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.

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.

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.

Pathological and Ultrastructural Characterization of an Edwardsiella ictaluri Triple hemR Mutant.

Enteric septicemia of catfish, which is caused by Edwardsiella ictaluri, is detrimental to farmed Channel Catfish Ictalurus punctatus. The hemin receptor HemR is involved in binding and uptake of heme into bacteria. Here, we explored pathological and ultrastructural changes in catfish fry that were immunized with a triple hemR mutant of E. ictaluri and challenged with wild-type E. ictaluri (EiWT) 28 d after immunization. Following immunization, pathological changes in the triple hemR-immunized fry were less severe compared to the EiWT-exposed control fry. Widely disseminated bacteria and severe necrosis in most organs, especially the kidney and spleen, were detected in both groups at days 4, 5, and 6. Multifocal granulomatous encephalitis with bacteria was seen in hemR-immunized fry at days 21 and 28 and in EiWT-exposed control fry at day 14. Phagocytic cells in the kidney and spleen of EiWT-exposed control fry contained more replicating bacteria compared to hemR-immunized fry. During the EiWT challenge of immunized fry, a robust immune response was observed in the triple hemR-immunized fry compared to the sham-vaccinated group. Many activated phagocytic cells were detected in the kidney and spleen with fragmented or no bacteria in the triple hemR-immunized fry. Our data suggested that virulence of triple hemR was lower and the onset of the lesions was delayed compared to EiWT. Additionally, triple hemR-immunized fry could mount an immune response and had milder lesions compared to the sham control after EiWT exposure.

Nephroblastoma in a Common Mudpuppy Necturus maculosus simultaneously Present with a Mollicute Bacterium of the Genus Acholeplasma.

In March 2017, a wild-caught female common mudpuppy Necturus maculosus from Iowa, USA, with an enlarged posterior abdomen was submitted for diagnostic assessment. The cause of the abdominal distension was a large fluid-filled abdominal mass, diagnosed as a nephroblastoma. Parasites and numerous bacteria were isolated and identified from the mudpuppy but were determined to be incidental. Samples of the neoplasm inoculated onto an American toad Anaxyrus americanus cell line (BufoTad) yielded cytopathic effect during several passages. However, standard molecular testing of the cell culture supernatant failed to identify any viruses. Next-generation sequencing identified the replicating agent as a bacterium of the genus Acholeplasma. Immunohistochemistry confirmed the presence of Acholeplasma within the nephroblastoma, including within tumor cells. This is the first report of nephroblastoma and the second report of neoplasia in this species. The results also suggest that certain bacteria of the genus Acholeplasma might be oncogenic.

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.

Vagococcus salmoninarum I-A chronic coldwater streptococcosis in broodstock brook trout (Salvelinus fontinalis) in Wisconsin, USA.

In 2018, Vagococcus salmoninarum was isolated from two lots of broodstock "coaster" brook trout (Salvelinus fontinalis) containing ~1,500 fish at the Iron River National Fish Hatchery, at which time it was identified as the causative agent of a chronic coldwater streptococcosis epizootic. Clinical signs included exophthalmia, lethargy, erratic swimming and loss of equilibrium. Female fish experienced disproportionately higher morbidity and mortality than male co-inhabitants, and routinely retained eggs following spawning. The most consistent gross clinical sign was heart pallor and turbid pericardial effusion. An attempted treatment using florfenicol was ineffective at halting the epizootic, which spanned more than a year and resulted in >50% mortality before remaining fish were culled. As there is no previous documentation of V. salmoninarum at this hatchery or in this species, it is still unclear what circumstances led to this epizootic. The inability to treat this chronic disease led to the loss of valuable broodstock, hampering ongoing fishery conservation efforts in the Great Lakes Basin.

Bluegill Picornavirus isolated from a mortality event involving Bluegill (Lepomis macrochirus) in the upper Mississippi River.

