Species-specific in situ hybridization confirms arrested development of Henneguya ictaluri in hybrid catfish (Channel Catfish × Blue Catfish) under experimental conditions, with notes on mixed-species infections in clinical cases of proliferative gill disease from Mississippi catfish aquaculture.

OBJECTIVE: Proliferative gill disease (PGD) in Channel Catfish Ictalurus punctatus and hybrid catfish (Channel Catfish × Blue Catfish I. furcatus) is attributed to the myxozoan Henneguya ictaluri. Despite evidence of decreased H. ictaluri transmission and impaired parasite development in hybrid catfish, PGD still occurs in hybrid production systems. Previous metagenomic assessments of clinical PGD cases revealed numerous myxozoans within affected gill tissues in addition to H. ictaluri. The objective of this study was to investigate the development and pathologic contributions of H. ictaluri and other myxozoans in naturally and experimentally induced PGD. METHODS: Henneguya species-specific in situ hybridization (ISH) assays were developed using RNAscope technology. Natural infections were sourced from diagnostic case submissions in 2019. Experimental challenges involved Channel Catfish and hybrid catfish exposed to pond water from an active PGD outbreak, and the fish were sampled at 1, 7, 10, 12, 14, 16, 18, and 20 weeks postchallenge. RESULT: Nine unique ISH probes were designed, targeting a diagnostic variable region of the 18S ribosomal RNA gene of select myxozoan taxa identified in clinical PGD cases. Partial validation from pure H. ictaluri, H. adiposa, H. postexilis, and H. exilis infections illustrated species-specific labeling and no cross-reactivity between different myxozoan species or the catfish hosts. After experimental challenge, mature plasmodia of H. ictaluri and H. postexilis formed in Channel Catfish but were not observed in hybrids, suggesting impaired or delayed sporogenesis in the hybridized host. These investigations also confirmed the presence of mixed infections in clinical PGD cases. CONCLUSION: Although H. ictaluri appears to be the primary cause of PGD, presporogonic stages of other myxozoans were also present, which may contribute to disease pathology and exacerbate respiratory compromise by further altering normal gill morphology. This work provides molecular confirmation and more resolute developmental timelines of H. ictaluri and H. postexilis in Channel Catfish and supports previous research indicating impaired or precluded H. ictaluri sporogony in hybrid catfish.

Molecular phylogenetic analysis of Neodiplostomum and Fibricola (Digenea, Diplostomidae) does not support host-based systematics.

Fibricola and Neodiplostomum are diplostomid genera with very similar morphology that are currently separated based on their definitive hosts. Fibricola spp. are normally found in mammals, while Neodiplostomum spp. typically parasitize birds. Previously, no DNA sequence data was available for any member of Fibricola. We generated nuclear ribosomal and mtDNA sequences of Fibricola cratera (type-species), Fibricola lucidum and 6 species of Neodiplostomum. DNA sequences were used to examine phylogenetic interrelationships among Fibricola and Neodiplostomum and re-evaluate their systematics. Molecular phylogenies and morphological study suggest that Fibricola should be considered a junior synonym of Neodiplostomum. Therefore, we synonymize the two genera and transfer all members of Fibricola into Neodiplostomum. Specimens morphologically identified as Neodiplostomum cratera belonged to 3 distinct phylogenetic clades based on mitochondrial data. One of those clades also included sequences of specimens identified morphologically as Neodiplostomum lucidum. Further study is necessary to resolve the situation regarding the morphology of N. cratera. Our results demonstrated that some DNA sequences of N. americanum available in GenBank originate from misidentified Neodiplostomum banghami. Molecular phylogentic data revealed at least 2 independent host-switching events between avian and mammalian hosts in the evolutionary history of Neodiplostomum; however, the directionality of these host-switching events remains unclear.

Kudoa hypoepicardialis and associated cardiac lesions in invasive red lionfish Pterois volitans in Grenada, West Indies.

