Search

Francisella orientalis is an important bacterial pathogen of marine and freshwater fish with worldwide distribution. Fish francisellosis is a severe subacute to chronic granulomatous disease, with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this disease. In this study, we evaluated the efficacy of a defined F. orientalis pathogenicity determinant protein A (pdpA) mutant (ΔpdpA) as a live attenuated immersion vaccine against subsequent immersion challenge with the wild-type organism. Immunized Nile tilapia Oreochromis niloticus were protected (45% relative percent survival) from the lethal challenges and presented significantly lower mortality than nonvaccinated and challenged treatments. Although serum IgM was significantly higher in immunized fish, similar bacterial loads were detected in vaccinated and nonvaccinated survivors. In conclusion, although the F. orientalis ΔpdpA is attenuated and effectively stimulated an adaptive immune response, the low relative percent survival and high bacterial persistence in survivors of immunized and challenged treatments indicates low suitability of ΔpdpA as a mucosal vaccine for tilapia under conditions used in this study.

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.

Tilapia tilapinevirus, or Tilapia Lake Virus (TiLV), is a RNA virus associated with mass morbidity and mortality in tilapia, leading to severe economic losses for global tilapia aquaculture. In this study, we investigated the persistence of TiLV in water by spiking sterile distilled water (SDW), freshwater collected from rearing fish tanks (FW) and natural pond water (PW) at 27°C as a representative of environmental water conditions with 0.6 ml of stock virus (3.18 × 10

The freshwater ornamental fish trade represents a major contributor to the livelihoods of many producers in Trinidad and Tobago, with stocks destined for local, regional and international markets. A review of clinical cases presented to the Aquatic Animal Health Unit at the University of the West Indies, School of Veterinary Medicine for the period September 2010 to December 2012 suggested that piscine mycobacteriosis may be widespread throughout the local ornamental fish industry. Thus, to determine the prevalence of mycobacteriosis in ornamental fish sold in pet stores, a total of 122 specimens were sourced from 24 retail suppliers across Trinidad. Fish were killed and internal organs were examined for lesions suggestive of granulomas. All wet-mount slides were acid-fast stained, regardless of the presence or absence of observed granuloma-like lesions. Histological analysis was performed on one randomly selected whole specimen from each facility. Mycobacterium sp. was identified using real-time PCR detecting the 16S rRNA gene in tissue samples. Associations between parasitism, facility biosecurity and presence of positive animals were determined. The prevalence of Mycobacterium sp. infection was 61 ± 7% (74/122), with positive specimens being acquired from 54.2% (13/24) of facilities examined. Further, 100% of facilities did not employ optimum biosecurity measures.

Edwardsiella piscicida is an emergent global fish pathogen with a wide host range, although host associations driving genetic diversity remain unclear. This study investigated the genetic and virulence diversity of 37 E. piscicida isolates recovered from 10 fish species in North America. Multilocus sequence analysis (MLSA) was conducted using concatenated alignments of the gyrB, pgi and phoU sequences. MLSA clustered the tested isolates into six discrete clades. In light of recent disease outbreaks in cultured salmonids, the virulence of each clade was evaluated in Chinook salmon Oncorhynchus tshawytscha fingerlings following intracoelomic challenge of ~106 CFU/fish. Challenged and control fish were monitored for 21d, and microbiological and histological examination was performed on dead and surviving fish. Peak mortality occurred 3-5 days post-challenge (dpc) regardless of isolate or genetic group. Edwardsiella piscicida was recovered from all moribund and dead animals. At 21 dpc, fish challenged with isolates from clades II, III and IV presented cumulative mortality ≥83.3%, whereas isolates from clade I, V and VI resulted in cumulative mortality ≤71.4%. This study suggests an underlying genetic basis for strain virulence and potential host associations. Further investigations using other fish models and variable challenge conditions are warranted.

Systemic phaeohyphomycosis caused by the dematiaceous mould Veronaea botryosa is an important emergent disease affecting captive sturgeons (Acipenser spp.). The disease, colloquially known as "fluid belly," causes morbidity and mortality in adult animals resulting in significant economic losses to the aquaculture industry. Advancements in therapeutic and prophylactic protocols have been partially hampered by the lack of basic protocols to grow and manipulate the fungus in the laboratory. In this study, microbroth kinetic protocols were established to analyse V. botryosa growth in seven nutrient media at different temperatures. Generated area under the curve (AUC) indicates that potato flake dextrose broth (PFD-B) and Sabouraud dextrose broth (SD-B) incubated at 25°C provided the greatest growth. The generated protocol was then used to test the susceptibility of V. botryosa isolates to natamycin, a macrolide polyene antifungal agent used as a food preservative. SD-B and RPMI with l-glutamine (+RPMI-B) containing different concentrations of natamycin were inoculated with V. botryosa conidia and the generated growth curves were compared using cubic smoothing spline model. The non-inhibitory concentration and minimal inhibitory concentration (MIC; decrease of AUC by 90% compared with control) were determined to be1 μg/mL and 16 μg/mL of natamycin in SD-B media. To gain an understanding of the tissue distribution of natamycin in white sturgeon, pharmacokinetics was tested. Based on pharmacokinetic parameters determined in this study and targeting a blood concentration 16 μg/mL for 24 h, an intravenous dose1 g/kg would be needed, making the use of this drug unrealistic. The information presented in this study can be used to investigate susceptibility of pathogenic fungus to antimicrobials and disinfectants as well as support future therapeutic protocols against emerging fungal diseases like fluid belly.

Streptococcosis is an infectious bacterial disease of both homeotherms and poikilotherms. Among the Streptococcus species that infect marine animals, Streptococcus agalactiae has the broadest host spectrum, including different aquatic organisms in freshwater and marine environments. The common dolphin (Delphinus delphis) is categorized as Endangered in the Mediterranean Sea. There are few reports of a streptococcal infection of D. delphis, caused by Streptococcus phocae and Streptococcus iniae. Here we report the isolation and identification of S. agalactiae in a stranded, wild male common dolphin that was found dead in September 2020 on the seashore next to the city of Bat-Yam, Israel. The carcass was fresh with a moderate nutritional status and with no apparent fishing gear or other anthropogenic-related signs. A post-mortem examination did not reveal an apparent cause of death, but further laboratory analysis demonstrated a S. agalactiae bacterial presence in urine, lungs and pericardial fluid that was characterized as type Ia-ST7 by whole genome sequencing. Interestingly, this isolate was found to be almost identical to another isolate recently recovered from a wild sandbar shark (Carcharhinus plumbeus) in the same area in Israel, the eastern Mediterranean Sea.

