Wes Baumgartner

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.

Edwardsiellosis Caused by Edwardsiella ictaluri in Laboratory Populations of Zebrafish Danio rerio

We report the first cases of Edwardsiella ictaluri causing epizootics in laboratory populations of Zebrafish Danio rerio. Edwardsiella ictaluri is primarily recognized as a disease of catfish species and is known to cause an economically important bacterial disease of farm-raised catfish in the USA and abroad; however, it has been isolated on occasion from 10 other genera of nonictalurid fishes. We isolated E. ictaluri from moribund Zebrafish held in quarantine at two different universities in two states and from a research facility in a third state between February 23 and December 6, 2011. Edwardsiellosis in Zebrafish can be described as a severe systemic disease characterized by tissue necrosis and the presence of large numbers of extracellular and intracellular bacteria, often within macrophages. The kidneys (pronephros and mesonephros), spleen, nares, and forebrain were the most commonly and severely affected tissues. In outbreaks, mortality was acute and numerous fish died over a 1-2 week period. Mortality continued until the majority of the population was lost, at which time the remaining fish were euthanized. In addition to these cases, four cultures of bacteria isolated from Zebrafish by another diagnostic laboratory were submitted to the Louisiana Aquatic Diagnostic Laboratory for identification and were confirmed as E. ictaluri. In total, eight cultures of E. ictaluri from Zebrafish from Louisiana, Massachusetts, Pennsylvania, and Florida were identified. The isolates were confirmed as E. ictaluri by biochemical phenotype, API 20E (bioMérieux), and amplification and sequencing of a portion of the 16S rRNA gene. Edwardsiella ictaluri isolates from Zebrafish are believed to comprise a unique group and were differentiated from catfish isolates by exhibiting weaker motility, autoaggregation in broth, a different plasmid profile (two plasmids of 4.0 and 3.5 kb), a different API 20E code (4204000), and lack of lipopolysaccharide recognition with Mab Ed9.

Determination of Florfenicol Dose Rate in Feed for Control of Mortality in Nile Tilapia Infected with Streptococcus iniae

A dose titration study was conducted to determine the dosage of florfenicol (FFC) in feed to control Streptococcus iniae-associated mortality in Nile tilapia Oreochromis niloticus. Six tanks were assigned to each of five treatments: (1) not challenged with S. iniae and fed unmedicated feed; (2) challenged with S. iniae by injection and fed unmedicated feed; (3) challenged with S. iniae and given FFC at 5 mg/kg of body weight (bw) in medicated feed; (4) challenged with S. iniae and given 10 mg FFC/kg bw; and (5) challenged with S. iniae and given 15 mg FFC/kg bw. Treatment was initiated the day after inoculation, and feed was administered for 10 d. Cumulative mortality was 0% in the unchallenged, untreated group; 35.8 +/- 4.4% (mean +/- SE) in the challenged, unmedicated group; 19.2 +/- 2.7% in the 5-mg/kg treated group, 12.5 +/- 3.8% in the 10-mg/kg group, and 2.5 +/- 1.1% in the 15-mg/kg group. The cumulative mortality was significantly less in each challenged, FFC-treated group than in the challenged, unmedicated controls (5 mg/ kg: P = 0.0156; 10 mg/kg: P = 0.0007; 15 mg/kg: P < 0.0001). The efficacy of the 10- and 15-mg/kg FFC dosages was studied in a separate dose confirmation study. Fish in all tanks were injected with S. iniae. At 4 h postinoculation, 10 tanks were assigned to each of three feed treatments: (1) unmedicated feed; (2) 10 mg FFC/kg bw; and (3) 15 mg FFC/kg bw. Cumulative mortality was 20.5 +/- 2.0% in the challenged, unmedicated group; 11.0 +/- 2.1% in the 10-mg/kg group; and 5.5 +/- 2.4% in the 15-mg/kg group. Mortality was significantly less in the medicated groups than in the challenged, unmedicated control group (10 mg/kg: P = 0.0270; 15 mg/kg: P = 0.0007). Fish in both studies were necropsied, cultured for bacteria, and examined for gross lesions. The minimum inhibitory concentration of FFC against S. iniae in both studies ranged from 0.5 to 1.0 microg/mL. Florfenicol was palatable, safe, and efficacious for control of Nile tilapia mortality due to S. iniae infection.

Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius

ABSTRACT A severe, sometimes fatal respiratory disease has been observed in captive ball pythons (Python regius) since the late 1990s. In order to better understand this disease and its etiology, we collected case and control samples and performed pathological and diagnostic analyses. Electron micrographs revealed filamentous virus-like particles in lung epithelial cells of sick animals. Diagnostic testing for known pathogens did not identify an etiologic agent, so unbiased metagenomic sequencing was performed. Abundant nidovirus-like sequences were identified in cases and were used to assemble the genome of a previously unknown virus in the order Nidovirales. The nidoviruses, which were not previously known to infect nonavian reptiles, are a diverse order that includes important human and veterinary pathogens. The presence of the viral RNA was confirmed in all diseased animals (n = 8) but was not detected in healthy pythons or other snakes (n = 57). Viral RNA levels were generally highest in the lung and other respiratory tract tissues. The 33.5-kb viral genome is the largest RNA genome yet described and shares canonical characteristics with other nidovirus genomes, although several features distinguish this from related viruses. This virus, which we named ball python nidovirus (BPNV), will likely establish a new genus in Torovirinae subfamily. The identification of a novel nidovirus in reptiles contributes to our understanding of the biology and evolution of related viruses, and its association with lung disease in pythons is a promising step toward elucidating an etiology for this long-standing veterinary disease. IMPORTANCE Ball pythons are popular pets because of their diverse coloration, generally nonaggressive behavior, and relatively small size. Since the 1990s, veterinarians have been aware of an infectious respiratory disease of unknown cause in ball pythons that can be fatal. We used unbiased shotgun sequencing to discover a novel virus in the order Nidovirales that was present in cases but not controls. While nidoviruses are known to infect a variety of animals, this is the first report of a nidovirus recovered from any reptile. This report will enable diagnostics that will assist in determining the role of this virus in the causation of disease, which would allow control of the disease in zoos and private collections. Given its evolutionary divergence from known nidoviruses and its unique host, the study of reptile nidoviruses may further our understanding of related diseases and the viruses that cause them in humans and other animals. Ball pythons are popular pets because of their diverse coloration, generally nonaggressive behavior, and relatively small size. Since the 1990s, veterinarians have been aware of an infectious respiratory disease of unknown cause in ball pythons that can be fatal. We used unbiased shotgun sequencing to discover a novel virus in the order Nidovirales that was present in cases but not controls. While nidoviruses are known to infect a variety of animals, this is the first report of a nidovirus recovered from any reptile. This report will enable diagnostics that will assist in determining the role of this virus in the causation of disease, which would allow control of the disease in zoos and private collections. Given its evolutionary divergence from known nidoviruses and its unique host, the study of reptile nidoviruses may further our understanding of related diseases and the viruses that cause them in humans and other animals.

Characterization of Isolates of Streptococcus agalactiae from Diseased Farmed and Wild Marine Fish from the U.S. Gulf Coast, Latin America, and Thailand

We examined Lancefield serogroup B Streptococcus isolates recovered from diseased, cultured hybrid Striped Bass (Striped Bass Morone saxatilis × White Bass M. chrysops) and wild and cultured Gulf Killifish Fundulus grandis from coastal waters of the U.S. Gulf of Mexico (Gulf coast) and compared those isolates to strains from tilapias Oreochromis spp. reared in Mississippi, Thailand, Ecuador, and Honduras and to the original Gulf coast strain identified by Plumb et al. ( 1974 ). The isolates were subjected to phylogenetic, biochemical, and antibiotic susceptibility analyses. Genetic analysis was performed using partial sequence comparison of (1) the 16S ribosomal RNA (rRNA) gene; (2) the sipA gene, which encodes a surface immunogenic protein; (3) the cspA gene, which encodes a cell surface-associated protein; and (4) the secY gene, which encodes components of a general protein secretion pathway. Phylogenies inferred from sipA, secY, and cspA gene sequence comparisons were more discriminating than that inferred from the 16S rRNA gene sequence comparison. The U.S. Gulf coast strains showed a high degree of similarity to strains from South America and Central America and belonged to a unique group that can be distinguished from other group B streptococci. In agreement with the molecular findings, biochemical and antimicrobial resistance analyses demonstrated that the isolates recovered from the U.S. Gulf coast and Latin America were more similar to each other than to isolates from Thailand. Three laboratory challenge methods for inducing streptococcosis in Gulf Killifish were evaluated-intraperitoneal (IP) injection, immersion (IMM), and immersion plus abrasion (IMMA)-using serial dilutions of S. agalactiae isolate LADL 97-151, a representative U.S. Gulf coast strain. The dose that was lethal to 50% of test fish by 14 d postchallenge was approximately 2 CFU/fish via IP injection. In contrast, the fish that were challenged via IMM or IMMA presented cumulative mortality less than 40% by 14 d postchallenge.

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.

Characterization of Histopathological and Ultrastructural Changes in Channel Catfish Experimentally Infected with Virulent Aeromonas hydrophila

A highly virulent clonal population of Aeromonas hydrophila (vAh) has been the cause of recent motile Aeromonas septicemia epizootic in channel catfish (Ictalurus punctatus) farms in the Southeastern United States. The pathology of the disease caused by vAh has not been studied well yet. Thus, our aim was to determine histopathological and ultrastructural changes in channel catfish following vAh challenge. To accomplish this, catfish fingerlings were challenged with vAh (strain ML09-119) by bath. Six fish per each time point were collected at 1, 3, 5, 6, 24, and 48 h for light microscopy, and six fish were collected at 48 h for transmission electron microscopy (TEM). The first pathological lesions were detected in the spleen and stomach at 1 h post-challenge (HPC) while intestine, gills, kidney, and liver lesions were observed at 24 and 48 HPC. Histopathological examination revealed degenerative changes, necrosis, extensive edema, and inflammation in internal organs. The TEM showed severe tissue destruction with multiple bacterial cells secreting outer membrane vesicles, especially in spleen and gills and far number in the stomach. Degenerated bacterial cells were observed in the intestinal lumen and the phagosomes of phagocytic kidney cells. We identified, for the first time, degranulate eosinophilic granular cells, and dendritic cells like (DC-like) cells in the necrotic intestinal epithelium. These findings suggest that vAh rapidly proliferated and spread through the catfish organs following bath challenge.

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.

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

Abstract:  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.

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

ABSTRACT 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 (Ei&Dgr;hemR) was constructed, and its virulence and immune protection were determined in catfish fingerlings and fry. The results indicated that the Ei&Dgr;hemR was attenuated completely in catfish fingerlings, but it was virulent in 14 day‐old catfish fry. To increase the attenuation of Ei&Dgr;hemR in fry, we introduced frdA and sdhC gene deletions to the mutant, yielding two double (Ei&Dgr;hemR&Dgr;frdA and Ei&Dgr;hemR&Dgr;sdhC) and one triple (Ei&Dgr;hemR&Dgr;frdA&Dgr;sdhC) 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 Ei&Dgr;hemR was safe and protective in catfish fingerlings, while Ei&Dgr;hemR&Dgr;frdA&Dgr;sdhC was much safer in catfish fry. HighlightsThe Ei&Dgr;hemR was attenuated in catfish fingerlings, but not in catfish fry.The triple mutant showed significantly less virulence and high protection in fry.The hemR gene is an important virulence factor for E. ictaluri.