Invasive red lionfish Pterois volitans (Linnaeus, 1758) represent an ongoing ecological threat within temperate and tropical waters. Relatively little is known regarding the overall health of P. volitans and their potential for spreading pathogens in non-native regions. Lionfish collected from inshore reefs of Grenada, West Indies, in 2019 and 2021 were identified as P. volitans based on cytochrome c oxidase subunit 1 barcoding. Gross and microscopic examination of tissues revealed myxozoan plasmodia in the hearts of 24/76 (31.6%) lionfish by histopathology or wet mount cytology. Further histopathologic examination revealed severe granulomatous inflammation and myofiber necrosis associated with developing plasmodia and presporogonic life stages. Fresh myxospores were morphologically and molecularly consistent with Kudoa hypoepicardialis, being quadrate in apical view with 4 valves and 4 equal polar capsules. The spore body was 5.1-7.9 (mean: 6.0) µm long, 8.1-9.8 (8.7) µm wide, and 6.9-8.5 (7.7) µm thick. Polar capsules were 2.3-2.7 (2.5) µm long and 0.9-1.6 (1.3) µm wide. 18S small subunit rDNA sequences were 99.81-99.87% similar to sequence data from the original description of the species. Novel 28S large subunit rDNA and elongation factor 2 data, which did not match any previously reported species, were provided. This is the first account of a myxozoan parasite of P. volitans, a new host record and locality for K. hypoepicardialis, and one of few reports describing pathogen-associated lesions in invasive lionfish.

New data on Henneguya postexilis Minchew, 1977, a parasite of channel catfish Ictalurus punctatus, with notes on resolution of molecular markers for myxozoan phylogeny.

Previous morphological and histological data are supplemented with molecular and ultrastructural data for a Henneguya sp. isolated from farm-raised channel catfish Ictalurus punctatus in Mississippi, USA. Myxospores were cryptic, encapsulated within a thin layer of epithelium in the gill lamellae with spore measurements consistent with the original description of Henneguya postexilis Minchew, 1977. Myxospores were 42.7-49.1 µm in total length with spore bodies 12.1-17.2 × 3.6-4.8 × 2.9-3 µm. Polar capsules were of unequal length, with the longer capsule being 4.4-6.7 × 1.1-1.6 µm and the shorter capsule being 4.4-6.4 × 1.1-1.6 µm. Polar tubules had 6-8 turns. Caudal processes were 25.7-38.1 µm in length. Spores were encapsulated in a thin layer of epithelium in the gill lamellae. Molecular data from the most commonly used markers for myxozoan identification and phylogeny, partial 18S small subunit ribosomal gene (SSU), partial 28S large subunit ribosomal gene (LSU), and elongation factor 2 (EF2) were generated for H. postexilis. Additionally, novel data for LSU and EF2 were generated for archived myxozoan specimens from farm-raised catfish (H. mississippiensis, H. ictaluri, H. exilis, H. adiposa, H. sutherlandi, H. bulbosus, Unicauda fimbrethilae), as well as archived specimens from wild fish (H. laseeae [from Pylodictis olivaris], Hennegoides flockae [from Aphredoderus sayanus], Myxobolus cloutmani [from Cycleptus elongatus]. These include the first EF2 sequence data for the genera Hennegoides and Unicauda. Phylogenetic analyses using these data placed H. postexilis in well supported clades with other ictalurid-infecting Henneguya species. Phylogenetic signal assessments on these analyses suggest that while SSU provided the greatest phylogenetic signal, LSU yielded comparable signal, supporting previous work implying this region may be underutilised in reconstructing myxobolid phylogenies.

First Isolation of a Novel Aquatic Flavivirus from Chinook Salmon (Oncorhynchus tshawytscha) and Its In Vivo Replication in a Piscine Animal Model.

The first isolation of a flavivirus from fish was made from moribund Chinook salmon (Oncorhynchus tshawytscha) from the Eel River, California, USA. Following the observation of cytopathic effect in a striped-snakehead fish cell line, 35-nm virions with flaviviral morphology were visualized using electron microcopy. Next-generation sequencing and rapid amplification of cDNA ends obtained the complete genome. Reverse transcriptase quantitative PCR (RT-qPCR) confirmed the presence of viral RNA in formalin-fixed tissues from the wild salmon. For the first time, in vivo replication of an aquatic flavivirus was demonstrated following intracoelomic injection in a Chinook salmon model of infection. RT-qPCR demonstrated viral replication in salmon brains up to 15 days postinjection. Infectious virus was then reisolated in culture, fulfilling Rivers' postulates. Only limited replication occurred in the kidneys of Chinook salmon or in tissues of rainbow trout (Oncorhynchus mykiss). The proposed salmon flavivirus (SFV) has a 10.3-kb genome that encodes a rare dual open reading frame, a feature uncharacteristic of classical flaviviruses. Phylogenetic analysis places SFV in a basal position among a new subgroup of recently recognized aquatic and bat flaviviruses distinct from the established mosquito-borne, tick-borne, insect-only, and unknown-vector flavivirus groups. While the pathogenic potential of the virus remains to be fully elucidated, its basal phylogeny and the in vivo infection model will allow SFV to serve as a prototype for aquatic flaviviruses. Ongoing field and laboratory studies will facilitate better understanding of the potential impacts of SFV infection on ecologically and economically important salmonid species.IMPORTANCE Chinook salmon are a keystone fish species of great ecological and commercial significance in their native northern Pacific range and in regions to which they have been introduced. Threats to salmon populations include habitat degradation, climate change, and infectious agents, including viruses. While the first isolation of a flavivirus from wild migrating salmon may indicate an emerging disease threat, characterization of the genome provides insights into the ecology and long evolutionary history of this important group of viruses affecting humans and other animals and into an expanding group of recently discovered aquatic flaviviruses.

Henneguya michiganensis n. sp. (Cnidaria: Myxosporea) from the gills of muskellunge Esox masquinongy Mitchill(Esociformes: Esocidae).

Henneguya Thélohan, 1892 is the second most species rich genus of myxozoans, with reports from freshwater and marine fish worldwide. In the Great Lakes region of North America, muskellunge Esox masquinongy is an important game fish species that serves as an apex predator in the ecosystems of many inland lakes. The myxozoan fauna of esocid fish, especially muskellunge, remains largely understudied. During fish health assessments, muskellunge were examined for parasitic infections and myxozoan pseudocysts were observed on gill clip wet mounts. When ruptured under pressure, the intralamellar pseudocysts released thousands of myxospores consistent with those of the genus Henneguya. The myxospores were 67.3-96.6 (79.1 ± 5.9) µm in total length. The spore body was 18.6-22.6 (20.9 ± 1.0) µm × 5.4-6.9 (6.3 ± 0.4) µm in valvular view and 3.5-4.0 (3.8 ± 0.3) µm wide in sutural view. The two pyriform polar capsules positioned at the anterior of the spore body were 6.4-7.7 (7.0 ± 0.4) µm × 1.8-2.1 (2.0 ± 0.1) µm and each contained a tightly coiled polar filament with 9-10 turns. Two tapering caudal processes extended from the posterior of the spore body and were 47.3-75.6 (58.3 ± 5.8) µm in length. Histologically, large intralamellar polysporic plasmodia were surrounded by plump pillar cells and a distinct layer of plasma. Mild inflammation was present peripherally, with small numbers of necrotic germinative cells and intraplasmodial phagocytes internally. Ribosomal 18S rRNA gene sequence data were obtained from three gill pseudocysts. The three ~2000-bp sequences were identical, but shared no significant similarity with any publicly available sequence data. Phylogenetic analyses demonstrated sequence data from this Henneguya fell within a well-supported clade of Henneguya spp. reported from northern pike Esox lucius in Europe. Based on the distinct morphological, histological and molecular data, this species is designated as Henneguya michiganensis n. sp. from muskellunge in Michigan, USA.

Characterisation of myxozoan fauna of western mosquitofish, Gambusia affinis (Baird and Gerard) (Cyprinodontiformes: Poeciliidae), inhabiting experimental catfish ponds in Mississippi, USA.

Characterising myxozoan taxa parasitising fish hosts in catfish aquaculture ponds is crucial to understanding myxozoan community dynamics in these diverse and complex ecological systems. This work investigated the myxozoan fauna of the western mosquitofish, Gambusia affinis, a common, incidental species found in catfish aquaculture ponds in the southeastern United States. 598 fish were sampled in May of 2018 and 2019 from the pond facility of the Thad Cochran National Warmwater Aquaculture Center in Stoneville, Mississippi, USA. Fish were examined microscopically using wet mount preparations of fresh tissue and histology for myxozoans. 18S rRNA gene sequences were amplified from myxospores obtained at necropsy. Updated morphologic, histologic, and 18S rRNA gene sequence features are provided for Henneguya gambusi, Myxobolus pharyngeus, and Myxidium phyllium. Two potentially novel myxozoans were observed during this survey, an undocumented Myxobolus sp. associated with chondrolysis of bones throughout the body and a putative Myxobilatus sp. observed histologically in the renal tubules, ureters, and urinary bladder. However, inadequate samples were obtained for proper species descriptions. Lastly, the life cycle of M. pharyngeus, which is thought to utilize the oligochaete worm Dero digitata as their definitive host, was putatively confirmed by 18S rRNA sequence matching to actinospore stages from oligochaetes in catfish ponds in Mississippi. This work provides novel and expanded morphologic, histologic, molecular and biologic data of five myxozoan parasites of G. affinis, expanding our knowledge of myxozoan diversity in catfish aquaculture ponds.

Insights into myxozoan composition and physiology revealed by histochemical properties of myxospores.

Myxozoa (phylum Cnidaria) are a diverse group of metazoan parasites that predominately infect fish. Little is known regarding the composition and physiology of their myxospore life stage. The objective of this work was to investigate the composition of myxospores and extrasporogonic stages of nine myxozoan species infecting various teleost fish using histochemical staining techniques. Thirty histochemical stains were applied to formalin-fixed, paraffin-embedded tissues processed routinely for light microscopic evaluation. The polar capsules were the most consistent stain target across the taxa examined. Polar capsule staining with Alizarin red, von Kossa and methyl green-pyronin suggests the presence of intracapsular calcium and phosphate, which may contribute to polar filament discharge or pathogenesis of host invasion. The shell valves and suture lines of most myxozoans were stained with Luna and phosphotungstic acid haematoxylin stains, consistent with the presence of chitin and microfibrils, respectively. Vacuoles were consistently highlighted by diastase-susceptible periodic acid-Schiff and Grocott's methenamine silver staining, indicating glycogen. Other histochemical stains exhibited inconsistent staining across the taxa, suggesting differences in myxospore composition potentially reflective of physiologic variations and tissue tropisms. This work provides some information on conserved features and taxa-associated composition of myxospores and lends insight into myxozoan physiology and host-parasite interactions.

Characterisation of Myxobolus stellatus n. sp. (Cnidaria: Myxobolidae) infecting the cranial nerves and ganglia of the spotfin hatchetfish Thoracocharax stellatus (Kner) (Characiformes: Gasteropelecidae) from Colombia.

A previously undescribed Myxobolus sp. was isolated from the cranial nerves and ganglia of the spotfin hatchetfish Thoracocharax stellatus (Kner) that exhibited neurologic signs following importation from Colombia. Associated plasmodia formed space-occupying masses within nerves, compressing neuronal cell bodies and causing axonal degeneration. Myxospores from these fish were morphologically and molecularly distinct from other myxobolids infecting the central nervous system of characins. In valvular view, spores are pyriform with a rounded posterior and tapering anterior aspect. Myxospore bodies are 17.0-19.4 (mean 18.4) µm long and 8.2-9.3 (mean 8.8) µm wide. Polar capsules are asymmetrical and pyriform with a neck-like projection at the apical end. The small polar capsule measures 4.3-5.9 × 2.2-3.1 (mean 5.0 × 2.6) µm, while the large polar capsule measures 9.1-10.7 × 4.9-6.3 (mean 9.9 × 5.4) µm wide. The sequence generated for the small subunit rRNA (18S) gene did not directly match any sequences available on GenBank, but demonstrated 92% nucleotide similarity to Myxobolus axelrodi Camus, Dill, Rosser, Pote & Griffin, 2017 infecting Paracheirodon axelrodi (Schultz). This study provides the first morphological, histological and molecular characterisation of Myxobolus stellatus n. sp. from the spotfin hatchetfish.

Necroulcerative dermatitis associated with Myxobolus dermatoulcerans n. sp. (Cnidaria: Myxobolidae) in red-bellied piranha, Pygocentrus nattereri Kner (Characiformes: Serrasalmidae), from Peru.

A group of red-bellied piranha, Pygocentrus nattereri Kner, recently imported from Peru exhibited multifocal, cutaneous ulcerations with exposure of the underlying musculature. Skin scrapes yielded moderate numbers of myxospores morphologically consistent with Myxobolus Bütschli, 1882. Myxospores from these fish were morphologically and molecularly distinct from other myxobolids infecting piranha. Myxospores are pyriform to capsular with a rounded posterior and slightly rounded to tapering anterior aspect in valvular view. Myxospore bodies are 14.3-17.8 (mean 16.1) µm long and 7.6-10.3 (mean 8.9) µm wide. Polar capsules are symmetrical, slender, elongate, and measure 7.4-10.2 (mean 9.2) µm long and 2.1-3.7 (mean 3.0) µm wide. Sequence generated for the 18S rRNA gene had no direct matches to any sequence available on GenBank but demonstrated less than 89% nucleotide similarity to various published and unpublished Myxobolus spp. from Piaractus brachypomus (Cuvier) and Colossoma macropomum (Cuvier). This paper provides the morphological and molecular characterisation of Myxobolus dermatoulcerans n. sp. from red-bellied piranha and describes associated pathological lesions.

Ithyoclinostomum yamagutii n. sp. (Digenea: Clinostomidae) in the great blue heron Ardea herodias L. (Aves: Ardeidae) from Mississippi, USA.

With only six recognised genera, the family Clinostomidae Lühe, 1901 remains a global research interest of parasitologists and ecologists. Recent efforts have focused on providing molecular data to investigate species diversity, elucidate life-cycles, and make inferences on the group's evolutionary history. Of the clinostomid genera, the monotypic Ithyoclinostomum Witenberg, 1926 has remained more enigmatic compared to the commonly encountered Clinostomum Leidy, 1856. Recent morphological and molecular evidence from metacercariae suggests a second Ithyoclinostomum species may exist in freshwater cichlids in Central America and Mexico. In a recent survey of great blue herons Ardea herodias L. from commercial catfish production farms in Mississippi, USA, two specimens of an abnormally large (> 20 mm) clinostomid were encountered in the oesophagus of a single bird. These specimens were identified as an Ithyoclinostomum sp. morphologically distinct from the only nominal species Ithyoclinostomum dimorphum (Diesing, 1850). Using morphological and molecular data these adult specimens were confirmed as conspecific with the larval metacercariae previously described from Central America and Mexico and represent the novel species, Ithyoclinostomum yamagutii n. sp.

Arrested Development of Henneguya ictaluri (Cnidaria: Myxobolidae) in ♀ Channel Catfish × â™‚ Blue Catfish Hybrids.

Henneguya ictaluri is the etiologic agent of proliferative gill disease (PGD) in farm-raised Channel Catfish Ictalurus punctatus and hybrid catfish in the southeastern United States, and significant annual losses are attributed to this disease. Research suggests that H. ictaluri infection dynamics in Blue Catfish I. furcatus and hybrid catfish (Channel Catfish × Blue Catfish) differ from those in Channel Catfish. Two separate infectivity trials were conducted to investigate H. ictaluri development in Channel Catfish, Blue Catfish, and their hybrids. On two separate occasions with two different year-classes, fish were exposed to pond water containing H. ictaluri actinospores and sampled weekly for 12 weeks (trial 1) or 14 weeks (trial 2). In trial 1, the presence of H. ictaluri was evaluated histologically and by quantitative PCR of fish tissues, including gills, blood, anterior kidney, brain, heart, liver, posterior kidney, spleen, and stomach. Henneguya ictaluri DNA was detected in significantly higher concentrations throughout multiple organ systems in the Channel Catfish compared to the hybrid catfish and Blue Catfish, with the gills having higher quantities. Myxospores were observed in Channel Catfish gill tissue at 8 weeks postexposure. No myxospores were observed in Blue Catfish or hybrid catfish. The second trial focused on gills only and yielded similar results, with Channel Catfish having significantly greater H. ictaluri DNA quantities than hybrids or Blue Catfish across all time points. Myxospores were observed in Channel Catfish beginning at 6 weeks postexposure and were found in 36% (58/162) of Channel Catfish sampled for molecular and histological analysis during weeks 6-14. Myxospores in hybrid catfish were sparse, with single pseudocysts observed in two hybrid catfish (1.2%) at 14 weeks postexposure. These results imply arrested development of H. ictaluri in hybrid catfish. As such, culture of hybrid catfish may be an effective management strategy to minimize the burden of PGD.

Morphological, molecular and phylogenetic characterisation of Eimeria macyi Wheat, 1975 (Apicomplexa: Eimeriidae) in the eastern red bat Lasiurus borealis (Müller) from Mississippi, USA.

