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.

Cross-protective efficacy of a live-attenuated Edwardsiella ictaluri vaccine against heterologous Edwardsiella piscicida isolates in channel and channel × blue catfish hybrids.

Edwardsiella piscicida is a growing problem for catfish aquaculture in the southeastern United States, particularly in channel (Ictalurus punctatus) x blue (I. furcatus) catfish hybrids. Research has shown E. piscicida isolates recovered from farmed catfish in Mississippi form at least five discrete phyletic groups, with no apparent differences in virulence in channel and hybrid catfish. Laboratory trials have shown a live-attenuated E. ictaluri vaccine (340X2) cross-protects against at least one E. piscicida isolate (S11-285) in channel and hybrid catfish, although it is unknown if this protection exists for other E. piscicida variants. To this end, channel and hybrid catfish were immunized by immersion with E. ictaluri 340X2. Thirty days later, fish were challenged by intracoelomic injection with representative E. piscicida variants from each phyletic group. Relative percent survival (RPS) for hybrids ranged from 54.7% to 77.8%, while RPS in channels ranged from 80.5% to 100%. A second study investigated whether channel and hybrid catfish exposed to heterologous E. piscicida isolates were similarly protected against wild-type E. ictaluri. Fish were exposed by bath immersion to representative E. piscicida isolates from each phyletic group. Thirty days post-immunization, fish were challenged by immersion with wild-type E. ictaluri isolate S97-773. Regardless of variant, previous exposure to heterologous E. piscicida isolates significantly improved survival following E. ictaluri challenge. These findings suggest the presence of shared and conserved antigens among E. piscicida and E. ictaluri that could be exploited by application of polyvalent or cross-protective vaccines.

High-Resolution, 3D Imaging of the Zebrafish Gill-Associated Lymphoid Tissue (GIALT) Reveals a Novel Lymphoid Structure, the Amphibranchial Lymphoid Tissue.

The zebrafish is extensively used as an animal model for human and fish diseases. However, our understanding of the structural organization of its immune system remains incomplete, especially the mucosa-associated lymphoid tissues (MALTs). Teleost MALTs are commonly perceived as diffuse and scattered populations of immune cells throughout the mucosa. Yet, structured MALTs have been recently discovered in Atlantic salmon (Salmo salar L.), including the interbranchial lymphoid tissue (ILT) in the gills. The existence of the ILT was only recently identified in zebrafish and other fish species, highlighting the need for in-depth characterizations of the gill-associated lymphoid tissue (GIALT) in teleosts. Here, using 3-D high-resolution microscopy, we analyze the GIALT of adult zebrafish with an immuno-histology approach that reveals the organization of lymphoid tissues via the labeling of T/NK cells with an antibody directed to a highly conserved epitope on the kinase ZAP70. We show that the GIALT in zebrafish is distributed over at least five distinct sub-regions, an organization found in all pairs of gill arches. The GIALT is diffuse in the pharyngeal part of the gill arch, the interbranchial septum and the filaments/lamellae, and structured in two sub-regions: the ILT, and a newly discovered lymphoid structure located along each side of the gill arch, which we named the Amphibranchial Lymphoid Tissue (ALT). Based on RAG2 expression, neither the ILT nor the ALT constitute additional thymi. The ALT shares several features with the ILT such as presence of abundant lymphoid cells and myeloid cells embedded in a network of reticulated epithelial cells. Further, the ILT and the ALT are also a site for T/NK cell proliferation. Both ILT and ALT show structural changes after infection with Spring Viraemia of Carp Virus (SVCV). Together, these data suggest that ALT and ILT play an active role in immune responses. Comparative studies show that whereas the ILT seems absent in most neoteleosts ("Percomorphs"), the ALT is widely present in cyprinids, salmonids and neoteleosts, suggesting that it constitutes a conserved tissue involved in the protection of teleosts via the gills.

Genetic characterization of heterologous Edwardsiella piscicida isolates from diverse fish hosts and virulence assessment in a Chinook salmon Oncorhynchus tshawytscha model.

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

Genetic variability of Edwardsiella piscicida isolates from Mississippi catfish aquaculture with an assessment of virulence in channel and channel × blue hybrid catfish.

The bacterium Edwardsiella piscicida causes significant losses in global aquaculture, particularly channel (Ictalurus punctatus) × blue (I. furcatus) hybrid catfish cultured in the south-eastern United States. Emergence of E. piscicida in hybrid catfish is worrisome given current industry trends towards increased hybrid production. The project objectives were to assess intraspecific genetic variability of E. piscicida isolates recovered from diseased channel and hybrid catfish in Mississippi; and determine virulence associations among genetic variants. Repetitive extragenic palindromic sequence-based PCR (rep-PCR) using ERIC I and II primers was used to screen 158 E. piscicida diagnostic case isolates. A subsample of 39 E. piscicida isolates, representing predominant rep-PCR profiles, was further characterized using BOX and (GTG)5 rep-PCR primers, virulence gene assessment and multilocus sequence analysis (MLSA) targeting housekeeping genes gyrb, pgi and phoU. The MLSA provided greater resolution than rep-PCR, revealing 5 discrete phylogroups that correlated similarly with virulence gene profiles. Virulence assessments using E. piscicida representatives from each MLSA group resulted in 14-day cumulative mortality ranging from 22% to 54% and 63 to 72% in channel and hybrid fingerlings, respectively. Across all phylogroups, mortality was higher in hybrid catfish (p < .05), supporting previous work indicating E. piscicida is an emerging threat to hybrid catfish aquaculture in the south-eastern United States.

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.

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.

Virulence and immunogenicity of blue catfish alloherpesvirus in channel, blue and blue × channel hybrid catfish.

Blue catfish alloherpesvirus (BCAHV) is a novel virus isolated from the blue catfish (Ictalurus furcatus). To date, the ultrastructure, virulence and immunogenicity of BCAHV have not been reported. Given the importance of blue catfish in producing channel ♀ (I. punctatus) × â™‚ blue (I. furcatus) catfish hybrids and the increasing demand for hybrid catfish in the US catfish industry, the susceptibility of blue, channel and hybrid catfish to BCAHV was assessed. Further, the cross-protective potential of BCAHV against Ictalurid herpesvirus 1 (IcHV1) was investigated in channel and hybrid catfish that survive BCAHV exposure. Neutralization assays revealed BCAHV is refractive (neutralization index [NI] = 0) to anti-IcHV1 monoclonal antibody Mab 95, compared to IcHV1 (NI = 1.8). Exposure of blue catfish fingerling to 1.3 × 105 TCID50 /L BCAHV produced cumulative mortality of 51.67 ± 0.70% and pathologic changes similar to those of channel catfish virus disease. No mortality was observed in channel or hybrid catfish. Twenty-eight days post-challenge, surviving channel and hybrid catfish were exposed to 9.4 × 104 TCID50 /L IcHV1 (LC50 dose), resulting in 100% relative per cent survival compared to naïve cohorts. These data provide baseline information for BCAHV and lay the groundwork for future studies. Data also identify BCAHV as a potential vaccine candidate against IcHV1. Based on host range and immunogenicity evaluations, in addition to genome sequence data from previous studies, BCAHV should be given consideration as a new species of Ictalurivirus.

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.

Red seabream iridovirus associated with cultured Florida pompano Trachinotus carolinus mortality in Central America.

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