Detection and diagnosis of Panulirus argus virus 1 in captive spiny lobsters using qPCR in conjunction with histopathology and transmission electron microscopy.

Panulirus argus virus 1 (PaV1) is the first and only naturally occurring pathogenic virus described in the Caribbean spiny lobster, Panulirus argus. PaV1 infection in decapod species that commonly co-occur with P. argus, including the spotted spiny lobster Panulirus guttatus, has not been previously described. In 2016, 14 Caribbean and 5 spotted spiny lobsters were collected near Summerland Key, Florida, to supplement the resident population of the Audubon Aquarium of the Americas in New Orleans, Louisiana. After 5 months in quarantine, Caribbean and spotted spiny lobsters began to exhibit clinical signs of lethargy and dying in the molt. Initial histologic evaluation revealed intranuclear inclusion bodies in circulating hemocytes in the spongy connective tissue of the epidermis, suggesting a viral infection. Samples of hepatopancreas and hemolymph from deceased Caribbean and spotted spiny lobsters tested negative for white spot syndrome virus and positive for PaV1 using real-time quantitative polymerase chain reaction (qPCR). Intranuclear, eosinophilic to amphophilic, Cowdry type A inclusion bodies observed primarily within fixed phagocytes and circulating hemocytes in the hepatopancreas of freshly euthanized Caribbean spiny lobsters were consistent with PaV1 infection. Transmission electron microscopy revealed that hemocytes associated with hepatopancreatic tubules contained viral inclusions with location, size, and morphology consistent with previously described PaV1 infection. These findings highlight the significance of using molecular diagnostics in conjunction with histopathology and electron microscopy in the investigation and diagnosis of PaV1 in spiny lobsters. Further study is required to investigate the relationship of PaV1-associated mortality events and microscopic lesions in the spotted spiny lobster.

Streptococcus dysgalactiae: A Pathogen of Feral Populations of Silver Carp from a Fish Kill Event.

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.

Experimental inoculation of Louisiana red swamp crayfish Procambarus clarkii with white spot syndrome virus (WSSV).

The red swamp crayfish Procambarus clarkii represents an important aquaculture species responsible for over half of all commercial aquaculture profits in Louisiana, USA. White spot syndrome virus (WSSV) is highly pathogenic in crustacean species and induces mass mortality in aquaculture operations worldwide. Natural outbreaks of WSSV occur yearly in cultured populations of crayfish in Louisiana. The goal of this study was to better understand the infectivity of WSSV in P. clarkii, by determining the minimum lethal dose necessary to initiate infection and to measure the resulting cumulative mortality following infection with different doses. A real time quantitative PCR (qPCR) method was used to detect WSSV in DNA extracted from gill tissue to ensure P. clarkii study populations were WSSV-free before the start of trials. Viable viral particles were isolated from naturally infected P. clarkii gill tissue and quantified using a novel digital PCR approach. Three infectivity trials were performed, and WSSV inocula were created by serial dilution, generating 5 treatments per trial. Five crayfish (weighing ~25 g) per dilution per trial received viral inoculations. Mortality was monitored daily for the duration of the trial in order to construct a median lethal dose (LD50) curve, and probit regression analysis was used to determine LD50 concentrations of viral particles. Knowledge of the infectivity of WSSV in native crayfish populations is of critical importance to the management of the commercial crayfish aquaculture industry in Louisiana. This is the first study to investigate the infectivity and to determine the LD50 of the Louisiana strain of WSSV in native crayfish.

Improved Broth Microdilution Method for Antimicrobial Susceptibility Testing of Francisella Noatunensis Orientalis.

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.

Variations in prevalence of viral, bacterial, and rhizocephalan diseases and parasites of the blue crab (Callinectes sapidus).

Prevalence of blue crab diseases and parasites has not been consistently monitored in the Gulf of Mexico. To establish current prevalence levels and to more fully understand population dynamics, commercial landing trends, and effects of future natural and anthropogenic disasters on animal health, we measured the prevalence of white spot syndrome virus (WSSV), Loxothylacus texanus, shell disease, and Vibrio spp. in blue crabs collected from Louisiana in 2013 and the beginning of 2014. We used PCR to detect WSSV and L. texanus infections, visual gross diagnosis for L. texanus externae and shell disease, and standard microbiological culture techniques and biochemical testing for Vibrio spp. We found no crabs infected with WSSV or L. texanus. Absence of L. texanus parasitization was expected based on the sampled salinities and the sampling focus on large crabs. Shell disease was present at a level of 54.8% and was most prevalent in the winter and summer and least prevalent in the spring. Vibrio spp. were found in the hemolymph of 22.3% of the crabs and prevalence varied by site, season, and sex. Additionally, three of 39 crabs tested were infected with reo-like virus.

Prevalence and distribution of three protozoan symbionts in blue crab (Callinectes sapidus) populations across Louisiana, USA.

