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.

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.

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.

Validation of environmental DNA sampling for determination of Ceratonova shasta (Cnidaria: Myxozoa) distribution in Plumas National Forest, CA.

Ceratonova shasta is the etiological agent of myxozoan-associated enteronecrosis in North American salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and a freshwater fabriciid annelid. The success and survival of annelids can be enhanced by flow moderation by dams, and through the erosion of fine sediments into stream channels following wildfires. In this study, the presence of C. shasta environmental/ex-host DNA (eDNA) in river water and substrate samples collected from areas affected by recent fire activity in California, USA, was investigated. Additionally, DNA loads in the environment were compared to C. shasta infection in sentinel-exposed rainbow trout (Oncorhynchus mykiss). Significant associations between C. shasta detection in environmental samples and location within a wildfire perimeter (p = 0.002), between C. shasta detection in sentinel fish and exposure location within a wildfire perimeter (p = 0.015), and between C. shasta detection in fish and locations where water temperature was above the median (p < 0.001) were observed. Additionally, a higher prevalence of C. shasta infection in fish was detected where C. shasta was also detected in environmental samples (p < 0.001). Results suggest that pathogen eDNA sampling can be used as a non-invasive, rapid, specific, and sensitive method for establishing risk of C. shasta infection in wild populations. Knowledge of the complete life cycle of the target parasite, including ecology of each host, can inform the choice of eDNA sampling strategy. Environmental DNA sampling also revealed a novel species of Ceratonova, not yet observed in a host.

Identification of Chryseobacterium spp. isolated from clinically affected fish in California, USA.

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.

Distribution and Prevalence of Myxobolus cerebralis in Postfire Areas of Plumas National Forest: Utility of Environmental DNA Sampling.

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.

Co-infection of Acipenserid herpesvirus 2 (AciHV-2) and Streptococcus iniae in cultured white sturgeon Acipenser transmontanus.

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.

Diversity of Veronaea botryosa from different hosts and evaluation of laboratory challenge models for phaeohyphomycosis in Acipenser transmontanus.

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.

Respiratory arsenate reductase as a bidirectional enzyme.

The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

HYPERMUCOVISCOUS KLEBSIELLA PNEUMONIAE ISOLATES FROM STRANDED AND WILD-CAUGHT MARINE MAMMALS OF THE US PACIFIC COAST: PREVALENCE, PHENOTYPE, AND GENOTYPE.

Emergent hypermucoviscous (HMV) strains of Klebsiella pneumoniae have been reported in multiple marine mammal species; however, there is limited information regarding the epidemiology and pathogenesis of this infection in these species. We determined the prevalence of HMV K. pneumoniae in wild-caught and stranded marine mammal populations on the US Pacific Coast. Samples were collected from 270 free-ranging California sea lions (CSLs; Zalophus californianus) captured at three discrete sampling sites and from 336 stranded marine mammals of various species. We recovered HMV K. pneumoniae only from CSLs, with a prevalence of 1.5% (4 of 275) in stranded animals, compared with 1.1% (3 of 270) in wild-caught animals. We assessed the phenotypic and genotypic variability of recovered HMV K. pneumoniae isolates recovered from CSLs ( n=11) and of archival HMV and non-HMV isolates from stranded marine mammals ( n=19). All but two HMV isolates were of the K2 serotype, whereas none of the non-HMV isolates belonged to this serotype. Of the HMV isolates, 96% (24 of 25) were PCR positive for the HMV-associated gene p- rmpA, whereas 92% (23 of 25) were PCR positive for p- rmpA2. Genetic fingerprinting by repetitive extragenic palindromic PCR showed four discrete clusters, demonstrating genotypic variability that loosely correlated with phenotype. Antimicrobial susceptibility testing revealed all isolates from stranded CSLs were susceptible to ceftiofur, indicating this antimicrobial agent is an appropriate choice for treatment of HMV K. pneumoniae infections in stranded CSLs. Our culture assay could reliably detect HMV K. pneumoniae from concentrations as low as 102 colony-forming units per milligram of feces. We identified the presence of HMV K. pneumoniae in both wild-caught and stranded CSLs from the US Pacific Coast and highlight the need for further studies to evaluate the potential impact of this pathogen on marine mammal health.