In November 2017, oöcysts of the coccidian Eimeria macyi Wheat, 1975 were isolated from the faeces of a single eastern red bat Lasiurus borealis Müller in Lowndes County, Mississippi, USA. Sporulated oöcysts, morphologically consistent with previous accounts of E. macyi in other chiropterans, were spherical to sub-spherical in shape with a highly mamillated outer wall that appears bi-layered. Oöcysts allowed to sporulate in 2.5% potassium dichromate at ambient temperature (c.23°C) for 7 days were 17-25 × 15-20 (20.7 × 17.9) µm. Micropyle and oöcyst residuum were absent with one to two polar granules scattered among sporocysts. The four ovoid sporocysts were 7-12 × 6-8 (9.9 × 7.1) µm. Stieda bodies were prominent and sub-Stieda bodies were present. Two sporozoites were reflexed within each sporocyst. Nuclear 18S rRNA gene, plastid 23S rRNA gene and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were sequenced from sporulated oöcysts and compared to other molecular data of Eimeria spp. from rodent and chiropteran hosts. No sequence data in the NCBI database matched E. macyi. Phylogenetic analyses of the sequence data of the 18S rRNA and 23S rRNA genes placed E. macyi within a clade containing Eimeria spp. from rodents and basal to a clade populated by sequences derived from Eimeria spp. of rodents and bats. This account represents a new host record of E. macyi in an eastern red bat and a new geographic locality. Additionally, the cox1 sequence data of Eimeria macyi represents the first mitochondrial sequence of an Eimeria sp. in bats.

A morphological, molecular, and histopathological redescription of Henneguya nyongensis Fomena & Bouix, 1996 (Cnidaria: Myxobolidae) infecting the gills of Peter's elephantnose fish, Gnathonemus petersii (Günther) (Osteoglossiformes: Mormyridae), imported from Nigeria.

A Henneguya sp., morphologically resembling Henneguya nyongensis Fomena & Bouix, 1996, was isolated from the gills of Peter's elephantnose fish, Gnathonemus petersii Günther, imported from Nigeria. Plasmodia were located between lamellae and within the gill epithelium, often leading to lamellar fusion. Although slightly smaller, the myxospores from these fish were morphologically consistent with H. nyongensis. In valvular view, spores are elongate, pyriform with a rounded posterior and tapering caudal processes. Myxospore bodies are 9.6-12.3 (mean 11.2) µm long and 4.0-4.7 (mean 4.3) µm wide. Polar capsules are pyriform, elongate, 4.5-5.2 (4.7) µm long and 1.3-1.6 (1.4) µm wide, with a characteristic neck-like structure at the apical end. Sequence generated for the 18S small subunit rRNA gene did not directly match any sequences available on GenBank, but demonstrated 91% nucleotide similarity to an unpublished Henneguya sp. infecting Mormyrus kannume Forsskål. Herein, the description of H. nyongensis is supplemented with new data on histopathology, molecular characterisation, and expanded host and geographical range.

Eimeria spp. (Apicomplexa: Eimeriidae) from great horned owls, Bubo virginianus (Gmelin) (Aves: Strigiformes) from Arkansas and Oklahoma, USA, with novel molecular information on Eimeria bubonis Cawthorn & Stockdale, 1981.

Although nine nominal species of Eimeria Schneider, 1875 have been reported from owls (Strigiformes Wagler), molecular data are available for only one, Eimeria megabubonis Upton, Campbell, Weigel & McKown, 1990. This species has been reported from the great horned owl, Bubo virginianus (Gmelin) at the type-locality of Kansas, USA, and the additional localities of Oklahoma and Mississippi, USA. Eimeria bubonis Cawthorn & Stockdale, 1981, also from B. virginianus, is represented in the literature only by its original description from Saskatchewan, Canada. The present work reports a novel host locality of E. bubonis from B. virginianus in Arkansas, USA. Additionally, novel molecular data from the 18S ribosomal RNA gene and the cytochrome c oxidase subunit 1 mitochondrial gene is provided for E. bubonis. Phylogenetic analysis using the newly generated and previously published sequence data for Eimeria spp. is also presented.

Francisella marina sp. nov., Etiologic Agent of Systemic Disease in Cultured Spotted Rose Snapper (Lutjanus guttatus) in Central America.