Louisiana has one of the largest blue crab (Callinectes sapidus) fisheries in the USA, but little is known about blue crab diseases, parasites, and symbionts in this area. In 2013-2014, large juvenile and adult blue crabs were collected at 4 diverse sites to determine the prevalence of the protozoan symbionts associated with black gill disease (Lagenophrys callinectes), buckshot crabs (Urosporidium crescens), and bitter crab disease (Hematodinium perezi). A high aggregate prevalence of L. callinectes (93.2%) was identified across all seasons at all 4 collection sites regardless of salinity. A moderately low aggregate prevalence of U. crescens (22.4%) was identified across all seasons and sites. Prevalence of U. crescens depended on site salinity, with only 10% of infections detected at sites with <6.3 ppt salinity, and no infections detected at the low salinity site. While L. callinectes and U. crescens are commensal parasites of blue crabs, infections can result in unmarketable and unappealing meat. In the Louisiana fishery, H. perezi has been blamed circumstantially for adult mortalities in the low salinity nearshore fishing grounds. Despite this, H. perezi was not detected in any of the large crabs sampled, even from the low salinity sites. The prevalence data reported here for these 3 protozoans are the first to include blue crabs sampled seasonally at multiple locations along the Louisiana coast over the period of a year.

Disease, parasite, and commensal prevalences for blue crab Callinectes sapidus at shedding facilities in Louisiana, USA.

Blue crab diseases, parasites, and commensals are not well studied in the Gulf of Mexico, and their prevalence rates have only been sporadically determined. Commercial soft shell shedding facilities in Louisiana experience high mortality rates of pre-molt crabs, and some of these deaths may be attributable to diseases or parasites. During the active shedding season in 2013, we determined the prevalence of shell disease, Vibrio spp., Lagenophrys callinectes, and Hematodinium perezi at 4 commercial shedding facilities along the Louisiana coast. We also detected Ameson michaelis and reo-like virus infections. Shell disease was moderately prevalent at rates above 50% and varied by shedding facility, collection month, and crab size. Vibrio spp. bacteria were prevalent in the hemolymph of 37% of the pre-molt crabs. Lagenophrys callinectes was highly prevalent in the pre-molt crabs, but because it is a commensal species, it may not cause high mortality rates. Hematodinium perezi was absent in all pre-molt crabs.

Betanodavirus meningoencephalitis in an Atlantic blue marlin.

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus (Nodaviridae, Betanodavirus). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin (Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans.

Epidemiological Cutoff Values for Standard Broth Microdilution Susceptibility Testing of Flavobacterium columnare Isolated from Fishes.

Flavobacterium columnare, the causative agent of columnaris disease in a large variety of freshwater fish, is a major problem in commercial aquaculture. A limited number of antimicrobial therapies are available to control this disease; therefore, these agents must be used judiciously. To facilitate effective monitoring for changes in susceptibility, the Clinical Laboratory Standards Institute (CLSI) has a standard broth microdilution test method specific for F. columnare. However, there are no CLSI-approved criteria (termed epidemiological cutoff values [ECVs]) to interpret results. Nevertheless, researchers have developed provisional ECVs based on testing by one laboratory. To satisfy CLSI data requirements, three laboratories used the standard method to generate additional antimicrobial susceptibility data against ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, oxolinic acid, oxytetracycline, sulfadimethoxine/ormetoprim, and sulfamethoxazole-trimethoprim using 109 F. columnare isolates. The new data combined with previously published data from 120 F. columnare isolates were analyzed and ECVs proposed to CLSI. Of the 10 antimicrobials, ECVs were approved for ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid, and oxytetracycline, which were published in the 2020 edition of the CLSI document VET04 performance standards. These ECVs will help microbiologists categorize decreased antimicrobial susceptibility among F. columnare and will help in surveillance efforts to ensure judicious antimicrobial use.

Epidemiological Cutoff Values for Standard Broth Microdilution and Disk Diffusion Susceptibility Testing of Aeromonas hydrophila Isolated from Fish.

Aeromonas hydrophila and other closely related Aeromonas species cause motile aeromonad septicemia, a common fish disease. The disease affects many aquaculture sectors potentially requiring antimicrobial treatments. Therefore, researchers and laboratory diagnosticians need criteria called epidemiological cutoff values (ECVs) to determine whether a bacterial isolate has developed decreased susceptibility to an antimicrobial. To generate ECVs for this bacterium, we assembled a diverse collection of 245 isolates previously identified as A. hydrophila from fish. Using rpoD sequencing, we confirmed that 97 of the 245 isolates were A. hydrophila. We allocated the isolates among three laboratories and tested their susceptibility against eight antimicrobials using standard Clinical and Laboratory Standards Institute (CLSI) disk diffusion and broth microdilution methods. The resulting frequency distributions were statistically analyzed to determine wild-type cutoff estimates, which, along with scatterplots, were used to estimate potential ECVs. In collaboration with the CLSI, aquaculture working group, we proposed ECVs for six of the eight antimicrobials tested. Subsequently, the CLSI Subcommittee on Veterinary Antimicrobial Susceptibility Testing reviewed our data and approved the ECVs to be added to the 2020 edition of the VET04 performance standards for antimicrobial susceptibility testing of aquatic bacteria.

Interaction of Francisella asiatica with tilapia (Oreochromis niloticus) innate immunity.

Members of the genus Francisella are facultative intracellular bacteria that cause important diseases in a wide variety of animals worldwide, including humans and fish. Several genes that are important for intramacrophage survival have been identified, including the iglC gene, which is found in the iglABCD operon in the Francisella sp. pathogenicity island (FPI). In the present study, we examined the interaction of wild-type Francisella asiatica and a Delta iglC mutant strain with fish serum and head kidney-derived macrophages (HKDM). Both the wild-type and the mutant strains were resistant to killing by normal and heat-inactivated sera. The wild-type F. asiatica is able to invade tilapia head kidney-derived macrophages and replicate vigorously within them, causing apoptosis and cytotoxicity in the macrophages at 24 and 36 h postinfection. The Delta iglC mutant, however, is defective for survival, replication, and the ability to cause cytotoxicity in HKDM, but the ability is restored when the mutant is complemented with the iglC gene. Uptake by the HKDM was mediated partially by complement and partially by macrophage mannose receptors, as demonstrated by in vitro assays. Light and electron microscopy analysis of the infected macrophages revealed intracellular bacteria present in a tight vacuole at 2 h postinoculation and the presence of numerous bacteria in spacious vacuoles at 12 h postinfection, with some bacteria free in the cytoplasm.

In vitro and in vivo efficacy of florfenicol for treatment of Francisella asiatica infection in tilapia.

Francisella asiatica is a recently described, Gram-negative, facultative intracellular fish pathogen, known to be the causative agent of francisellosis in warm-water fish. Francisellosis outbreaks have increased in frequency among commercial aquaculture operations and have caused severe economic losses in every case reported. The lack of effective treatments for piscine francisellosis led us to investigate the potential efficacy of florfenicol for inhibition of F. asiatica in vitro and as an oral therapeutic agent in vivo. The MIC of florfenicol for F. asiatica, as determined by the broth dilution method, was 2 µg/ml, which indicates its potential efficacy as a therapeutic agent for treatment of francisellosis. The intracellular susceptibility of the bacterium to florfenicol in tilapia head kidney-derived macrophages (THKDM) was also investigated. Addition of florfenicol to the medium at 10 µg/ml was sufficient to significantly reduce bacterial loads in the THKDM in vitro. Cytotoxicity assays done in infected THKDM also demonstrated drug efficacy in vivo, as determined by lactate dehydrogenase (LDH) release. Levels of LDH released from infected THKDM were significantly lower in macrophages treated with florfenicol (P < 0.001) than in untreated cells. In medicated-feed trials, fish were fed 15 mg of florfenicol/kg of fish body weight for 10 days, and the feeding was initiated at either 1, 3, or 6 days postchallenge. Immersion challenges resulted in 30% mean percent survival in nontreated fish, and fish receiving medicated feed administered at 1 and 3 days postinfection showed higher mean percent survival (100% and 86.7%, respectively). A significant decrease (P < 0.001) in bacterial numbers (number of CFU/g of spleen tissue) was observed in treated groups compared to nontreated infected fish at both 1 and 3 days postchallenge. There were no differences in bacterial burden in the spleens between fish treated 6 days postchallenge and untreated controls. In conclusion, if florfenicol is administered during early stages of infection, it has the potential for effectively treating piscine francisellosis, including the capacity for intracellular penetration and bacterial clearance.

Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella noatunensis subsp. orientalis.

Members of the genus Francisella are small Gram-negative facultative intracellular bacteria that cause francisellosis in a wide variety of fish species worldwide. F. noatunensis subsp. orientalis has been recently described as a warm-water pathogen of tilapia Oreochromis spp. In this study, a quantitative real-time polymerase chain reaction (qPCR) TaqMan probe assay was developed to rapidly and accurately detect and quantify F. noatunensis subsp. orientalis from fish tissue. The target region of the assay was the F. tularensis iglC gene homologue previously found in F. noatunensis subsp. orientalis. Probe specificity was confirmed by the lack of signal and cross-reactivity with 12 common fish pathogens, 2 subspecies of F. tularensis, F. noatunensis subsp. noatunensis, and tilapia tissue. The range of linearity was determined to be 50 fg to 1.4 mg, and the lower limit of detection was 50 fg of DNA (equivalent to approximately 25 genome equivalents) per reaction. A similar sensitivity was observed with DNA extracted from a mixture of F. noatunensis subsp. orientalis and fish tissue. The assay was also able to detect and quantify F. noatunensis subsp. orientalis from the spleens of experimentally infected tilapia. No signal was observed in the control groups. In conclusion, we have developed a highly sensitive and specific assay that can be used for the specific identification and quantification of F. noatunensis subsp. orientalis.