A mortality event involving an estimated 1,000 adult bluegills (Lepomis macrochirus) was observed in an ice-covered backwater lake of the upper Mississippi River near Alma, Wisconsin, in December of 2017. Macroscopic signs of disease included abdominal distension due to fluid accumulation within the internal organs as well as external and internal haemorrhaging. Histological evaluation revealed chronic peritonitis with peritoneal adhesions in all fish examined. Kidney, spleen and ascites fluid samples were collected from diseased bluegills and examined for the presence of pathogens. Bluegill picornavirus (BGPV) was isolated using tissue cell culture methods utilizing a recently developed, uncharacterized bluegill fry cell line (BF-4), and the presence of this virus was confirmed through molecular identification. The current geographic range, known susceptible hosts as well as historical epizootics associated with BPGV is discussed. The ability of BGPV to cause significant mortality in wild fish further emphasizes the importance of monitoring both wild and hatchery populations for this pathogen.

Effects of short- and long-term administration of nonsteroidal anti-inflammatory drugs on osteotomy healing in dogs.

OBJECTIVE: To determine the influence of short-term administration of carprofen on bone healing in dogs. STUDY DESIGN: Randomized controlled experimental study. ANIMALS: Eighteen purpose-bred sexually mature hound dogs. METHODS: Tibial osteotomies were performed, and dogs were divided into three groups: no carprofen (n = 6), 2-week administration of carprofen at 2.2 mg/kg twice daily (n = 6), and 8-week administration of carprofen at 2.2 mg/kg twice daily (n = 5). Bone healing was evaluated radiographically at 4 and 8 weeks postoperatively. Postmortem, fracture healing was assessed via biomechanical testing (three-point bending), histological cartilage:callus ratio, and bone mineral density (BMD) with quantitative computed tomography. RESULTS: No biomechanical difference was detected between dogs that received no carprofen and those that received 2 weeks of carprofen or between those that received 2 weeks vs 8 weeks of carprofen. Stiffness (P = .035) and maximum stress (P = .042) were higher in dogs that received no carprofen than in those that received 8 weeks of carprofen. Radiographic healing did not differ between dogs without carprofen and those with 2-week administration of carprofen (P = .9923). However, tibias of dogs without carprofen and those with 2-week administration of carprofen were more healed compared with those in the 8-week-carprofen group at 4 and 8 weeks after surgery (P = .0011). No treatment effect was detected by cartilage:callus ratio or BMD. CONCLUSION: Long-term administration of carprofen had a negative effect on bone healing compared with short-term or no administration of carprofen. CLINICAL SIGNIFICANCE: Nonsteroidal anti-inflammatory drugs should be used cautiously in dogs at risk for delayed bone healing, and administration should be discontinued beyond the perioperative period in dogs with fractures or osteotomies.

The virulence and immune protection of Edwardsiella ictaluri HemR mutants in catfish.

Edwardsiella ictaluri is a Gram-negative facultative intracellular rod, causing enteric septicemia of catfish (ESC). Several heme uptake systems have been described in bacterial pathogens, most of which involve outer membrane proteins (OMPs). We have shown recently that heme/hemoglobin receptor family protein (HemR) is significantly up-regulated in E. ictaluri under iron-restricted conditions. In this work, our goal was to construct E. ictaluri HemR mutants and assess their virulence and immune protection potentials in catfish. To accomplish this, an in-frame deletion mutant (EiΔhemR) was constructed, and its virulence and immune protection were determined in catfish fingerlings and fry. The results indicated that the EiΔhemR was attenuated completely in catfish fingerlings, but it was virulent in 14 day-old catfish fry. To increase the attenuation of EiΔhemR in fry, we introduced frdA and sdhC gene deletions to the mutant, yielding two double (EiΔhemRΔfrdA and EiΔhemRΔsdhC) and one triple (EiΔhemRΔfrdAΔsdhC) mutants. Results indicated that two double HemR mutants did not exhibit increased attenuation, but the triple HemR mutant showed significantly less virulence and high protection in fry (p < 0.05). Histological examination of fry tissues vaccinated with the triple mutant displayed similar inflammation to that of wild-type infected fry, but much less necrosis and far fewer bacteria were observed. Immunohistochemistry (IHC) result indicated fewer numbers of bacteria around blood vessel and in the hematopoietic tissue in fry infected with triple mutant compared to control group infected with E. ictaluri wild-type. Our data indicated that EiΔhemR was safe and protective in catfish fingerlings, while EiΔhemRΔfrdAΔsdhC was much safer in catfish fry.

Fin healing and regeneration in sturgeon.

Pectoral fin healing in fin spines and rays were examined in juvenile Atlantic sturgeon Acipenser oxyrinchus oxyrinchus following three different sampling techniques (n = 8-9 fish per treatment): entire leading fin spine removed, a 1-2 cm portion removed near the point of articulation, or a 1-2 cm portion removed from a secondary fin ray. Also, to determine whether antibiotic treatment influences healing, an additional group of fish (n = 8) was not given an injection of an oxytetracycline (OTC)-based antibiotic following removal of the entire leading fin spine. Following fin sampling, fish from different treatments were mixed equally between three large (4,000 I) recirculating systems and fin-ray healing and mortality were monitored over a 12 month period. To assess healing, blood samples were collected at 4 months to measure immune system responses, radiographs were taken at 4, 8 and 12 months to assess the degree of calcification in regions of damaged fins and fins were analyzed histologically at 12 months. Fish grew from a mean weight of 1.8 to 3.2 kg during the experiment and survival was near 100% in all treatments, with only one fish dying of unknown causes. Leukocyte counts, an indication of health status and survival were similar among treatments and in groups with or without antibiotic injection. Radiographs revealed mineralization took longer in fish with the entire leading fin spine removed and was the slowest near the point of articulation, presumably due to the greater structural support for the pectoral fin at this location. Histological sampling indicated spines and rays had similar healing patterns. Following injury, an orderly matrix of collagen bundles and many evenly spaced scleroblasts were present, transitioning to Sharpey fibres, with concentric layers forming lamellar bone. Healing and mineralization were characterized as periosteal osteogenesis and included embedded osteocytes surrounded by an osteoid seam. Chondroid formation was apparent in a few fractures not associated with treatments. The duration of time for external wound healing and internal mineralization of spines and rays depended on the fin treatment, with the slowest healing observed in fish with the most tissue removed, the entire leading fin spine.

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.

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 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.

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics.

Identification of Langerhans-like cells in the immunocompetent tissues of channel catfish, Ictalurus punctatus.

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) that have a critical role in bridging innate and adaptive immune responses in vertebrates. Dendritic cells have been characterized morphologically and functionally in the teleost fish models such as rainbow trout, salmonids, medaka, and zebrafish. The presence of DCs with remarkable similarities to human Langerhans cells (LCs) has been described in the spleen and anterior kidney of salmonids and rainbow trout. However, there is no evidence of the presence of DCs and their role in channel catfish immunity. In this study, we assessed DC-like cells in the immunocompetent tissues of channel catfish by immunohistochemistry (IHC), flow cytometry and transmission electron microscopy (TEM). We identified Langerin/CD207+ (L/CD207+) cells in the channel catfish anterior kidney, spleen and gill by IHC. Moreover, we described the cells that resembled mammal LC DCs containing Birbeck-like (BL) granules in channel catfish spleen, anterior and posterior kidneys and gill by TEM. Our data suggest that cells with DC-like morphology in the immune related organs of catfish may share morphological and functional properties with previously reported DCs in teleost fish and mammals. More detailed knowledge of the phenotype and the function of catfish DCs will not only help gain insight into the evolution of the vertebrate adaptive immune system but will also provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use.

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.

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.