Warm-water piscine francisellosis is a granulomatous bacterial disease caused by Francisella orientalis (Fo). The disease has been detected in a wide range of fish species globally, causing mortalities as high as 90% and significant economic losses. Currently there are no commercially available vaccines and few treatment options exist. In the current study, two novel recombinant vaccines were prepared using diatom-expressed IglC or bacterial-expressed GroEL proteins. The vaccine antigens were emulsified with either nanoparticles or a commercially available oil-based adjuvant. Nile tilapia, Oreochromis niloticus, fingerlings were immunized intracoelomically with the recombinant IglC or GroEL vaccines, diatoms alone or phosphate buffer saline. Approximately 840-degree days post-vaccination, fish were challenged via immersion with 106 CFU/mL of wild-type Fo. Twenty-one days post challenge (dpc), the highest relative percent survival was recorded in the IglC-Montanide group (75%), compared to 53%, 50%, 22%, 19% and 16% in the IglC-nanoparticles, GroEL-Montanide, GroEL-nanoparticles, diatoms-Montanide and diatoms-nanoparticles groups, respectively. Protection correlated with significantly higher specific antibody responses in the IglC-Montanide group. Moreover, a significantly lower bacterial load was detected in spleen samples from the IglC-Montanide survivor tilapia compared to the other experimental groups. This is the first report of recombinant vaccines against piscine francisellosis in tilapia. The Fo vaccines described in our study may facilitate development of a safe, cost-effective and highly protective vaccine against francisellosis in farmed tilapia.

Streptococcus iniae is a zoonotic pathogen and one of the major aetiologic agents of streptococcosis. In White Sturgeon Acipenser transmontanus, S. iniae infection typically presents as a necrotizing and heterophilic myositis, causing 30-50% mortality in infected fish. To gain a better understanding of the pathogenesis of streptococcosis in White Sturgeon, and to identify the experimental route of infection that most closely mimics the natural disease, fingerlings were challenged with a single dose of 1.3 × 108 cells/fish of S. iniae that was administered via intracoelomic/intraperitoneal (IC) or intramuscular (IM) routes. Acute mortalities were present only in the IM-challenged fish, with first mortality occurring 4 d postchallenge and the mortality rate reaching 18.3% after 9 d. The challenged fish presented erratic swimming, ulcerative skin lesions, and hemorrhages in the liver and swim bladder. Streptococcus iniae was recovered from the kidney and brain tissues of moribund and dead fish. Histopathologic analysis of fish that died acutely revealed massive proliferation of bacteria in the muscle at the injection site and within vascular organs such as the heart and spleen, with variable amounts of tissue necrosis including a necrotizing myositis. Fish that died closer to 9 d postchallenge demonstrated more pronounced multifocal to locally extensive granulomatous inflammation of skeletal muscle at the injection site, liver, kidney, and spleen. No mortality, clinical signs, or gross changes were observed in the control or IC-challenged fish. Postmortem evaluation of 10 survivors in each treatment was performed to determine carrier status in the brain and posterior kidney tissues. The prevalence of S. iniae in survivors was 10% and 0% in the IM- and IC-challenged groups, respectively. The results from this study suggest that IM-injection challenge methods are suitable for inducing streptococcosis in White Sturgeon, and they may be the preferred method for studying the pathogenesis of the naturally occurring disease in this species.

The Gram‐negative bacterium, Flavobacterium psychrophilum, is endemic to California, USA, where it is an important pathogen in salmonid aquaculture, especially in rainbow trout (Oncorhynchus mykiss). Disease outbreaks caused by F. psychrophilum in rainbow trout fingerlings can approach 90% mortality, resulting in millions of dollars of economic losses annually. The focus of this study was to investigate the genetic diversity of 49 F. psychrophilum isolates collected from disease outbreaks in 17 salmonid hatcheries in California, USA, from 2015 to 2018 using multilocus sequence typing. Results suggest California F. psychrophilum isolates are diverse, representing 11 distinct sequence types (STs), three of which were previously undescribed. Still, the majority of genotyped isolates (n = 41) belonged to a single clonal complex (CC), CC‐ST10, which is the largest CC worldwide and has been linked to disease outbreaks on several continents. Results of this study provide evidence of marked intraspecific genetic diversity of F. psychrophilum from California. The biological significance of this genetic variability is unclear but could have implications for future vaccine development and treatments. Further studies investigating the virulence, antigenic, and antimicrobial susceptibility profiles of F. psychrophilum are warranted to better understand the epizootiology of this pathogen in the Western United States.

Erysipelothrix piscisicarius is an emergent pathogen in fish aquaculture, particularly in the ornamental fish trade. Very little is known on the biology of this pathogen; however, the recurrence of infection and disease outbreaks after removing the fish from a system and disinfecting the tank suggest its environmental persistence. Moreover, biofilm lifestyle in E. piscisicarius has been suspected but not previously shown. The purpose of this study was to investigate the formation of biofilms on an abiotic surface in Erysipelothrix spp. We used hydroxyapatite-coated plastic pegs to demonstrate the attachment, growth, and persistence of E. piscisicarius on abiotic surfaces in both fresh and marine environments and to investigate the susceptibility of this pathogen to different disinfectants that are used in the aquaculture industry. E. piscisicarius formed biofilms that persisted significantly longer than planktonic cells did in both freshwater and saltwater over a period of 120 h (P = 0.004). The biofilms were also more resistant to disinfectants than the planktonic cells were. Hydrogen peroxide was the most effective disinfectant against E. piscisicarius, and it eradicated the biofilms and planktonic cells at the recommended concentrations. In contrast, Virkon and bleach were able to eradicate only the planktonic cells. This information should be taken into consideration when developing biosecurity protocols in aquaculture systems, aquariums, and private collections.

Outbreaks of an infectious disease affecting cultured white sturgeon (Acipenser transmontanus) were investigated. Clinical signs included erratic swimming, arching of the back and mortality. Necropsy findings included poorly demarcated yellow to dark-red and friable lesions in the epaxial muscle, ulcerative skin lesions and haemorrhages in the swim bladder and coelomic wall. Histological evaluation revealed areas of necrotizing and heterophilic myositis with aggregates of bacterial cocci. The lumen of blood vessels in the dermis, under ulcerated areas, and in the posterior kidney, was occluded by fibrin thrombi. Aggregates of Gram-positive cocci were observed in the muscle lesions and within the fibrin thrombi in the dermis and kidney. Genetically homogeneous Streptococcus iniae strains were recovered from affected fish from different outbreaks. The isolates shared high degree of similarity at gene locus (gyrB) with previously characterized S. iniae from cultured fish in California, confirming the emergence of this particular strain of S. iniae in US aquaculture.

White Sturgeon Acipenser transmontanus are cultured for human consumption as well as for conservation purposes. In this study, two commercially available portable devices for measuring glucose and lactate were compared to a benchtop analyzer and blood reference intervals were generated using heparin plasma collected from 43 healthy White Sturgeon yearlings. The generated normal ranges were used to compare plasma values collected from Veronaea botryosa-infected White Sturgeon at 10, 20, and 30 d postchallenge (dpc). In the 43 healthy yearlings, significantly different glucose and lactate values were obtained when comparing the portable devices to the benchtop analyzer. In the portable devices, blood glucose showed a consistent bias of 12.3 mg/dL and blood lactate showed a proportional bias. The detected blood glucose values in infected sturgeon were significantly different from those in noninfected controls when using the benchtop analyzer at 20 and 30 dpc and when using the portable device at 20 dpc. However, blood glucose in infected individuals and controls was within the reference interval on either device. No significant difference was noted for lactate measurements in infected sturgeon. The results of this study indicate that portable devices for blood glucose and blood lactate evaluation in White Sturgeon may be useful in a setting where relative values are an acceptable approximation of absolute values.