Whole-Genome Sequence of Photobacterium damselae subsp. piscicida Strain 91-197, Isolated from Hybrid Striped Bass (Morone sp.) in the United States.

Photobacterium damselae subsp. piscicida is a causative bacterium of fish pasteurellosis, which has caused serious economic damage to aquaculture farms worldwide. Here, the whole-genome sequence of P. damselae subsp. piscicida 91-197, isolated in the United States, suggests that this genome consists of two chromosomes and two plasmids.

Complete Genome Sequence of Photobacterium damselae subsp. piscicida Strain OT-51443 Isolated from Yellowtail (Seriola quinqueradiata) in Japan.

Pseudotuberculosis caused by infection of Photobacterium damselae subsp. piscicida has caused serious economic damages to aquaculture farms worldwide. Here, the whole-genome sequence of P. damselae subsp. piscicida strain OT-51443, isolated in Japan, was determined and suggests that this genome consists of two chromosomes and five plasmids.

Draft Genome Sequences of Edwardsiella ictaluri Strains LADL11-100 and LADL11-194 Isolated from Zebrafish Danio rerio.

Here, we report the draft genome sequences of Edwardsiella ictaluri strains LADL11-100 and LADL11-194, two isolates from natural outbreaks of edwardsiellosis in the zebrafish Danio rerio, as well as the sequences of the plasmids carried by the zebrafish strain of E. ictaluri.

Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida.

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.

Phenotypic and genotypic heterogeneity among Streptococcus iniae isolates recovered from cultured and wild fish in North America, Central America and the Caribbean islands.

Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.

Edwardsiellosis caused by Edwardsiella ictaluri in laboratory populations of Zebrafish Danio rerio.

We report the first cases of Edwardsiella ictaluri causing epizootics in laboratory populations of Zebrafish Danio rerio. Edwardsiella ictaluri is primarily recognized as a disease of catfish species and is known to cause an economically important bacterial disease of farm-raised catfish in the USA and abroad; however, it has been isolated on occasion from 10 other genera of nonictalurid fishes. We isolated E. ictaluri from moribund Zebrafish held in quarantine at two different universities in two states and from a research facility in a third state between February 23 and December 6, 2011. Edwardsiellosis in Zebrafish can be described as a severe systemic disease characterized by tissue necrosis and the presence of large numbers of extracellular and intracellular bacteria, often within macrophages. The kidneys (pronephros and mesonephros), spleen, nares, and forebrain were the most commonly and severely affected tissues. In outbreaks, mortality was acute and numerous fish died over a 1-2 week period. Mortality continued until the majority of the population was lost, at which time the remaining fish were euthanized. In addition to these cases, four cultures of bacteria isolated from Zebrafish by another diagnostic laboratory were submitted to the Louisiana Aquatic Diagnostic Laboratory for identification and were confirmed as E. ictaluri. In total, eight cultures of E. ictaluri from Zebrafish from Louisiana, Massachusetts, Pennsylvania, and Florida were identified. The isolates were confirmed as E. ictaluri by biochemical phenotype, API 20E (bioMérieux), and amplification and sequencing of a portion of the 16S rRNA gene. Edwardsiella ictaluri isolates from Zebrafish are believed to comprise a unique group and were differentiated from catfish isolates by exhibiting weaker motility, autoaggregation in broth, a different plasmid profile (two plasmids of 4.0 and 3.5 kb), a different API 20E code (4204000), and lack of lipopolysaccharide recognition with Mab Ed9.

Genetic analysis and antimicrobial susceptibility of Francisella noatunensis subsp. orientalis (syn. F. asiatica) isolates from fish.

Francisella noatunensis subsp. orientalis (syn. F. asiatica) (Fno) is an emergent fish pathogen that causes acute to chronic disease in a wide variety of freshwater, brackish and marine fish. Due to the emergent nature of this bacterium, established protocols to measure antimicrobial susceptibility are lacking. In this project we compare three different methods to examine the antimicrobial susceptibility (Etest, broth microdilution and disk diffusion) of 10 different isolates of Fno from two different fish species and four different geographic outbreaks from 2006 to 2010. PCR mediated genomic fingerprinting (rep-PCR) performed on the different isolates confirmed genetic homogeneity amongst the different isolates. The in vitro susceptibility data presented here provides important baseline data for future research monitoring the development of antibiotic resistance among Fno isolates as well as provides invaluable data for the development of potential therapeutics.