Betanodavirus meningoencephalitis in an Atlantic blue marlin.

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

Flavobacterium covae is the predominant species of columnaris-causing bacteria impacting the Channel Catfish industry in the southeastern United States.

OBJECTIVE: Columnaris disease is a leading cause of disease-related losses in the catfish industry of the southeastern United States. The term "columnaris-causing bacteria" (CCB) has been coined in reference to the four described species that cause columnaris disease: Flavobacterium columnare, F. covae, F. davisii, and F. oreochromis. Historically, F. columnare, F. covae, and F. davisii have been isolated from columnaris disease cases in the catfish industry; however, there is a lack of knowledge of which CCB species are most prevalent in farm-raised catfish. The current research objectives were to (1) sample columnaris disease cases from the U.S. catfish industry and identify the species of CCB involved and (2) determine the virulence of the four CCB species in Channel Catfish Ictalurus punctatus in controlled laboratory challenges. METHODS: Bacterial isolates or swabs of external lesions from catfish were collected from 259 columnaris disease cases in Mississippi and Alabama during 2015-2019. The DNA extracted from the samples was analyzed using a CCB-specific multiplex polymerase chain reaction to identify the CCB present in each diagnostic case. Channel Catfish were challenged by immersion with isolates belonging to each CCB species to determine virulence at ~28°C and 20°C. RESULT: Flavobacterium covae was identified as the predominant CCB species impacting the U.S. catfish industry, as it was present in 94.2% (n = 244) of diagnostic case submissions. Challenge experiments demonstrated that F. covae and F. oreochromis were highly virulent to Channel Catfish, with most isolates resulting in near 100% mortality. In contrast, F. columnare and F. davisii were less virulent, with most isolates resulting in less than 40% mortality. CONCLUSION: Collectively, these results demonstrate that F. covae is the predominant CCB in the U.S. catfish industry, and research aimed at developing new control and prevention strategies should target this bacterial species. The methods described herein can be used to continue monitoring the prevalence of CCB in the catfish industry and can be easily applied to other industries to identify which Flavobacterium species have the greatest impact.

Canine thyroid carcinomas: A review with emphasis on comparing the compact subtype of follicular thyroid carcinomas and medullary thyroid carcinomas.

Canine thyroid carcinomas are relatively common malignant endocrine neoplasms in dogs derived from either thyroid follicular cells (forming follicular thyroid carcinomas) or medullary cells (parafollicular, C-cells; forming medullary thyroid carcinomas). Older and recent clinical studies often fail to discriminate between compact cellular (solid) follicular thyroid carcinomas and medullary thyroid carcinomas, which may skew conclusions. The compact subtype of follicular thyroid carcinomas appears to be the least differentiated subtype of follicular thyroid carcinomas and needs to be differentiated from medullary thyroid carcinomas. This review includes information on the signalment, presentation, etiopathogenesis, classification, histologic and immunohistochemical diagnosis, clinical management, and biochemical and genetic derangements of canine follicular and medullary carcinomas, and their correlates with human medicine.

A practical protocol to prepare paraffin-embedded whole tick histology sections.

Ticks are important ectoparasites that are capable of transmitting multiple classes of pathogens and are currently linked with many emerging tick-borne diseases worldwide. With increasing occurrences of tick-borne diseases in both humans and veterinary species, there is a continuous need to further our understanding of ticks and the pathogens they transmit. Whole tick histology provides a full scope of the tick internal anatomy, allowing researchers to examine multiple organs of interest in a single section. This is in contrast to other techniques that are more commonly utilized in tick-borne disease research, such as electron microscopy and light microscopy of individual organs. There is a lack of literature describing a practical technique to process whole tick histologic sections. Therefore, the current study aims to provide researchers with a workable protocol to prepare high quality paraffin-embedded whole tick histology sections. Amblyomma americanum adults were used as an example species for this study. After a series of pilot experiments using a combination of various fixatives, softening agents and processing techniques, we elected to compare two common fixatives, 10% neutral-buffered formalin (NBF) and Bouin's solution for whole ticks. Equal numbers of A. americanum unfed adults (n = 10/fixative) were processed identically and their whole tick histology coronal sections were individually scored. Higher scores were assigned to whole tick sections that contained more internal organs that are crucial for tick-borne disease research (e.g. salivary glands and midgut), high integrity of tissues and exoskeleton on the section, and good fixation and staining quality of the tissues. The mean total scores for Bouin's-fixed ticks were significantly higher compared to NBF-fixed ticks (p = 0.001). To further assess our preferred technique, we also demonstrated the feasibility of producing high quality whole tick sections for three other common tick species of medical importance (Rhipicephalus sanguineus, Ixodes scapularis, and Dermacentor variabilis) using Bouin's solution. While this technique may require further optimization for other tick species, we described a feasible protocol that uses commonly available tools, reagents and standard histologic equipment. This should allow any investigator to easily make adjustments to this protocol as needed based on their experimental goals.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1A IN ARCHIVAL TISSUE USING RNASCOPE® IN SITU HYBRIDIZATION.

