Arginine metabolism in the Edwardsiella ictaluri- channel catfish macrophage dynamic

Edwardsiella ictaluri encodes a urease operon and an arginine decarboxylase (AdiA) that are required for virulence in head kidney derived macrophages (HKDM). The urease produces ammonia in amounts sufficient to alter environmental pH from acid to neutral. A hypothetical model was proposed, involving arginine metabolism in E. ictaluri infected HKDM, focusing on bacterial urease, AdiA, a second arginine decarboxylase (SpeA), and agmatinase (SpeB). Using fluorescence based ratiometric pH determination of E. ictaluri in live HKDM, it was shown that E. ictaluri modulates HKDM phagosome pH to above six. Urease and AdiA mutants failed to up-regulate vacuole pH, while vacuole pH for the SpeA and SpeB mutants was similar to the wild-type. These mutants could also replicate in HKDM similar to wild type E. ictaluri. These data show that urease and AdiA are required for phagosome pH neutralization. To determine the source of urea for E. ictaluri’s urease, an arginase inhibitor, L-norvaline, was used to partially block HKDM urea production. In arginase inhibited HKDM, E. ictaluri could not neutralize phagosome pH, nor could it replicate. