Making Science Computable Using Evidence-Based Medicine on Fast Healthcare Interoperability Resources: Standards Development Project.

BACKGROUND: Evidence-based medicine (EBM) has the potential to improve health outcomes, but EBM has not been widely integrated into the systems used for research or clinical decision-making. There has not been a scalable and reusable computer-readable standard for distributing research results and synthesized evidence among creators, implementers, and the ultimate users of that evidence. Evidence that is more rapidly updated, synthesized, disseminated, and implemented would improve both the delivery of EBM and evidence-based health care policy. OBJECTIVE: This study aimed to introduce the EBM on Fast Healthcare Interoperability Resources (FHIR) project (EBMonFHIR), which is extending the methods and infrastructure of Health Level Seven (HL7) FHIR to provide an interoperability standard for the electronic exchange of health-related scientific knowledge. METHODS: As an ongoing process, the project creates and refines FHIR resources to represent evidence from clinical studies and syntheses of those studies and develops tools to assist with the creation and visualization of FHIR resources. RESULTS: The EBMonFHIR project created FHIR resources (ie, ArtifactAssessment, Citation, Evidence, EvidenceReport, and EvidenceVariable) for representing evidence. The COVID-19 Knowledge Accelerator (COKA) project, now Health Evidence Knowledge Accelerator (HEvKA), took this work further and created FHIR resources that express EvidenceReport, Citation, and ArtifactAssessment concepts. The group is (1) continually refining FHIR resources to support the representation of EBM; (2) developing controlled terminology related to EBM (ie, study design, statistic type, statistical model, and risk of bias); and (3) developing tools to facilitate the visualization and data entry of EBM information into FHIR resources, including human-readable interfaces and JSON viewers. CONCLUSIONS: EBMonFHIR resources in conjunction with other FHIR resources can support relaying EBM components in a manner that is interoperable and consumable by downstream tools and health information technology systems to support the users of evidence.

Diagnosis of piscine francisellosis in Largemouth Bass from a public display exhibit in north-central Florida, USA.

OBJECTIVE: The Largemouth Bass Micropterus salmoides is an important freshwater fish that is native to the southeastern United States and is cultured for conservation, food, and for the sports fishing industry. Francisella orientalis is a globally distributed bacterial pathogen of warmwater fish species and is associated with granulomatous inflammation and high mortalities. Outbreaks of piscine francisellosis in the United States have been reported in only a few fish species. This study describes three case presentations of francisellosis in Largemouth Bass from a public display system in north-central Florida. Additionally, laboratory-controlled immersion challenges using an F. orientalis isolate from tilapia Oreochromis spp. evaluate susceptibility of Largemouth Bass fingerlings to F. orientalis infection and mortality through this exposure route. METHODS: Necropsy, histologic examination, immunohistochemistry, bacterial recovery and culture, and quantitative polymerase chain reaction were used as diagnostic tools to evaluate both the affected display fish and the immersion-challenged fingerlings. RESULT: Although the display fish and immersion-challenged fingerlings presented with nonspecific clinical signs, gross and histological changes were indicative of granulomatous disease. Immunohistochemical and molecular testing methods confirmed F. orientalis infection in affected fish. CONCLUSION: The three case presentations described here mark the first reporting of naturally occurring piscine francisellosis in Largemouth Bass that were held in a public display exhibit. Additionally, causality was proven in the Largemouth Bass fingerlings through the immersion challenges. These findings demonstrate susceptibility through immersion-based exposure and assert that francisellosis should be considered among the list of differential diagnoses for Largemouth Bass with granulomatous disease.

Striking a match between FHIR-based patient data and FHIR-based eligibility criteria.

Inputs and Outputs: The Strike-a-Match Function, written in JavaScript version ES6+, accepts the input of two datasets (one dataset defining eligibility criteria for research studies or clinical decision support, and one dataset defining characteristics for an individual patient). It returns an output signaling whether the patient characteristics are a match for the eligibility criteria. Purpose: Ultimately, such a system will play a "matchmaker" role in facilitating point-of-care recognition of patient-specific clinical decision support. Specifications: The eligibility criteria are defined in HL7 FHIR (version R5) EvidenceVariable Resource JSON structure. The patient characteristics are provided in an FHIR Bundle Resource JSON including one Patient Resource and one or more Observation and Condition Resources which could be obtained from the patient's electronic health record. Application: The Strike-a-Match Function determines whether or not the patient is a match to the eligibility criteria and an Eligibility Criteria Matching Software Demonstration interface provides a human-readable display of matching results by criteria for the clinician or patient to consider. This is the first software application, serving as proof of principle, that compares patient characteristics and eligibility criteria with all data exchanged using HL7 FHIR JSON. An Eligibility Criteria Matching Software Library at https://fevir.net/110192 provides a method for sharing functions using the same information model.

Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California.

OBJECTIVE: The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS: Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT: Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION: The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

Pathology, microbiology, and genetic diversity associated with Erysipelothrix rhusiopathiae and novel Erysipelothrix spp. infections in southern sea otters (Enhydra lutris nereis).

Erysipelothrix spp., including E. rhusiopathiae, are zoonotic bacterial pathogens that can cause morbidity and mortality in mammals, fish, reptiles, birds, and humans. The southern sea otter (SSO; Enhydra lutris nereis) is a federally-listed threatened species for which infectious disease is a major cause of mortality. We estimated the frequency of detection of these opportunistic pathogens in dead SSOs, described pathology associated with Erysipelothrix infections in SSOs, characterized the genetic diversity and antimicrobial susceptibility of SSO isolates, and evaluated the virulence of two novel Erysipelothrix isolates from SSOs using an in vivo fish model. From 1998 to 2021 Erysipelothrix spp. were isolated from six of >500 necropsied SSOs. Erysipelothrix spp. were isolated in pure culture from three cases, while the other three were mixed cultures. Bacterial septicemia was a primary or contributing cause of death in five of the six cases. Other pathology observed included suppurative lymphadenopathy, fibrinosuppurative arteritis with thrombosis and infarction, bilateral uveitis and endophthalmitis, hypopyon, petechia and ecchymoses, mucosal infarction, and suppurative meningoencephalitis and ventriculitis. Short to long slender Gram-positive or Gram-variable bacterial rods were identified within lesions, alone or with other opportunistic bacteria. All six SSO isolates had the spaA genotype-four isolates clustered with spaA E. rhusiopathiae strains from various terrestrial and marine animal hosts. Two isolates did not cluster with any known Erysipelothrix spp.; whole genome sequencing revealed a novel Erysipelothrix species and a novel E. rhusiopathiae subspecies. We propose the names Erysipelothrix enhydrae sp. nov. and Erysipelothrix rhusiopathiae ohloneorum ssp. nov. respectively. The type strains are E. enhydrae UCD-4322-04 and E. rhusiopathiae ohloneorum UCD-4724-06, respectively. Experimental injection of tiger barbs (Puntigrus tetrazona) resulted in infection and mortality from the two novel Erysipelothrix spp. Antimicrobial susceptibility testing of Erysipelothrix isolates from SSOs shows similar susceptibility profiles to isolates from other terrestrial and aquatic animals. This is the first description of the pathology, microbial characteristics, and genetic diversity of Erysipelothrix isolates recovered from diseased SSOs. Methods presented here can facilitate case recognition, aid characterization of Erysipelothrix isolates, and illustrate assessment of virulence using fish models.

Enhancement of protective efficacy of innate immunostimulant based formulations against yolk sac infection in young chicks.

This study was conducted to characterize and compare the protective effects of various innate immune stimulants against yolk sac infection (YSI) caused by an avian pathogenic Escherichia coli in young chicks. The immune stimulants were administered alone or in various combinations of unmethylated CpG oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (Poly I:C), and avian antimicrobial peptides (AMPs). Routes included in ovo or in ovo followed by a subcutaneous (S/C) injection. CpG alone and in combination with Poly I:C, truncated avian cathelicidin (CATH)-1(6-26), avian beta defensin (AvBD)1, and CATH-1(6-26) + AvBD1, were administered in ovo to 18-day-old embryonated eggs for gene expression and challenge studies. Next, CpG alone and the potentially effective formulation of CpG + Poly I:C, were administrated via the in ovo route using 40 embryonated eggs. At 1 day post-hatch, half of each group also received their respective treatments via the S/C route. Four hours later, all chicks were challenged using E. coli strain EC317 and mortalities were recorded for 14 d. The first challenge study revealed that amongst the single use and combinations of CpG with different innate immune stimulants, a higher protection and a lower clinical score were offered by the combination of CpG + Poly I:C. The second challenge study showed that this combination (CpG + Poly I:C) provides an even higher level of protection when a second dose is administered via the S/C route at 1 day post-hatch. The current research highlights the efficacy of a combination of CpG + Poly I:C administered either in ovo or in ovo along with a S/C injection and its potential use as an alternative to antibiotics against yolk sac infection in young chicks.

Silica nanoparticles are novel aqueous additive mitigating heavy metals toxicity and improving the health of African catfish, Clarias gariepinus.

Silica nanoparticles (SiNPs) are among the non-toxic nanoparticles (NPs) that have magnetic capabilities. It is hypothesized that SiNPs may be able to reduce toxic effects exerted by a mixture of lead (Pb) and mercury (Hg) in African catfish Clarias gariepinus. The in vitro magnetic potential of SiNPs to absorb Pb and Hg was tested. Fish (N = 240) were divided into four groups in triplicates for 30 days. The first group served as control and the second group (SiNPs) was exposed to 1/10 of 96 h LC50 of SiNPs (14.45 mg/L). The third group (HMM) was exposed to 1/10 of 96-h LC50 of a mixture of mercury chloride (HgCl2) and lead chloride (PbCl2) equal to 0.04 mg/ L and 23.1 mg/L. The fourth group (SiNPs+ HMM) was exposed to a suspension composed of SiNPs, HgCl2, and PbCl2 at the same concentrations as the third group. Results showed that fish exposed to heavy metals revealed the following consequences; a significant decrease in hematological, immunological (complement-3 and nitric oxide), and antioxidants (total antioxidant capacity, glutathione peroxidase, superoxide dismutase, and catalase) indices, down-regulation of IL-1ß, IL-8, TGF-ß, NF-κß, HSP70, and Hepcidin genes, the highest mortality rate (48.33%), higher values of alkaline phosphatase, alanine, and aspartate aminotransferases, urea, creatinine, and branchial malondialdehyde, marked up-regulation of CC chemokine and CXC chemokines, and high HMs residues levels in muscles. Extensive pathology showed degeneration with diffuse vacuolation of hepatopancreatic cells and hemorrhage in the HMM group. Interestingly, the exposed group to SiNPs and HMM demonstrated a decline of HMs concentration in fish muscles and modulated the abovementioned parameters with the regeneration of histological alterations of liver and gills. Based on the study outcomes, we highlight the importance and the safety of SiNPs as a novel aqueous additive to alleviate HMs toxicity and recommend using SiNPs for enhancing fish performance for sustaining aquaculture without adverting safety of human health by their little accumulation in muscular tissue.

Development of a quantitative polymerase chain reaction assay for detection of the aetiological agents of piscine lactococcosis.

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.

Development and efficacy of Streptococcus iniae live-attenuated vaccines in Nile tilapia, Oreochromis niloticus.

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%.

Isolation and characterization of Lactococcus garvieae from rainbow trout, Onchorhyncus mykiss, from California, USA.

Lactococcus garvieae is an emergent bacterial pathogen of salmonid fish in North America that causes acute infections particularly at water temperatures above 15°C. During 2020, L. garvieae was detected in rainbow trout, Onchorhyncus mykiss, cultured in Southern California and the Eastern Sierras. Infected fish exhibited high mortalities and nonspecific clinical signs of lethargy, erratic swimming, dark skin pigmentation, and exophthalmia. Macroscopic changes included external and internal hemorrhages, mainly in the eyes, liver, coelomic fat, intestine, and brain. Histological examination revealed splenitis, branchitis, panophthalmitis, hepatitis, enteritis, and coelomitis, with variable degrees of tissue damage among evaluated fish. Pure colonies of L. garvieae were isolated from infected trout and specific PCR primers for L. garvieae confirmed the preliminary diagnosis. Multilocus sequence analysis showed that the strains recovered from diseased trout represent a novel genetic group. Isolates were able to form biofilms within 24 h that increased their resistance to disinfection by hydrogen peroxide. Laboratory challenge methods for inducing lactococcosis in steelhead trout, O. mykiss, were evaluated by intracoelomic injection with serial dilutions of L. garvieae. The median lethal dose 21 days post challenge was ∼20 colony-forming units/fish. Experimentally infected trout presented similar clinical signs, gross changes, and microscopic lesions as those with natural disease, fulfilling Koch's postulates and demonstrating the high virulence of the recovered strains.

Genetics and pathology associated with Klebsiella pneumoniae and Klebsiella spp. isolates from North American Pacific coastal marine mammals.

Southern sea otters (SSO: Enhydra lutris nereis) are a federally-listed threatened subspecies found almost exclusively in California, USA. Despite their zoonotic potential and lack of host specificity, K. pneumoniae and Klebsiella spp. have largely unknown epizootiology in SSOs. Klebsiella pneumoniae is occasionally isolated at necropsy, but not from live SSOs. Hypermucoviscous (HMV) K. pneumoniae strains are confirmed pathogens of Pacific Basin pinnipeds, but have not been previously isolated from SSOs. We characterized the virulence profiles of K. pneumoniae isolates from necropsied SSOs, evaluated killing of marine mammal K. pneumoniae following in vitro exposure to California sea lion (CSL: Zalophanus californianus) whole blood and serum, and characterized lesion patterns associated with Klebsiella spp. infection in SSOs. Four of 15 SSO K. pneumoniae isolates were HMV and all were recovered from SSOs that stranded during 2005. Many K. pneumoniae infections were associated with moderate to severe pathology as a cause of death or sequela. All HMV infections were assessed as a primary cause of death or as a direct result of the primary cause of death. Klebsiella-infected SSOs exhibited bronchopneumonia, tracheobronchitis and/or pleuritis, enteritis, Profilicollis sp. acanthocephalan peritonitis, septic peritonitis, and septicemia. All SSO HMV isolates were capsular type K2, the serotype most associated with HMV infections in CSLs. Multiplex PCR revealed two distinct virulence gene profiles within HMV isolates and two within non-HMV isolates. In vitro experiments investigating CSL whole blood and serum killing of K. pneumoniae suggest that HMV isolates are more resistant to serum killing than non-HMV isolates.

Isolation, Identification and Characterization of a Novel Megalocytivirus from Cultured Tilapia (Oreochromis spp.) from Southern California, USA.

In spring 2019, diseased four-month-old tilapia (Oreochromis spp.) from an aquaculture farm in Southern California, USA were received for diagnostic evaluation with signs of lethargy, anorexia, abnormal swimming, and low-level mortalities. At necropsy, non-specific external lesions were noted including fin erosion, cutaneous melanosis, gill pallor, and coelomic distension. Internal changes included ascites, hepatomegaly, renomegaly, splenomegaly, and multifocal yellow-white nodules in the spleen and kidney. Cultures of spleen and kidney produced bacterial colonies identified as Francisella orientalis. Homogenized samples of gill, brain, liver, spleen, and kidney inoculated onto Mozambique tilapia brain cells (OmB) developed cytopathic effects, characterized by rounding of cells and detaching from the monolayer 6-10 days post-inoculation at 25 °C. Transmission electron microscopy revealed 115.4 ± 5.8 nm icosahedral virions with dense central cores in the cytoplasm of OmB cells. A consensus PCR, targeting the DNA polymerase gene of large double-stranded DNA viruses, performed on cell culture supernatant yielded a sequence consistent with an iridovirus. Phylogenetic analyses based on the concatenated full length major capsid protein and DNA polymerase gene sequences supported the tilapia virus as a novel species within the genus Megalocytivirus, most closely related to scale drop disease virus and European chub iridovirus. An intracoelomic injection challenge in Nile tilapia (O. niloticus) fingerlings resulted in 39% mortality after 16 days. Histopathology revealed necrosis of head kidney and splenic hematopoietic tissues.

Genetic characterization of Flavobacterium columnare isolates from the Pacific Northwest, USA.

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.

Quantitative PCR for detection and quantification of Veronaea botryosa in fish and environmental samples.

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 ß-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.

Making science computable: Developing code systems for statistics, study design, and risk of bias.

The COVID-19 crisis led a group of scientific and informatics experts to accelerate development of an infrastructure for electronic data exchange for the identification, processing, and reporting of scientific findings. The Fast Healthcare Interoperability Resources (FHIR®) standard which is overcoming the interoperability problems in health information exchange was extended to evidence-based medicine (EBM) knowledge with the EBMonFHIR project. A 13-step Code System Development Protocol was created in September 2020 to support global development of terminologies for exchange of scientific evidence. For Step 1, we assembled expert working groups with 55 people from 26 countries by October 2020. For Step 2, we identified 23 commonly used tools and systems for which the first version of code systems will be developed. For Step 3, a total of 368 non-redundant concepts were drafted to become display terms for four code systems (Statistic Type, Statistic Model, Study Design, Risk of Bias). Steps 4 through 13 will guide ongoing development and maintenance of these terminologies for scientific exchange. When completed, the code systems will facilitate identifying, processing, and reporting research results and the reliability of those results. More efficient and detailed scientific communication will reduce cost and burden and improve health outcomes, quality of life, and patient, caregiver, and healthcare professional satisfaction. We hope the achievements reached thus far will outlive COVID-19 and provide an infrastructure to make science computable for future generations. Anyone may join the effort at https://www.gps.health/covid19_knowledge_accelerator.html.

Efficacy of nano zinc oxide dietary supplements on growth performance, immunomodulation and disease resistance of African catfish Clarias gariepinus.

Zinc (Zn) is an important trace element in fish diets that is required for growth, immunity and antioxidant defense mechanisms. The current study assessed the effects of both organic and nanoparticle zinc oxide (ZnO and ZnO-NPs, respectively) on growth performance, immune response and the antimicrobial effect against Pseudomonas aeruginosa in African catfish Clarias gariepinus. Fish were fed either a control diet or diets supplemented with organic ZnO at concentrations of 20 and 30 mg kg-1 or ZnO-NPs at concentrations of 20 and 30 mg kg-1. After 60 d, a subset of the fish was injected intraperitoneally with 3 × 107 CFU ml-1 of P. aeruginosa. Results showed that body weight gain, feed conversion ratio and specific growth rates were significantly increased in ZnO-NPs20 compared to all other groups. The dietary supplementation with 20 mg kg-1 of ZnO-NPs improved the antioxidant status of fish. Moreover, IgM, lysozyme and nitric oxide showed a significant increase in the fish which received the ZnO-NPs20-supplemented diet. A significant upregulation of growth and stress-related genes was seen in the ZnO-NPs20-supplemented group compared to other groups. However, there was no significant difference in the expression of immune-related genes among ZnO-NPs20, ZnO-NPs30 and ZnO30 groups. These findings highlight the potential use of nano-ZnO for improving growth performance, antioxidant status, immunological status and antibacterial activity against P. aeruginosa in African catfish.

Development of IglC and GroEL recombinant vaccines for francisellosis in Nile tilapia, Oreochromis niloticus.

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.

Outbreaks of severe myositis in cultured white sturgeon (Acipenser transmontanus L.) associated with Streptococcus iniae.

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.

Genetic Diversity of Cyprinid Herpesvirus 3 from Different Geographical Locations during 1999-2019 in the United States of America.

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.

Larva of greater wax moth Galleria mellonella is a suitable alternative host for the fish pathogen Francisella noatunensis subsp. orientalis.

BACKGROUND: Francisella noatunensis subsp. orientalis (Fno) is the etiological agent of francisellosis in cultured warm water fish, such as tilapia. Antibiotics are administered to treat the disease but a better understanding of Fno infection biology will inform improved treatment and prevention measures. However, studies with native hosts are costly and considerable benefits would derive from access to a practical alternative host. Here, larvae of Galleria mellonella were assessed for suitability to study Fno virulence. RESULTS: Larvae were killed by Fno in a dose-dependent manner but the insects could be rescued from lethal doses of bacteria by antibiotic therapy. Infection progression was assessed by histopathology (haematoxylin and eosin staining, Gram Twort and immunohistochemistry) and enumeration of bacteria recovered from the larval haemolymph on selective agar. Fno was phagocytosed and could survive intracellularly, which is consistent with observations in fish. Virulence of five Fno isolates showed strong agreement between G. mellonella and red Nile tilapia hosts. CONCLUSIONS: This study shows that an alternative host, G. mellonella, can be applied to understand Fno infections, which will assist efforts to identify solutions to piscine francisellosis thus securing the livelihoods of tilapia farmers worldwide and ensuring the production of this important food source.