Francisella noatunensis subsp. orientalis (Fno) is a Gram-negative, pleomorphic, facultative intracellular bacterial pathogen affecting a variety of cultured and wild fish species. Outbreaks of piscine francisellosis in warmwater fish have been documented worldwide; however, reports of Fno from Central America have been limited to a single documented outbreak in cultured tilapia in Costa Rica in 2007. From 2015 to 2017, Fno was consistently recovered from disease outbreaks in Nile tilapia Oreochromis niloticus cultivated in floating cages in Lake Yojoa, Honduras. Mortality rates during these outbreaks ranged from 50 to 85%. Fno was isolated by aerobic culture on selective media and identity confirmed by Fno-specific PCR. Repetitive extragenic palindromic PCR analysis revealed that the case isolates were genetically homogeneous with archived strains recovered from epizootics in cultured tilapia from Costa Rica and Mexico, suggesting the same strain of Fno was responsible for these otherwise unrelated fish kills. The current study provides only the second report of Fno in Central America and characterizes the first Fno outbreak in cultured fish in Honduras.

Native and introduced fish can serve as reservoirs for pathogens of cultured fish species. In the current study, 351 archived western mosquitofish Gambusia affinis collected from experimental catfish production ponds in Mississippi, USA, were surveyed histologically to evaluate their potential as vectors for fish pathogens. In addition to epitheliocystis and multiple metazoan parasites, 8 fish had widespread basophilic colonies of small Gram-positive rods associated primarily with stroma supporting the skeletal muscle and bone, as well as connective tissue components of other tissues and organ systems, such as perivascular adventitia and basement membranes. These findings were consistent with spaC-type Erysipelothrix sp. infections in ornamental fish cultured in the USA. The 16S rRNA, gyrase B (gyrB), and surface protective antigen (spa) genes were amplified and sequenced from bacterial colonies excised from paraffin-embedded tissue sections using laser capture microdissection. Molecular data confirmed the identity of a spaC-type Erysipelothrix sp., which grouped phylogenetically with spaC-type Erysipelothrix sp. from diseased ornamental fish. Given the significance of commercial catfish aquaculture in the southeastern USA and the widespread distribution of mosquitofish in catfish ponds throughout the region, infectivity trials with channel catfish Ictalurus punctatus were conducted. Catfish fingerlings were exposed to a spaC-type Erysipelothrix sp. isolate by intracoelomic injection and gavage. No mortality was observed in catfish exposed by either route, and surviving fish demonstrated no significant histopathologic lesions, suggesting channel catfish have low susceptibility to the bacteria. Further research is warranted to investigate the susceptibility of other cultured fish species to this emergent fish pathogen.

In August 2018, a series of large fish kills involving only Silver Carp Hypophthalmichthys molitrix occurred on the Mississippi River in northern Louisiana. Clinical signs observed in moribund animals included erratic swimming behavior, such as spiraling and spinning at the surface. A moribund specimen was captured by dip net near the surface at Lake Providence Landing in East Carroll Parish, northern Louisiana, and was submitted for analysis. An aseptic necropsy was performed, and diagnostic procedures, including bacteriology, parasitology, histopathology, virology, and electron microscopy, revealed that a gram-positive coccus was the primary pathogen. Pure cultures of the organism were obtained from the brain, and it was the predominant colony type isolated from the spleen, kidney, and liver. Bacterial sepsis caused by the gram-positive coccus and involving multiple organ systems was diagnosed histologically. Bacterial colonization and necrotic lesions were seen in the spleen, liver, kidney, heart, eye, and brain. Numerous cocci were observed dividing intracellularly in phagocytic cells of the kidney and brain by transmission electron microscopy. The organism was identified as Streptococcus dysgalactiae ssp. dysgalactiae by conventional biochemical methods and subsequently by the API 20 Strep system. The identity of the pathogen was later confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequencing of the 16S ribosomal RNA gene. Multilocus sequence analysis clustered this isolate along with two other S. dysgalactiae isolates from fish in a divergent phyletic group that was separate from other S. dysgalactiae ssp. dysgalactiae isolates from terrestrial animals, implying a possible novel clade that is pathogenic for fish.

Erysipelothrix piscisicarius is an emerging bacterial pathogen and the aetiologic agent of piscine erysipelosis, a recently recognized disease of ornamental fish. However, little is known regarding the dynamics of infection in fish. The purpose of this study was to gain a better understanding of the pathogenesis of piscine erysipelosis in the tiger barb (Puntigrus tetrazona) by investigating tissue tropisms and responses to bacterial dissemination following immersion challenge with a virulent strain recovered from diseased fish. The challenge resulted in 83% mortality by day 16. Erysipelothrix piscisicarius DNA was first detected in the skin using quantitative PCR, and bacteria were visualized in association with microscopic lesions on day 4. By day 8, E. piscisicarius DNA was further detected in intestines, hearts, spleens, gills and skin; parenchymal organs were largely spared. The data suggest a primary cutaneous portal of entry and tropism for collagenous tissues, particularly those within vascular walls. Initial spread occurs directly from the dermis into interstitial areas of skeletal muscle, then centrally to the peritoneum and coelomic cavity following collagenous tissue pathways. Although histopathology revealed widespread bacterial dissemination over time, the severity of skin and muscle lesions with high levels of bacterial DNA identifies these tissues as primary targets of infection.

Streptococcus iniae is a re-emerging bacterial pathogen in freshwater and marine aquaculture worldwide. There are no commercial vaccines available for S. iniae in the United States, and autogenous vaccines are restricted to inactivated whole-cell preparations with limited protection against heterogenous strains. Live-attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they induce robust cellular and humoral immunity, and may provide longer lasting protection through less stressful routes of administration. We investigated whether accumulation of mutations in S. iniae by serial passage in the presence of rifampin can generate immunogenic LAV conferring protection against challenge with heterologous wild-type (WT) S. iniae strains in Nile tilapia (Oreochromis niloticus). Three lineages of rifampin-resistant S. iniae strains were generated from three genetically distinct parent strains (n = 9) by multiple passages in increments of Rifamycin SV sodium salt. Growth in liquid media, extent of capsulation, antimicrobial susceptibility, survival in Nile tilapia whole blood, and cytotoxicity in an O. mossambicus endothelial cell line were compared between the passaged and WT strains. Nile tilapia challenges were used to assess strain virulence, generation of anti-S. iniae IgM, and the protection conferred by LAV candidates against virulent S. iniae. Rifampin-resistant strains demonstrated changes in growth rate and cytotoxicity in endothelial cells, as well as significant reductions in whole blood survival (p 0.05). Selected strains also showed attenuated virulence in the Nile tilapia challenge model, and anti-S. iniae IgM generated against these strains demonstrated cross-reactivity against heterologous bacteria. Immunization by intracoelomic injection induced protection against a virulent WT strain of S. iniae, with relative percent survival up to 95.05%.

Systemic phaeohyphomycosis, aka ‘fluid belly’, is one of the most important emergent diseases in sturgeon Acipenser spp. aquaculture. The etiologic agent is the saprobic, dematiaceous fungus Veronaea botryosa. Effective vaccines and chemotherapeutic treatments are currently unavailable. Additionally, the fungus is a slow-growing organism, taking from 10-15 d for colonies to be observed in agar media. To this end, a specific quantitative PCR (qPCR) targeting the V. botryosa β-tubulin gene was developed and validated. The specificity of the assay to V. botryosa was initially confirmed in silico and in vivo against common fungal fish pathogens, including closely related members of the order Chaetothyriales (Exophiala spp.) and other black pigmented fungi (Alternaria spp. and Cladosporium spp.), as well as tissues from uninfected sturgeon. The assay possessed high clinical specificity (100%) and clinical sensitivity (74%) in detecting V. botryosa DNA in splenic tissues from laboratory-infected sturgeon. Using V. botryosa genomic DNA as a template, the limit of detection was equivalent to 10 conidia, and the method was found suitable for the detection of fungal DNA in fresh and formalin-fixed tissues. In addition, the presence of non-target DNA from white sturgeon did not influence assay sensitivity. The developed qPCR assay is a sensitive, specific, and rapid diagnostic method for the detection and quantification of V. botryosa DNA from white sturgeon tissues.

Flavobacterium columnare is the causative agent of columnaris disease. Previous work has demonstrated a high degree of genetic variability among F. columnare isolates, identifying 4 genetic groups (GGs) with some host associations. Herein, a total of 49 F. columnare isolates were characterized, the majority of which were collected from 15 different locations throughout the US Pacific Northwest. Most isolates were collected from 2015-2018 and originated from disease outbreaks in salmonid hatcheries and rearing ponds, sturgeon hatcheries and ornamental fish. Other isolates were part of collections recovered from 1980-2018. Initial identification was confirmed by F. columnare species-specific qPCR. Study isolates were further characterized using a multiplex PCR that differentiates between the 4 currently recognized F. columnare GGs. Multiplex PCR results were supported by repetitive sequence-mediated PCR fingerprinting and gyrB sequence analysis. F. columnare GG1 was the most prevalent (83.7%, n = 41/49), represented by isolates from salmonids (n = 32), white sturgeon (n = 2), channel catfish (n = 1), ornamental goldfish (n = 1), koi (n = 3), wild sunfish (n = 1) and 1 unknown host. Six isolates (12.2%, n = 6/49) were identified as GG3, which were cultured from rainbow trout (n = 3) and steelhead trout (n = 3). Two isolates were identified as GG2 (4.1%, n = 2/49) and were from ornamental fish. No GG4 isolates were cultured in this study. The biological significance of this genetic variability remains unclear, but this variation could have significant implications for fish health management. The results from this study provide baseline data for future work developing strategies to ameliorate columnaris-related losses in the US Pacific Northwest.

Streptococcus iniae is a Gram-positive, opportunistically zoonotic bacterium infective to a wide variety of farmed and wild fish species worldwide. Outbreaks in wild fish can have detrimental environmental and cultural impacts, and mortality events in aquaculture can result in significant economic losses. As an emerging or re-emerging pathogen of global significance, understanding the coalescing factors contributing to piscine streptococcosis is crucial for developing strategies to control infections. Intraspecific antigenic and genetic variability of S. iniae has made development of autogenous vaccines a challenge, particularly where the diversity of locally endemic S. iniae strains is unknown. This study genetically and phenotypically characterized 11 S. iniae isolates from diseased wild and farmed fish from North America, Central America, and the Caribbean. A multilocus sequence analysis (MLSA) scheme was developed to phylogenetically compare these isolates to 84 other strains of Streptococcus spp. relevant to aquaculture. MLSA generated phylogenies comparable to established genotyping methods, and isolates formed distinct clades related to phenotype and host species. The endothelial Oreochromis mossambicus bulbus arteriosus cell line and whole blood from rainbow trout Oncorhynchus mykiss, Nile tilapia Oreochromis niloticus, and white sturgeon Acipenser transmontanus were used to investigate the persistence and virulence of the 11 isolates using in vitro assays. In vivo challenges using an O. niloticus model were used to evaluate virulence by the intragastric route of infection. Isolates showed significant differences (p < 0.05) in virulence and persistence, with some correlation to genogroup, establishing a basis for further work uncovering genetic factors leading to increased pathogenicity.

Chinook Salmon Oncorhynchus tshawytscha is a keystone fish species in the Pacific Northwest. In 2019, unusual mortalities occurred in two different populations of cultured fingerlings from the same facility in California, USA. The systems consist of outdoor, enclosed, flow-through freshwater tanks that are maintained at 18 ± 1°C. Clinical signs and gross findings were only observed in one population and included abnormal swimming, inappetence, lethargy, skin discoloration, and the presence of multifocal nodular and ulcerative skin lesions. Microscopic lesions were infrequent and consisted of severe, locally extensive granulomatous dermatitis and myositis and mild, multifocal, granulomatous branchitis, myocarditis, and hepatitis. Intracellular acid-fast organisms were observed within areas of granulomatous myositis. Posterior kidney swabs were collected and inoculated in nutrient-rich and selective agar media and incubated at 25°C for 2 weeks. Visibly pure bacterial colonies were observed 7-10 d postinoculation. Partial sequences of 16S rRNA initially identified the recovered bacteria as members of the genus Mycobacterium. However, marked variability was observed among Mycobacterium spp. isolates by using repetitive extragenic palindromic polymerase chain reaction fingerprinting. Amplification and sequencing of the ribosomal RNA internal transcribed spacer, 65-kDa heat shock protein, and RNA polymerase β-subunit gene of the cultured isolates identified M. salmoniphilum and M. chelonae, discrete members of the M. chelonae-abscessus complex, isolated from diseased Chinook Salmon fingerlings.

Tilapia lake virus (TiLV) is regarded as one of the most important pathogens in tilapia aquaculture worldwide. Despite this, little is known regarding disease pathogenesis and immune responses to infection. The main objective of this study was to investigate the tissue distribution, histopathological changes, and immune response of fish exposed to TiLV. Nile tilapia (Oreochromis niloticus) maintained at 25 ± 2 °C were challenged with TiLV via intragastric-gavage. At 0.5, 1, 3, 5, 7, 10 and 15 days post-challenge (dpc), six fish per treatment were euthanized and subjected to complete necropsy. TiLV exposed fish presented 45% cumulative mortality at the end of the study. Gross lesions included cutaneous petechiae and ecchymoses, scale losses, skin ulcers, and exophthalmia. Mild multifocal hepatocellular degeneration and necrosis was observed as early as 3 dpc occasionally accompanied by syncytial formation, intracytoplasmic inclusion bodies, and inflammatory infiltrates of lymphocytes at subsequent time points. Necrosis of epithelial cells of the gastric glands and intestinal glands was also observed as early as 5 dpc. Intestinal samples showed reactive in situ hybridization signals as early as 1 dpc. No other lesions were observed in the brain or other organs. Histological changes were associated with viral dissemination and disease progression, as evidenced by increased TiLV detection in the intestine, gills, liver and spleen. Highest TiLV abundance was detected 7 dpc in gills, intestine, and liver showing an average of 6 LOG genome equivalent per ng of total RNA. Different transcript abundance was detected for the pro-inflammatory cytokine interleukin-1β and interferon-induced myxovirus resistance protein gene in the mucosal sites (gills and intestine). Interferon regulatory transcription factor 3 transcript was more abundant in systemic organs (liver and spleen) while the expression in gills and intestine showed mixed expression at different time points. On the other hand, transforming growth factor β expression patterns differed amongst the tissues with a trend towards downregulation of the gene in liver and gills, and a trend towards upregulation in the spleen and intestine. Overall, these results demonstrate the intestinal routes as a main port of entry for TiLV, which subsequently spreads systematically throughout the fish body.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is a viral pathogen responsible for mass mortalities of carp worldwide. In this study, we compared the sensitivity and specificity of ELISA and quantitative PCR (qPCR) methods for the diagnosis of KHV in experimentally infected koi Cyprinus carpio over an 11 mo period. Koi were exposed to KHV at 18 ± 1°C (permissive temperatures for KHV disease) in laboratory-controlled conditions. At 21 d post challenge, the temperature in the system was decreased to 30°C (non-permissive temperature for KHV disease). Finally, KHV ELISA specificity was confirmed using cyprinid herpesvirus 1-exposed koi through a period of 3 mo. This study demonstrates that the combination of ELISA and gill qPCR should be recommended in the diagnosis of KHV exposure of suspected carrier-state fish.

Edwardsiella spp., Streptococcus spp., and Francisella noatunensis subsp. orientalis are some of the most important fish pathogens affecting global tilapia, Oreochromis spp., aquaculture. In Costa Rica, the aquaculture industry is dominated by freshwater‐cultured Nile tilapia, Oreochromis niloticus, which are raised in all seven national provinces. At present, little is known regarding the diversity of pathogens present in these facilities, and definitive identification of agents associated with disease outbreaks are rare. To evaluate the prevalence of common bacterial pathogens in these systems, this study used multiplex quantitative polymerase chain reaction (qPCR) assays targeting Edwardsiella, Streptococcus, and Francisella species as a diagnostic and surveillance tool. In 2017, seven different tilapia hatcheries were visited, and 350 fingerlings were subjected to necropsy and molecular diagnostic evaluation. Fish exhibiting gross signs of disease were subjected to histological and microbiological analysis. For the first time, Edwardsiella anguillarum was recovered and molecularly confirmed from diseased tilapia in Costa Rica. In addition, F. noatunensis subsp. orientalis was identified in a region of Costa Rica where it had not been previously reported Edwardsiella spp., Streptococcus spp. y Francisella noatunensis subsp. orientalis son algunos de los patógenos de peces más importantes que afectan a la tilapia mundial, Oreochromis spp. y la acuicultura. En Costa Rica, la industria de la acuicultura está dominada por la tilapia del Nilo cultivada en agua dulce, Oreochromis niloticus, que se cría en las siete provincias nacionales. En la actualidad se sabe poco sobre la diversidad de patógenos presentes en esas instalaciones, y la identificación definitiva de los agentes asociados a los brotes de enfermedades es poco frecuente. Para evaluar la prevalencia de patógenos bacterianos comunes en estos sistemas, en el presente estudio se utilizaron ensayos cuantitativos múltiples de reacción en cadena de la polimerasa (qPCR) dirigidos a las especies Edwardsiella, Streptococcus y Francisella como instrumento de diagnóstico y vigilancia. En 2017, se visitaron siete criaderos diferentes de tilapia y se sometió a 350 alevines a una necropsia y a una evaluación de diagnóstico molecular. Los peces que presentaban signos graves de enfermedad se sometieron a análisis histológicos y microbiológicos. Por primera vez, se recuperó y se confirmó molecularmente la presencia de Edwardsiella anguillarum en tilapias enfermas en Costa Rica. Además, se identificó F. noatunensis subsp. orientalis en una región de Costa Rica en la que no se había notificado anteriormente. Escuela Medicina Veterinaria

Myxobolus cerebralis is a myxozoan parasite and the etiological agent of whirling disease in salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and the benthic freshwater oligochaete worm Tubifex tubifex (Oligochaeta: Tubificidae). Wildfires can lead to the erosion of fine sediments into stream channels and have been implicated as promoting environmental conditions that are suitable for the survival and success of T. tubifex, whose presence in turn can affect the prevalence of M. cerebralis. Analysis of environmental DNA (eDNA) has the potential to be a powerful tool for evaluating the presence of microorganisms, for which direct observation is impossible. We investigated the presence of M. cerebraliseDNA in river water and river sediment samples collected from areas affected by recent fire activity in Plumas National Forest, California. We compared eDNA loads in the environment to M. cerebralis infection in T. tubifex and sentinel-exposed Rainbow Trout Oncorhynchus mykiss and the presence of T. tubifex lineages in the same environment. For the latter, we developed a multiplex quantitative PCR assay for detection of T. tubifex lineages I, III, and V. Lineage IIIT. tubifex and M. cerebralis (eDNA as well as DNA extracted from fish and worm tissues) were detected only in samples obtained from areas affected by the Moonlight wildfire. The association between M. cerebralis infection in sentinel-exposed fish and eDNA detection in environmental samples only approached significance at a P-value of 0.056. However, given the difference in relative effort between the two sampling methods (host versus nonhost environment), our data suggest that eDNA sampling of water and substrate is a promising approach for surveillance of myxozoan fish parasites.

Tilapia lake virus (TiLV) is a single-stranded negative-sense RNA virus associated with high morbidity and mortality in tilapia. Although the susceptibility of different fish species against TiLV has been investigated, there is limited information of the infectivity of this emerging virus in ornamental African cichlids, which share a genetic background with tilapia. Here, our results revealed that TiLV could cause disease in ornamental Malawi cichlid, Aulonocara spp. Severe morbidity and mortality occurred 5–11 days post-coelomic injection of 1 × 105 TCID50/mL, with a cumulative mortality of 56.25%, 11 days post-challenge. The moribund fish exhibit clinical signs of infection, including coelomic distension, abnormal swimming, exophthalmos, fin rot, and lethargy. Most affected fish also present gross changes, including hyperemic and sometimes ulcerative skin lesions, particularly in the head. Histopathological examination revealed syncytial cells formation, necrosis and lymphocytic inflammation in the liver, with eosinophilic inclusion body in hepatocytes, and necrosis and lymphocytic inflammation in spleen and anterior kidneys. Further, myocardial and intestinal necrosis with infiltration of lymphocytes in the cardiac muscle and intestinal lamina propria were observed in TiLV challenged fish. The infectivity of TiLV in ornamental African cichlids was confirmed by virus isolation in E-11 cells. Cytopathic effect (CPE) developed within three days of inoculation of TiLV challenged fish brain homogenates. The presence of TiLV nucleic acid was further demonstrated in liver, anterior kidney, and heart of the challenged fish by in situ hybridization and reverse transcription-quantitative polymerase chain reaction assay. Altogether, these findings suggest that ornamental African cichlids could be a potential carrier for TiLV and highlight the need for additional assessment to determine the role of other fish species in the transmission of TiLV.

The globally distributed bacterium Streptococcus iniae is responsible for outbreaks of disease resulting in high mortality in a wide range of economically important freshwater and marine fish species. Despite the significance of S. iniae, our understanding of its transmission and infection dynamics remains incomplete. Biofilms are important for the survival and pathogenesis of many bacteria, but there is a paucity of information on their role in the ex-host persistence of S. iniae. This study aimed to compare biofilm formation by isolates representing different S. iniae genotypes and to investigate the effect of biofilm formation on environmental persistence and resistance to common disinfectants and antimicrobials. Eleven clinical isolates of S. iniae representing 4 distinct genetic groups and diverse host types were assessed for their ability to form biofilms. Planktonic bacteria or mature biofilms were exposed to in vitro aquatic microcosms of different temperatures to quantify the number of culturable bacteria in each system over time. The minimum biofilm eradication concentration (MBEC) assay® system was used to determine biofilm resistance to 18 antimicrobials and 4 disinfectants commonly used in food producing animals and aquaculture, respectively. All isolates formed biofilms within 72 h. Bacteria remained culturable notably longer in the biofilm form compared to the planktonic, with a significant impact from temperature and salinity (p

Severe clinical mycobacteriosis with consistent ocular lesion localization was diagnosed in a population of 800 juvenile tank-reared Cobia (Rachycentron canadum) which experienced a sudden increase in mortality approximately 5 months after arriving into Trinidad and Tobago from Florida, USA. Moderate daily mortality (15-20 animals per day) persisted for just over 1 month. Moribund fish displayed circling behaviour and had an open-mouth gape upon death. Fish consistently presented with bilateral exophthalmia, corneal cloudiness and hyphema. Non-branching acid-fast rods were detected in aqueous humour touch preparations. Histological analysis revealed severe bilateral intra-ocular granulomatous responses in all specimens. Mycobacterium sp. was identified using a real-time PCR assay detecting the RNA polymerase β-subunit (rpoB) gene in different tissue samples. Specimens did not present with characteristic granulomatous responses usually seen in viscera. To the best of our knowledge, this represents only the third documentation of piscine mycobacterial infection presenting with only localized ocular lesions, and the second documented case of mycobacteriosis in cobia. It is, however, the first documentation of an ocular presentation of mycobacteriosis in a marine species and is the first documentation of such a presentation in cobia.

Since 2012, low-to-moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram-positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (

Veronaea botryosa has been identified as a pathogen of cultured white sturgeon Acipenser transmontanus. In 2015, samples from 19 white sturgeon were received for diagnosis, of which 14 cultured positive for V. botryosa. Intraspecific variability among V. botryosa isolates from different clinically affected hosts and geographic regions was investigated using repetitive extragenic palindromic PCR fingerprinting (rep-PCR). The rep-PCR profiles of 16 V. botryosa isolates from a human, sea turtles, and cultured fish were distinct from those of other phaeoid fungi belonging to the genera Cladophialophora and Exophiala. To gain a better understanding of the pathogenesis of V. botryosa mycosis, 5 laboratory challenge methods were evaluated in white sturgeon fingerlings. Intramuscular (IM) and intracoelomic (IC) injection challenges produced cumulative mortalities of 13.3% (8/60) and 3.3% (2/60), respectively, and V. botryosa was recovered from 100% (10/10) of dead fingerlings. Affected fish exhibited abnormal orientation and/or failure to maintain neutral buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerated skin. After 6 wk, surviving fish were euthanized, and samples of liver were taken for mycological evaluation. Viable fungus was detected in 90% and 100% of fish surviving IM and IC challenge, respectively. No V. botryosa-associated mortality was detected in other groups challenged by immersion, immersion with abrasion, or orally. Both IM and IC challenge routes appear suitable for the induction of V. botryosa infection in white sturgeon and can serve as models for the study of disease pathogenesis associated with this emergent pathogen.

Department of Medicine & Epidemiology School of Veterinary Medicine University of California-Davis

A mortality event in cultured white sturgeon Acipenser transmontanus (Richardson, 1836) sub-adults was investigated. After transfer between farms, high mortality was observed in fish, associated with back arching, abnormal swimming, and ulcerative skin lesions. Necropsy of moribund individuals revealed hemorrhagic ascites and petechial hemorrhages in the coelomic peritoneum and serosa of internal organs. Acipenserid herpesvirus 2 (AciHV-2) was isolated from external tissue samples, then identified and genotyped by sequencing of the terminase and polymerase genes. In addition, Streptococcus iniae was recovered from internal organs of affected fish. Histologic changes were limited to interstitial hematopoietic areas of the kidney and consisted of small foci of necrosis accompanied by fibrin deposition, minimal inflammatory response, and small numbers of bacterial cocci compatible with streptococci. Identity was confirmed by partial sequencing of the 16S rRNA, rpoB, and gyrB genes. Genetic fingerprinting demonstrated a genetic profile distinct from S. iniae isolates recovered from previous outbreaks in wild and cultured fish in North America, South America, and the Caribbean. Although the isolates were resistant to white sturgeon complement in serum killing assays, in vivo challenges failed to fulfill Koch's postulates. However, the clinical presentation, coupled with consistent recovery of S. iniae and AciHV-2 from moribund fish, suggests viral and bacterial co-infection were the proximate cause of death. To our knowledge, this represents the first report of AciHV-2 and S. iniae co-infection in cultured white sturgeon.

Streptococcus iniae and S. agalactiae are both economically important Gram positive bacterial pathogens affecting the globally farmed tilapia (Oreochromis spp.). Historically, control of these bacteria in tilapia culture has included biosecurity, therapeutants and vaccination strategies. Genetic gains in performance traits have been realized for Nile tilapia (Oreochromis niloticus) and interest in breeding for disease resistance has recently received attention. The goal of this study was three fold: 1) to verify previous results demonstrating heritability of S. iniae resistance in Nile tilapia families using increased numbers of fish per family; 2) to determine if realized genetic gain in resistance and/or susceptibility to S. iniae can be obtained following positive assortative mating between parents with high or low estimated breeding values (EBV); and 3) to determine if resistance to S. iniae and S. agalactiae capsular type Ib is genetically correlated. A total of 144 and 130 full sib families were challenged intraperitoneally with S. iniae and intramuscularly with S. agalactiae Ib, respectively. Cumulative mortality at test end was 46% for S. iniae and 68% for S. agalactiae. There was a high additive genetic component found for survival in fish injected with S. iniae (estimated heritability 0.52 ± 0.12) and with S. agalactiae (estimated heritability 0.38 ± 0.11). The S. iniae challenge results confirmed additive genetic variation in resistance of Nile tilapia to S. iniae. We also demonstrated via assortative mating that genetic gain for survival to S. iniae is possible. The genetic correlation between resistance to S. iniae and S. agalactiae Ib was not significantly different from zero (rg = − 0.30 ± 0.19). The lack of correlation suggests if resistance to both Streptococcus sp. is desired, selection for both traits must be simultaneous. Selection of fish to improve survival to Streptococcus sp. may require a thorough understanding of the type of pathogen prevalent in the region so that custom genetic material may be tailored to meet the needs of the individual farm and/or region.

Chryseobacterium spp. (Family Flavobacteriaceae) are emergent fish pathogens in Europe, Asia and North America. In 2016-2017, 7 bacterial isolates were recovered from posterior kidney or spleen of cultured diseased rainbow trout Oncorhynchus mykiss (n = 1), green sturgeon Acipenser medirostris (n = 1), white sturgeon A. transmontanus (n = 2), blue ram cichlid Mikrogeophagus ramirezi (n = 1), and returning fall Chinook salmon O. tshawytscha (n = 2) from different freshwater systems. Bacterial colonies were visible after 24-48 h incubation at 20°C on agar media. Isolates were Gram-negative, rod-shaped, catalase and oxidase positive. Amplification and partial sequence analysis of the 16S rRNA and gyrB genes allocated the microorganisms to the genus Chryseobacterium sharing 97.2-99.6% similarity to 6 described Chryseobacterium spp. at the 16S rRNA locus, and 87.8-99.1% similarity at gyrB. Phylogenetic analyses in conjunction with percent sequence identity suggest some of the recovered isolates may represent novel Chryseobacterium subspecies or species. The pathogenicity of 5 isolates was evaluated experimentally in rainbow trout (n = 60), brown trout Salmo trutta (n = 60) and white sturgeon (n = 36) in flow-through freshwater at 18°C. Approximately 107 CFU fish-1 was injected in the epaxial musculature of anesthetized animals. Limited mortality was observed and no bacteria were recovered from dead or moribund fish post-challenge. Thirty days post-challenge, survivors were euthanized and multiple tissues were collected and fixed for histological analysis. No consistent histopathological changes were observed in challenged or control fish. While results suggest the recovered Chryseobacterium spp. may be opportunistic pathogens, further research is warranted to better understand the role of these bacteria in fish disease.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is an important pathogen in common and koi carp Cyprinus carpio, varieties. Two main genotypes of KHV have been reported worldwide that are associated with Asian and European origins. In the USA, outbreaks of KHV diseases have been reported in different states since the early 1990s; however, the diversity of KHV is unknown. In the current study, 67 DNA samples that were extracted from clinical cases of koi tissues that were submitted for diagnosis during KHV outbreaks from 10 different states in the USA from 1999 to 2019 were used to investigate their genetic diversity. The thymidine kinase gene was amplified, sequenced, and used for phylogenetic analysis. Our results showed that the KHV isolates that were collected from the different states were clustered in the two known KHV genogroups, where 31 isolates belonged to the Asian genotype branch and 36 to the European genotype branch. The spatiotemporal analysis demonstrated fluctuation of KHV genotypes in the USA, as the main KHV genotype that was detected in koi in the USA from 1999 to 2013 was the European genotype, whereas the Asian KHV genotype appeared to emerge in the USA in 2008, increasing in incidence until 2019. The current study provides evidence on the genetic diversity of KHV in the USA. Future studies that evaluate the virulence of these genetically diverse isolates is warranted to obtain a better understanding of the epidemiology of this re-emerging pathogen. This may provide an improved awareness of the current status of KHV and help to control the disease in the koi population in the USA.

To gain a better understanding of the pathogenesis of tilapia lake virus (TiLV) infections in Nile tilapia (Oreochromis niloticus), fingerlings were challenged with a single dose of 1 × 104 TCID50 /fish of TiLV utilizing intracoelomic/intraperitoneal (ICch ) or intragastric (IGch ) routes. Acute mortalities were present in both groups, reaching 70 and 40% in ICch and IGch after 10 days, respectively. Challenged fish presented erratic swimming, lethargy, anorexia, exophthalmia and cutaneous petechiae and ecchymoses. Histological changes in challenged groups included syncytial formation, intracytoplasmic inclusion bodies and multifocal hepatocellular degeneration and necrosis. In addition, multifocal areas of mild proliferation of glial cells and lymphocytic perivascular cuffing were observed in the brain of exposed challenged groups. TiLV RNA was detected in gills and faeces of challenged fish using quantitative reverse transcriptase-PCR, as well as in the tank water holding challenged fish. Moreover, TiLV RNA was detected in scrolls obtained from formalin-fixed paraffin-embedded tissue blocks from challenged fish. Results from this study suggest that IG methods represent an additional method to study the pathogenesis of the disease in this species, as it results in infection and diseases as in naturally occurring cases and does not bypass important mucosal immune responses as injectable routes do.

Veronaea botryosa is a dematiaceous fungus and the causative agent of systemic mycosis known as “fluid belly’, one of the most important emergent diseases in sturgeon aquaculture. A better understanding of the pathogenesis of the disease requires knowledge of environmental conditions that promote colonization, infection and disease. In this study, both traditional cultured-based methods and qPCR assays were used to evaluate V. botryosa persistence in water microcosms at two different temperatures and salinities, as different environmental factors can predispose poikilotherm animals to infection. Results indicate that V. botryosa persisted significantly greater in fresh than marine water microcosms (p-values

A 25-week immersion challenge was conducted exposing Oreochromis mossambicus, Oreochromis aureus and Oreochromis urolepis hornorum to Francisella noatunensis subsp. orientalis (Fno). Two populations were compared for each fish species; 'resident fish' were defined as fish maintained in tanks since week 0 of challenge, whereas 'naïve fish' were defined as fish added to tanks once temperature in water reached26 °C at 21 weeks post-challenge. Fno genome equivalents (GEs) in water were similar in all treatments 1 h post-challenge; however, significantly lower Fno GEs were detected 2 weeks post-challenge in all tanks, and the only treatment with detectable Fno GE after 4 weeks of challenge were the O. mossambicus tanks. Twenty-one weeks post-challenge, naïve fish were stocked with 'resident' cohorts. Over a 4-week period, mortalities occurred consistently only in O. mossambicus naïve cohorts. Overall presence of granulomas in spleen of survivors was similar (55%) in all resident populations; however, in naïve populations, only O. mossambicus presented granulomas. Similarly, only O. mossambicus presented viable Fno in the spleen of survivors, and Fno GEs were only detected in O. mossambicus, and in resident O. aureus. In conclusion, the results of this study suggest different susceptibility of tilapia species to piscine francisellosis.

White abalone (Haliotis sorenseni) are critically endangered marine gastropods that are native to kelp forests in the northeastern Pacific. White abalone are highly susceptible to withering syndrome, a fatal bacterial disease caused by Candidatus Xenohaliotis californiensis (CaXc), an intracellular, order Rickettsiales prokaryote that is endemic throughout the white abalone's range in California and Mexico. Oxytetracycline (OTC) baths at a dose of 500 mg/L are successful in clearing CaXc infections from the gastrointestinal tract of infected abalone. The impact of OTC treatment on the diversity and stability of the gut microbiome in white abalone is unknown. The objectives of this study were two-fold: (1) to characterize the gastrointestinal microbiome of clinically-normal white abalone and (2) to compare the gastrointestinal microbiomes of OTC-treated white abalone to those of control animals. Gastrointestinal tracts from five OTC-treated individuals and five untreated controls were sampled at each time point: day 0, one day after the 21-day OTC treatment (day 22), and at 203 days post-treatment. Gastrointestinal tract microbiomes were analyzed after amplification and sequence of the 16S rRNA. Gastrointestinal microbiomes of untreated animals were dominated by three core bacterial phyla: Proteobacteria, Fusobacteria, and Bacteroidetes. Reduced Shannon α-diversity and absence of various phyla in the microbiome of OTC-treated animals were observed in samples at day 22. Bacterial profiles were improved in terms of α-diversity at 203 days but some bacterial phyla, mainly Fusobacteria, remained absent. All animals remained clinically normal throughout the study period and there was no significant difference in a condition index between the two groups. OTC treatment for withering syndrome appears to be clinically safe in white abalone.