Historically, piscine francisellosis in various warm-, temperate-, and cold-water fish hosts has been attributed to Francisella noatunensis From 2015 to 2016, an undescribed Francisella sp. was recovered during mortality events in cultured spotted rose snapper (Lutjanus guttatus) off the Pacific coast of Central America. Despite high mortality and emaciation, limited gross findings were observed in affected fish. Histological examination revealed multifocal granulomatous lesions, with the presence of numerous small, pleomorphic coccobacilli, predominantly in the peritoneum, spleen, kidneys, liver, pancreas, heart, and intestine. Sequencing of an ∼1,400-bp fragment of the 16S rRNA gene demonstrated these isolates to be most similar (99.9% identity) to Francisella sp. isolate TX077308 cultured from seawater in the Gulf of Mexico, while sharing <99% similarity to other Fransicella spp. Biochemical analysis, multilocus sequence comparisons of select housekeeping genes, repetitive extragenic palindromic PCR fingerprinting, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and fatty acid methyl ester analysis revealed marked differences between these isolates and other described members of the genus. Koch's postulates were fulfilled by experimental intracoelomic injection and immersion trials using Nile (Oreochromis niloticus) and blue (Oreochromis aureus) tilapia. Based on observed phenotypic and genotypic differences from recognized Francisella spp., the name Francisellamarina sp. nov. (NRRL B-65518) is proposed to accommodate these novel strains.IMPORTANCE Finfish aquaculture is the fastest growing global food production sector. Infectious disease, particularly emergent pathogens, pose a significant threat to established and nascent aquaculture industries worldwide. Herein, we characterize a novel pathogen isolated from mortality events in cultured spotted rose snapper in Central America. The bacteria recovered from these outbreaks were genetically and phenotypically dissimilar from other known Francisella spp. from fish, representing a previously unrecognized member of the genus Francisella, for which the name Francisella marina sp. nov. is proposed.

Clinostomum poteae n. sp. (Digenea: Clinostomidae), in the trachea of a double-crested cormorant Phalacrocorax auritus Lesson, 1831 and molecular data linking the life-cycle stages of Clinostomum album Rosser, Alberson, Woodyard, Cunningham, Pote & Griffin, 2017 in Mississippi, USA.

Clinostomum spp. (Digenea: Clinostomidae) are a group of trematodes commonly found in the buccal cavity and oesophagus of a variety of piscivorous birds. The metacercariae, colloquially known as "yellow grubs," have been reported from a diverse group of freshwater fishes worldwide. In the catfish farming region of the southeastern USA, piscivorous birds present a continuous challenge for aquaculturists in the form of fish depredation and the introduction of trematodes into these static, earthen pond systems. Clinostomum spp. are commonly encountered in farm-raised catfish. While generally considered pests of minimal importance, heavy infections can result in unmarketable fillets. Of the piscivorous birds that frequent catfish aquaculture operations in the southeastern US, the double-crested cormorant (Phalacrocorax auritus Lesson) is one of the most damaging, although reports of Clinostomum spp. from P. auritus are limited. In this study, adult trematodes morphologically consistent with Clinostomum sp. were found in the trachea of a double-crested cormorant captured in Lowndes Co., Mississippi, USA. These specimens differed from other recognised Clinostomum spp. in several key morphological characters. Moreover, sequence data of mitochondrial cytochrome c oxidase subunit 1 gene (cox1), nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene (nad1) and ribosomal internal transcribed spacer (ITS) regions did not match any known Clinostomum sp. for which sequence data are available. While genetically similar to C. marginatum and C. album Rosser, Alberson, Woodyard, Cunningham, Pote & Griffin, 2017 reported from the great egret Ardea alba L. in Mississippi, these adult clinostomids were larger in size and limited to the trachea, whereas both C. marginatum Rudolphi, 1819 and C. album are found in the oral cavity and esophagus. Given these distinct morphological and molecular characters we propose a new member of the genus, known hereafter as Clinostomum poteae n. sp. Additionally, larval stages in the life-cycle of C. album are morphologically and molecularly identified for the first time from ramshorn snails Planorbella trivolvis Say and fathead minnows Pimephales promelas Rafinesque.

New data on Neodiplostomum americanum Chandler and Rausch, 1947 (Digenea: Diplostomidae), in the Great Horned Owl Bubo virginianus Gmelin, 1788 and the Eastern Screech Owl Megascops asio Linnaeus, 1758 in Mississippi, USA.

Neodiplostomum americanum Chandler and Rausch, 1947 has been reported from six species of owls in North America. At present, there are no molecular data for this species and gene sequence data from Neodiplostomum Railliet, 1919 are limited. A freshly deceased specimen of the Great Horned Owl Bubo virginianus Gmelin, 1788 and a freshly deceased specimen of the Eastern Screech Owl Megascops asio Linnaeus, 1758 were collected in Oktibbeha County, Mississippi in 2014 and 2016, respectively. Neodiplostomum americanum were recovered from both hosts. Herein, updated morphological descriptions are supplemented with gene sequence data from conserved (18S, ITS1-5.8S, ITS2, and 28S rRNA) and fast-evolving (cytochrome c oxidase subunit 1 mtDNA) regions. Preliminary phylogenetic analysis of the genus based on cytochrome c oxidase subunit 1 gene sequence data supports the placement of N. americanum within a discrete phylogroup of the family Diplostomidae. The life history of N. americanum is unknown and currently limited to the description of the adult stage in avian hosts. The molecular data generated in this study offer insight into the phylogenetic placement of N. americanum within the Diplostomatidae and will aid in identifying different life stages in putative intermediate hosts.

Henneguya laseeae n. sp. from flathead catfish (Pylodictis olivaris) in the upper Mississippi River.

A novel species of Henneguya was isolated from flathead catfish (Pylodictis olivaris) captured in the upper Mississippi River near Lansing (Allamakee County), IA, and La Crosse (La Crosse County), WI. Designated Henneguya laseeae n. sp., this novel species is described using critical morphological features, histology, and 18S ribosomal RNA gene sequence. Ovoid cysts, ranging from 1200 to 1800 µm in width, tended to be at filament tips or in the distal third, often directly on the filament midline, but occasionally paramedian. Lanceolate-shaped myxospores were consistent with those of the genus Henneguya. The spore body was 16.2 ± 0.5 µm (mean ± standard deviation; range = 15.1-17.0 µm) in length, 6.0 ± 0.4 µm (5.1-6.6 µm) in width, and 4.7 ± 0.2 µm (4.4-4.9 µm) thick. The two polar capsules at the anterior of the spore body were 5.9 ± 0.3 µm (5.3-6.3 µm) in length and 1.8 ± 0.1 µm (1.6-2.1 µm) in width and contained six to seven turns in the polar filament. The caudal processes tapered to fine points and were 54.3 ± 2.9 µm (49.1-61.7 µm) in length. Total spore length was 70.4 ± 3.3 µm (64.5-79.4 µm). The spores and plasmodium of this species are of similar size and morphology to other species of Henneguya from ictalurid fishes. Additionally, the 18S rRNA gene sequences placed this isolate within a clade populated by Henneguya spp. from North American ictalurids. This is the first reported species of Henneguya from flathead catfish.

Myxobolus axelrodi n. sp. (Myxosporea: Myxobolidae) a parasite infecting the brain and retinas of the cardinal tetra Paracheirodon axelrodi (Teleostei: Characidae).

An investigation of mortalities in a group of cardinal tetras Paracheirodon axelrodi Meyers, 1936, a popular ornamental fish, revealed myxozoan parasites in ventricles of the brains in 3/10 fish and the ocular retina of a fourth. Parasite impacts were unclear, as additional histopathological findings were present, including bacterial dermatitis and meningitis. Ethanol-preserved specimens pooled from multiple fish were used for morphological characterization of myxospores. Elongate, teardrop myxospores were 20.5 ± 0.7-µm (mean ± SD; range = 19.0-21.8 µm) long, 6.6 ± 0.5-µm (5.7-7.9 µm) wide, and 5.1 ± 0.4-µm (4.8-5.9 µm) thick (valvular width). Two, unequally sized, apical, pyriform polar capsules were in the same plane as the sutural ridge. The larger measured 9.9 ± 0.8-µm (8.0-11.2 µm) long and 3.8 ± 0.3-µm (3.2-4.8 µm) wide. The smaller was 4.1 ± 0.3-µm (3.5-4.5 µm) long and 2.0 ± 0.1-µm (1.8-2.3 µm) wide. Identical 1912 bp 18S rRNA sequences were obtained from two pooled spore samples from tetra brains, which did not match any sequences in the NCBI nr/nt database. Phylogenetically, these parasites grouped loosely within a clade containing Myxobolus spp. from other South American characins and Unicauda spp. from siluriform catfish. Myxospores shared some morphological similarities with Myxobolus inaequus from the unrelated glass knifefish (Order: Gymnotiformes), but were genetically divergent (<85 % similarity) from other myxozoan parasites of South American characins and shared few morphological features or tissue predilection sites. Based on host and tissue tropism, spore morphology, and 18S rRNA sequencing, we report this isolate as a previously unknown species, Myxobolus axelrodi n. sp.