Hemorrhagic disease due to elephant endotheliotropic herpesvirus infection (EEHV-HD) is an important cause of calf mortality in managed and free-ranging Asian (Elephas maximus) and African elephant (Loxodonta spp.) populations. Consequently, infection has profound implications for elephant population growth and sustainability. The mechanisms of disease caused by EEHV (i.e., infection, dissemination, shedding, latency) are relatively undefined, in part because of a lack of robust validated assays for detecting viral gene products in relevant samples. To address this issue, we used RNAscope® in situ hybridization (ISH) based on EEHV1A DNA polymerase and terminase genes to detect EEHV1A RNA in archival formalin-fixed, paraffin-embedded Asian elephant heart and tongue from PCR-confirmed cases (n = 4) of EEHV-HD and Asian elephants (n = 2) that died from other causes. EEHV1A-positive cases had positive hybridization signal in endothelial cell nuclei of both tissues for both DNA polymerase and terminase. EEHV-negative cases lacked signal. In positive cases, the number of positive nuclei was manually assessed to provide an estimate of the viral load and compare sensitivity of the two probes. In all cases, heart had greater signal than tongue for both probes (Wilcoxon rank test; P ≤ 0.01). Overall, terminase hybridization signal was greater than DNA polymerase signal (Wilcoxon rank test; P ≤ 0.01). Results indicate RNAscope ISH is a valuable tool for detection of EEHV in archival samples and for confirming infection. Additionally, the terminase gene is the optimal target and heart is preferable to tongue for detection in cases of EEHV-HD. Results will inform future investigations of viral tropism in EEHV-HD cases due to EEHV1A.

EXPERIMENTAL ELUCIDATION OF THE LIFE CYCLE OF DREPANOCEPHALUS SPATHANS (DIGENEA: ECHINOSTOMATIDAE) WITH NOTES ON THE MORPHOLOGICAL PLASTICITY OF D. SPATHANS IN THE UNITED STATES.

The echinostomatid Drepanocephalus spathans (syn. Drepanocephalus auritus) parasitizes the double-crested cormorant Phalacrocorax auritus. In North America, the marsh rams-horn snail Planorbella trivolvis and ghost rams-horn snail Biomphalaria havanensis serve as snail intermediate hosts, both of which inhabit catfish aquaculture ponds in the southeastern United States. Studies have demonstrated D. spathans exposure can be lethal to juvenile channel catfish Ictalurus punctatus. Two studies were undertaken to elucidate the life cycle of D. spathans to establish a developmental time line. In both studies, D. spathans cercariae collected from naturally infected P. trivolvis individuals were used to infect channel catfish fingerlings, which were then fed to double-crested cormorants (DCCOs) that had been pharmaceutically dewormed. In study 1, laboratory-reared P. trivolvis and B. havanensis individuals were placed in aviary ponds with experimentally infected DCCO and examined bi-weekly for release of cercariae. Trematode eggs were observed in the feces of exposed birds 3 days post-infection. Birds were sacrificed 18 days post-exposure (dpe), and gravid adults morphologically and molecularly consistent with D. spathans were recovered. Snails from the aviary pond were observed shedding D. spathans cercariae 18-54 dpe. In study 2, trematode eggs were observed in the feces of exposed DCCOs beginning 8 dpe. Once eggs were observed, birds were allowed to defecate into clean tanks containing naïve laboratory-reared P. trivolvis individuals. Additionally, eggs from experimental DCCO feces were recovered by sedimentation and placed in an aquarium housing laboratory-reared P. trivolvis individuals. Birds in study 2 were sacrificed after 60 days, and gravid D. spathans specimens were recovered. Snails from the experimental DCCO tanks shed D. spathans cercariae 89-97 dpe. Lastly, trematode eggs were isolated and observed for the hatching of miracidia, which emerged on average after 16 days at ambient temperatures. No D. spathans adults were observed in control birds fed non-parasitized fish. This is the first experimental confirmation of the D. spathans life cycle, resolving previously unknown developmental time lines. In addition, the effects of fixation on adult trematode morphology were assessed, clarifying reports of pronounced morphological plasticity for D. spathans.

Mineralizing Pulmonary Elastosis in a Cat.

Mineralizing pulmonary elastosis (MPE) is a rare and unique phenomenon that has been reported in humans, typically secondary to recurrent pulmonary haemorrhage. MPE has a complex histopathological appearance, often containing iron-calcium deposits that can be mistaken as fungal organisms or other inorganic material. This report documents the first case of MPE in an animal species. A 10-year-old female domestic cat with respiratory failure was submitted for necropsy. The lungs were consolidated with severe pulmonary haemosiderosis, and widely disseminated granulomas surrounded large aggregates of hyphae-like structures. The pulmonary vasculature and airway smooth muscle were partially mineralized and fragmented. Histochemical stains revealed that the fungus-like material stained strongly with Prussian blue and alizarin red but only sparingly with von Kossa and negative with Gomori's methenamine silver stain. These findings are similar to those of MPE in humans. As most veterinary pathologists may not be familiar with MPE, it is important to avoid possible misinterpretation by recognizing its distinct features and the ancillary testing that may be required.

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.

Pathology associated with Odhneriotrema incommodum infection in wild-caught American alligators Alligator mississippiensis and assessment of potential first intermediate snail hosts.

AIMS: To assess pathological changes associated with natural infections of the trematode Odhneriotrema incommodum in wild-caught American alligators Alligator mississippiensis and assess potential first intermediate hosts. MATERIALS AND METHODS: Tongues from two wild-caught alligators were obtained from a commercial alligator processor in Port Gibson, Mississippi, USA. Tongues were subjected to gross parasitological examination and routine histological assessment. Eggs were expressed from adult trematodes collected from these tongues into distilled water, where they hatched into infectious miracidia. The snails Planorbella trivolvis, Physa gyrina, and Biomphalaria havanensis were exposed to these miracidia and observed for cercarial emergence for 200 days post-exposure. RESULTS: Histological assessment of alligator tongues revealed marked hemorrhage, necrosis, presence of bacteria, and inflammation at sites of Odhneriotrema incommodum attachment, differing from previous histological reports from controlled experimental studies. Cercarial emergence was not observed in snails exposed to infectious miracidia. CONCLUSIONS: Wild-caught alligators infected with Odhneriotrema incommodum exhibit more severe pathology than was previously noted from experimentally infected alligators. This adverse pathology may be associated with microbes present in eutrophic natural habitats that are absent from controlled environments used in experimental exposures. Impacts of this parasite in wild alligator populations are likely underestimated and damage associated with parasite attachment could increase host susceptibility to secondary infections. Given the importance of alligators as game animals and sustained demand for alligator products, further study into the role of O. incommodum on alligator health is warranted. Results of snail exposures to miracidia suggest these snail species are not suitable first intermediate hosts for this trematode and the true first intermediate host of O. incommodum remains unknown.

Two Novel Myxozoans from Pirate Perch Aphredoderus sayanus (Gilliams, 1824) in the Upper Mississippi River, Including the First North American Species of Hennegoides Lom, Tonguthai, & Dyková, 1991.

The pirate perch Aphredoderus sayanus is a relatively small fish species found in rivers throughout much of the eastern United States. Due to their cryptic nature, relatively little is known regarding their parasite fauna. A survey of pirate perch from the upper Mississippi River revealed 2 novel myxozoans. Hennegoides flockae n. sp. was observed in heavily infected gills where the lamellae featured irregular expansion by bulbous myxozoan polysporic plasmodia, typically affecting the middle to distal half of the filaments. When severe, infection of sequential filaments was such that the filaments were fused, forming what appeared as multicystic/lobular parasitic aggregates subdivided by fine epithelial cords. The total myxospore length of Hennegoides flockae was 35.4-46.4 (41.3 ± 3.3) and the spore body, asymmetrically ovoid in valvular view, was 15.4-18.7 (17.0 ± 0.7) × 7.1-8.7 (7.9 ± 0.4). Henneguya marcquenskiae n. sp. was observed in the liver with plasmodia present randomly and infrequently in the hepatocellular parenchyma. The total myxospore length for Henneguya marcquenskiae was 39.5-55.9 (48.4 ± 4.2), with the spore body being lanceolate, 13.9-16.5 (15.4 ± 0.7) × 7.1-9.0 (8.3 ± 0.5). Phylogenetic analysis of the SSU rRNA gene placed both Hennegoides flockae and Henneguya marcquenskiae as sisters to each other in a clade containing other Myxozoans known to infect the gills of esocids, percids, and centrarchids. These parasites represent the first reports of Henneguya and Hennegoides from pirate perch, with the latter being the first report of this genus outside of the Asian continent.

Morphological, Molecular, and Histopathological Data for Sebekia mississippiensis Overstreet, Self, and Vliet, 1985 (Pentastomida: Sebekidae) in the American Alligator, Alligator mississippiensis Daudin, and the Spotted Gar, Lepisosteus oculatus Winchell.

Novel molecular data from both mitochondrial ( cytochrome c oxidase subunit 1) and ribosomal regions ( 18S, ITS1-5.8S, ITS2, and 28S) are provided for Sebekia mississippiensis Overstreet, Self, & Vliet, 1985 , a pentastome infecting the American alligator, Alligator mississippiensis Daudin, 1801, and the spotted gar, Lepisosteus oculatus Winchell, 1864. Adult and nymphal pentastomes are described from the lungs and liver of the type host, A. mississippiensis, collected from Mississippi, while additional nymphs are described from the esophageal lining of L. oculatus specimens collected from Louisiana. This sequencing data will facilitate more accurate identification of various life cycle stages of S. mississippiensis, enabling future work to resolve many ambiguities in the literature regarding this species. Additionally, histopathological data are provided from both the definitive and intermediate hosts.

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.

Lesions caused by virulent Aeromonas hydrophila in farmed catfish ( Ictalurus punctatus and I. punctatus × I. furcatus) in Mississippi.

In 2009, a virulent strain of Aeromonas hydrophila caused severe disease and high mortalities (motile aeromonad septicemia variant [MASv]) in farmed channel catfish ( Ictalurus punctatus) and hybrid catfish ( I. punctatus × I. furcatus) in eastern Mississippi and Alabama. As is common in MAS, there is severe hemorrhagic dermatitis with ulceration, as well as abdominal hyperemia, petechiation, and mild ascites. Additional findings in MASv cases include panophthalmitis and orbital cellulitis, leading to ocular rupture, and brains are often hyperemic with mild random acute hemorrhage. In MASv infections, there is consistently also marked hemorrhage and edema in the submucosa and muscularis of the stomach, with lymphangitis and a few bacteria, plus splenomegaly with infarcts. Microscopically, spleens have necrosis of ellipsoids with macrophage infiltration and small numbers of bacteria; however, large infarcts are filled with bacteria. Other organs, such as liver, kidneys, and intestine, which are typically associated with MAS caused by various aeromonad species, are less affected. The findings in the stomach have not been reported in MAS in farmed catfish, to our knowledge, and the splenic changes are highly characteristic of MAS compared to infection with other gram-negative bacteria, including Edwardsiella ictaluri and other aeromonad species and strains.

Myxobolus lepomis n. sp. (Cnidaria: Myxobolidae), a gill myxozoan infecting Lepomis marginatus Holbrook and Lepomis miniatus Jordan (Perciformes: Centrarchidae), in the Big Thicket National Preserve, Texas, USA.

A parasitological survey of freshwater fishes in the Big Thicket National Preserve in southeast Texas revealed myxozoan infections in two species of sunfish, Lepomis marginatus Holbrook and Lepomis miniatus Jordan (Perciformes: Centrarchidae). Pseudocysts were elongate-oval, 988 × 485 µm (ex L. marginatus) and 800 × 606 µm (ex L. miniatus) and demonstrated a predilection to the edge of the primary gill lamellae. Myxospores consistent with the genus Myxobolus were oblong, 16.8-21.3 (19.0 ± 0.9) µm long, 7.0-8.8 (7.9 ± 0.5) µm wide and 5.3-6.1 (5.8 ± 0.3) µm thick (ex L. marginatus) and 17.2-20.3 (18.8 ± 0.7) µm long, 7.5-9.9 (8.7 ± 0.6) µm wide, and 6.8-7.2 (7.0 ± 0.2) µm thick (ex L. miniatus); with 2 pyriform polar capsules 8.3-9.8 (9.0 ± 0.5) µm long, 2.2-2.7 (2.5 ± 0.2) µm wide (ex L. marginatus) and 9.2-10.5 (10.0 ± 0.4) µm long, 2.2-3.0 (2.8 ± 0.2) µm wide (ex L. miniatus). Statistically, the measurements of spore body width, polar capsule length, and polar capsule width were significantly different between myxospores from L. marginatus and L. miniatus. However, intraspecific genetic variability between isolates at the 18S rRNA gene was negligible, with < 0.8% variability across > 2,000 bp of sequence. The isolates shared no significant sequence similarity with any myxozoan deposited in the GenBank nucleotide database. Phylogenetic analysis inferred from the 18S rRNA gene from both L. marginatus and L. miniatus placed the isolates within a clade of myxozoan parasites of perciform fishes. Based on shared tissue and host family tropism, overlapping morphological characters and high degrees of sequence conservation at the 18S rRNA gene, we propose these isolates as morphologically distinct, genetically conspecific representatives of M. lepomis n. sp. from the gills of L. marginatus and L. miniatus in the Big Thicket National Preserve in Texas, USA.

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.

Austrodiplostomum sp., Bolbophorus sp. (Digenea: Diplostomidae), and Clinostomum marginatum (Digenea: Clinostomidae) metacercariae in inland silverside Menidia beryllina from catfish aquaculture ponds, with notes on the infectivity of Austrodiplostomum sp. cercariae in channel catfish Ictalurus punctatus.

In the southeastern USA, catfish aquaculture is burdened by predation from piscivorous birds and the digenetic trematodes they carry. In addition to cultured ictalurid fish, other forage or incidental fish species inhabit catfish production ponds. Of these, the inland silverside Menidia beryllina was recently found to harbor larval metacercariae of several trematode species. Three species of metacercariae were reported, two of which represent the first morphological descriptions of an Austrodiplostomum sp. and Bolbophorus sp. metacercaria, respectively. A total of 15 silversides were collected from a commercial catfish pond and examined for trematode infection. These fish were parasitized by metacercariae of an Austrodiplostomum sp. (100 % prevalence) in the eyes and brain, a Bolbophorus sp. (86.7 % prevalence) in the musculature, and Clinostomum marginatum (33.3 % prevalence) in the musculature and fins. All three trematode species were characterized morphologically and molecularly by sequencing of the cytochrome c oxidase subunit 1 gene (CO1). In addition, the internal transcribed spacer 1 region (ITS1), 5.8S rRNA gene, and ITS2 region were determined for the Bolbophorus sp., which linked this metacercaria to a Bolbophorus sp. cercaria from a planorbid snail Planorbella trivolvis and an unnamed Bolbophorus sp. adult from the American white pelican Pelecanus erythrorhynchos. Furthermore, Biomphalaria havanensis snails were collected from the same pond and found actively shedding cercariae morphologically and molecularly consistent with a diplostomid cercaria reported from Bi . havanensis in catfish ponds in Mississippi, USA. Sequence comparisons deemed these cercariae conspecific to the Austrodiplostomum sp. from inland silverside described here. Channel catfish fingerlings were exposed to these cercariae at doses of 50 and 100 cercariae per fish. The infectivity of this Austrodiplostomum sp. in channel catfish was assessed at 10 and 20 days post exposure (dpe). Metacercariae were observed in both the eyes and brain of infected channel catfish, supporting molecular data that suggests the cercaria and metacercaria are both stages of a previously unidentified Austrodiplostomum sp. life cycle.