Mariculture of Florida pompano Trachinotus carolinus in Central America has increased over the last few decades and it is now a highly valued food fish. High feed costs and infectious diseases are significant impediments to the expansion of mariculture. Members of the genus Megalocytivirus (MCV), subfamily Alphairidovirinae, within the family Iridoviridae, are emerging pathogens that negatively impact Asian mariculture. A significant mortality event in Florida pompano fingerlings cultured in Central America occurred in October 2014. Affected fish presented with abdominal distension, darkening of the skin, and periocular hemorrhages. Microscopic lesions included cytomegalic 'inclusion body-bearing cells' characterized by basophilic granular cytoplasmic inclusions in multiple organs. Transmission electron microscopy revealed arrays of hexagonal virions (155-180 nm in diameter) with electron-dense cores within the cytoplasm of cytomegalic cells. Pathological findings were suggestive of an MCV infection, and the diagnosis was later confirmed by partial PCR amplification and sequencing of the viral gene encoding the myristylated membrane protein. The viral sequence revealed that the fingerlings were infected with an MCV genotype, red seabream iridovirus (RSIV), previously reported only from epizootics in Asian mariculture. This case underscores the threat RSIV poses to global mariculture, including the production of Florida pompano in Central America.

We examined Lancefield serogroup B Streptococcus isolates recovered from diseased, cultured hybrid Striped Bass (Striped Bass Morone saxatilis × White Bass M. chrysops) and wild and cultured Gulf Killifish Fundulus grandis from coastal waters of the U.S. Gulf of Mexico (Gulf coast) and compared those isolates to strains from tilapias Oreochromis spp. reared in Mississippi, Thailand, Ecuador, and Honduras and to the original Gulf coast strain identified by Plumb et al. ( 1974 ). The isolates were subjected to phylogenetic, biochemical, and antibiotic susceptibility analyses. Genetic analysis was performed using partial sequence comparison of (1) the 16S ribosomal RNA (rRNA) gene; (2) the sipA gene, which encodes a surface immunogenic protein; (3) the cspA gene, which encodes a cell surface-associated protein; and (4) the secY gene, which encodes components of a general protein secretion pathway. Phylogenies inferred from sipA, secY, and cspA gene sequence comparisons were more discriminating than that inferred from the 16S rRNA gene sequence comparison. The U.S. Gulf coast strains showed a high degree of similarity to strains from South America and Central America and belonged to a unique group that can be distinguished from other group B streptococci. In agreement with the molecular findings, biochemical and antimicrobial resistance analyses demonstrated that the isolates recovered from the U.S. Gulf coast and Latin America were more similar to each other than to isolates from Thailand. Three laboratory challenge methods for inducing streptococcosis in Gulf Killifish were evaluated-intraperitoneal (IP) injection, immersion (IMM), and immersion plus abrasion (IMMA)-using serial dilutions of S. agalactiae isolate LADL 97-151, a representative U.S. Gulf coast strain. The dose that was lethal to 50% of test fish by 14 d postchallenge was approximately 2 CFU/fish via IP injection. In contrast, the fish that were challenged via IMM or IMMA presented cumulative mortality less than 40% by 14 d postchallenge.

Tilapia Lake Virus (TiLV) is an emergent orthomyxo-like virus affecting the global tilapia (Oreochromis sp.) aquaculture industry. Mortality in affected farms can reach 80–90% in severe outbreaks. Effective vaccines and chemotherapeutic treatments are currently unavailable. The aim of this study was to identify the virucidal efficacy of two commonly used disinfectants, namely household bleach (free-chlorine) and a Buffered Povidone-iodine (PVP Iodine) complex, against TiLV. An archived TiLV isolate was cultured on an endothelial Oreochromis mossambicus bulbus arteriosus cell line (TmB). Bleach and PVP ranging from 10 to 100 mg/L (ppm) were diluted the day of the assay in autoclaved well water collected from an aquaculture facility in California, USA and added to TiLV cultures for 0.5, 1, or 24 h. At each respective timepoint, sodium thiosulphate or fetal bovine serum were added to inactivate the available iodine and free chlorine, respectively. All aliquots were then titrated on TmB cells to determine the TCID50/ml. Virucidal reductions in titer >4 log10 TCID50/ml were obtained with ≥20 ppm free chlorine and ≥ 50 ppm available iodine after just 30 min contact time. When contact time was increased to 24 h, ≥10 ppm free chlorine and ≥ 25 ppm available iodine were required to achieve virucidal reductions in titer >4 log10 TCID50/ml. This information should be taken into consideration when developing biosecurity protocols in tilapia aquaculture to prevent the spread of TiLV.

Edwardsiella ictaluri, a Gram-negative enteric bacterium, is the known etiological agent of enteric septicemia of catfish. In the last few years, different strains have been implicated as the causative agent of mortality events in cultured fish, including Nile tilapia Oreochromis niloticus L. Due to the emergent nature of edwardsiellosis in non-ictalurid fish, little is known about the dynamics of E. ictaluri infection in tilapia. The purpose of this study was to gain a better understanding of the pathogenesis of edwardsiellosis in tilapia by determining the median lethal and infective doses, tissue targets of infection, rate of bacterial dissemination, and the specific tissue response to E. ictaluri following an immersion challenge with bacterial strains recovered from outbreak events in tilapia. In addition to histopathology assessment, the bacterial burdens in several tissues of infected fish were determined over a 2 wk course of infection using quantitative real-time PCR (qPCR). The collected data suggest the cutaneous and oral routes as the main ports of entry for the organism, which later spreads hematogenously throughout the body. Even though histopathological assessment of infected fish revealed involvement of a wide range of tissues, the severity of the necrotizing and granulomatous lesions in the spleen and head kidney, with concomitant high levels of bacterial DNA in these organs determined by qPCR, identifies them as the main targets of infection.

In this project we optimized a minimal inhibitory concentration testing protocol for Francisella noatunensis orientalis. Thirty-three F. noatunensis orientalis isolates recovered from different fish species and locations were tested, and Escherichia coli ATCC 25922 was used as a quality control reference strain. A modified cation-adjusted Mueller Hinton broth supplemented with 2% IsoVitalex and 0.1% glucose (MMH) was tested at a pH of 6.4 ± 0.1, 7.1 ± 0.1, and 7.3 ± 0.1. Growth curves generated for F. noatunensis orientalis indicated that MMH at a pH of 6.4 ± 0.1 provided optimal growth. There were no significant differences in the growth curves obtained from isolates recovered from different fish species or from fresh or marine water. The pH of 6.4 ± 0.1 in the MMH media interfered with the inhibitory properties of the potentiated sulfonamides (ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole) when using the E. coli ATCC reference strain. Minimal inhibitory concentrations of eight antimicrobials (gentamicin, enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, and oxolinic acid) were similar for all F. noatunensis orientalis isolates. The in vitro susceptibility data provided here can provide a baseline for monitoring the development of antimicrobial resistance among F. noatunensis orientalis isolates, as well as provide valuable data in the development of potential therapeutics. Received October 27, 2015; accepted April 13, 2016.