fno) is a gram-negative intracellular bacterium identified in many fish species worldwide, including cultured Nile tilapia (Oreochromis niloticus) in Taiwan. To investigate the gene expression responses to Fno infection, we performed transcriptome analysis of the head kidney and spleen in Nile tilapia using RNA-seq. Total RNA was extracted from the head kidney and spleen of infected (Fno-injected) and uninfected (control) tilapia at 1-day and 2-days post-infection, and RNA-seq was performed using the Illumina HiSeq™ 4000 platform. After de novo assembly, a total of 106,534 transcripts were detected. These transcripts were annotated and categorized into a total of 7171 genes based on the KEGG pathway database. Differentially expressed genes (DEGs) were significantly (2-fold difference comparing Fno and PBS groups at each time point) enriched in the immune-related pathways, including the following: complement and coagulation cascades, cytokine-cytokine receptor interaction, hematopoietic cell lineage, lysosome, phagosome. We identified the upregulation of inflammatory cytokine-, apoptosis-, and neutrophil-related genes, and downregulation of complement- and lymphocyte-related genes. Additionally, we found the induction of natural resistance-associated macrophage protein 1 (NRAMP1) and heme responsive gene-1 (HRG1). Anemia of inflammation, caused by intracellular iron storage in spleen after Fno infection, was also observed. This study provides natural disease control strategies against Fno infection in tilapia. It is suggested that intercellular iron storage is a host protection strategy.

Comparison of the pathogenicity of Francisella orientalis in Nile tilapia (Oreochromis niloticus), Asian seabass (Lates calcarifer) and largemouth bass (Micropterus salmoides) through experimental intraperitoneal infection.

Francisella orientalis is a highly virulent, emerging bacterium that causes mass mortalities in tilapia. This pathogen also affects numerous other warm-water fish species, including three-line grunt, hybrid striped bass and various ornamental fish. This study sheds light on two new species of fish that are susceptible to F. orientalis. Asian seabass and largemouth bass showed variable levels of susceptibility in a bacterial challenge experiment. After intraperitoneally injected with a dose of 106  CFU/fish, a total of 64.28% and 21.42% mortalities were obtained in Asian seabass and largemouth bass, respectively. Meanwhile, Nile tilapia showed acute mortality of 100%. All fish showed typical lesions of francisellosis, including multifocal granulomas in the spleen and head kidney. Immunohistochemical analysis revealed strong positive signals inside the granulomas of all fish. The bacterial recovery in solid media from infected fish was highest in Nile tilapia (85.71%), followed by Asian seabass (35.71%) and largemouth bass (21.42%). PCR results tested 100% positive for Nile tilapia, and 78.57% and 21.42% for Asian seabass and largemouth bass, respectively. In conclusion, Asian seabass and largemouth bass are susceptible to this pathogen, which warrants new management strategies when employing predation polyculture systems of these species with tilapia.

Immune Responses and Protective Efficacy of a Formalin-Killed Francisella Noatunensis Subsp. Orientalis Vaccine Evaluated through Intraperitoneal and Immersion Challenge Methods in Oreochromis Niloticus.

Francisella noatunensis subsp. orientalis (Fno), an intracellular bacterium, causes systemic granulomatous diseases, resulting in high mortality and huge economic losses in Taiwanese tilapia farming. In this study, we tested the efficacy of a formalin-killed Fno vaccine in cultured tilapia. Fno was isolated from diseased tilapia, inactivated with formalin, and mixed with the mineral oil base adjuvant (MontanideTM ISA 763 AVG). A total of 300 tilapia were divided into two groups. The experimental group was intraperitoneally injected with 0.1 mL of vaccine, which was substituted with phosphate-buffered saline (PBS) in the control group. A booster was administered at 2 weeks post-immunization. Tilapia were challenged at 6 weeks post primary immunization by intraperitoneal (IP) injection and immersion methods. Mortality was recorded at 21 and 60 days. The results revealed that the vaccine induced a greater antibody titer and led to 71% and 76% of relative percent survival (RPS) after the IP and immersion challenge. The transcripts of proinflammatory cytokines and immune-related genes, including interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNFα), C-X-C motif chemokine ligand 8 (CXCL8), and interleukin-17C (IL-17C), were significantly upregulated after vaccination. Additionally, vaccinated fish had lower bacterial loads in the blood and lower granuloma intensities in the kidney, spleen, liver, and gill than control fish. The results in this study demonstrate that the inactivated Fno vaccine could be an essential resource in Taiwanese tilapia farming.

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan.

Francisella noatunensis subsp. orientalis is a causative agent of systemic granulomatous disease in tilapia. The present study was designed to understand the genetic and phenotypic diversities among Taiwanese Fno isolates obtained from tilapia (n = 17) and green Texas cichlid (Herichthys cyanoguttatus) (n = 1). The enzymatic profiles of the isolates were studied using the API ZYM system. Phylogenetic tree analysis of the 16S rRNA and housekeeping gene and pulsed-field gel electrophoresis (PFGE) were carried out to determine the genotypic characters of all isolates. The phylogenetic tree showed similarity of 99%-100% nucleotide sequences of 16S rRNA and housekeeping genes compared to the Fno references genes from GenBank database. Comparatively, the results revealed an identical profile of enzymatic and PFGE pattern which was distincted from that of F. philomiragia. To understand the pathogenicity, the isolates were intraperitoneal injected to tilapia the gross lesions were observed concomitant with natural outbreak. Median lethal dose upon Nile tilapia and red tilapia were 9.06 × 103 CFU/fish and 2.08 × 102 CFU/fish, respectively. Thus, our data provide understanding the epidemiology of Taiwanese Fno isolates, and help in development of future control and prevention.