Morphological, Histological, and Molecular Description of Unicauda fimbrethilae n. sp. (Cnidaria: Myxosporea: Myxobolidae) from the Intestinal Tract of Channel Catfish Ictalurus punctatus.

The channel catfish Ictalurus punctatus is a known host for 10 species of Henneguya, but few other myxozoan genera are described from this species. Unicauda is a genus of myxozoan parasites within the family Myxobolidae that consists of 10 valid species from freshwater fish. Herein, we describe a novel species of Unicauda from the intestinal tract of farm-raised channel catfish in Mississippi. Myxospores were consistent with the genus Unicauda but exhibited a unique branching at the terminal end of the caudal process that has not previously been reported. Myxospores measured 90.39 ± 14.97 µm (mean ± SD; range = 70.88-126.02 µm) in total length. The spherical spore body measured 7.31 ± 0.26 µm (6.75-7.84 µm) in length and 7.01 ± 0.63 µm (6.1-8.01 µm) in width. The 2 polar capsules measured 3.45 ± 0.33 µm (3.02-4.03 µm) in length and 2.65 ± 0.32 µm (2.18-3.11 µm) in width. The single caudal process measured 82.98 ± 14.97 µm (63.39-118.63 µm) in length from the base of the spore body to the end of the most terminal projection. Terminal projections measured 26.83 ± 8.8 µm (12.34-42.29 µm) in length and 0.95 ± 0.23 µm (0.52-1.6 µm) in width. The 18S rRNA gene sequence obtained did not match any published sequences. Given the uniqueness of the myxospore morphology, histological presentation, and gene sequence data, we describe this as an unreported species, Unicauda fimbrethilae n. sp.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Modulation of vacuolar pH is required for replication of Edwardsiella ictaluri in channel catfish macrophages.

Previous in vitro work demonstrated that Edwardsiella ictaluri produces an acid-activated urease that can modulate environmental pH through the production of ammonia from urea. Additional work revealed that expression of the E. ictaluri type III secretion system (T3SS) is upregulated by acidic pH. Both the urease and the T3SS were previously shown to be essential to intracellular replication. In this work, fluorescence microscopy with LysoTracker Red DND-99 (LTR) indicated that E. ictaluri-containing vacuoles (ECV) became acidified following ingestion by head kidney-derived macrophages (HKDM). In vivo ratiometric imaging demonstrated a lowered ECV pH, which fell to as low as pH 4 but subsequently increased to pH 6 or greater. Inhibition of vacuolar H(+)-ATPases by use of the specific inhibitor bafilomycin A1 abrogated both ECV acidification and intracellular replication in HKDM. Failure of an E. ictaluri urease knockout mutant to increase the ECV pH in the in vivo ratiometric assay suggests that ammonia produced by the urease reaction mediates the pH increase. Additionally, when the specific arginase inhibitor l-norvaline was used to treat E. ictaluri-infected HKDM, the ECV failed to neutralize and E. ictaluri was unable to replicate. This indicates that the HKDM-encoded arginase enzyme produces the urea used by the E. ictaluri urease enzyme. Failure of the ECV to acidify would prevent both upregulation of the T3SS and activation of the urease enzyme, either of which would prevent E. ictaluri from replicating in HKDM. Failure of the ECV to neutralize would result in a vacuolar pH too low to support E. ictaluri replication.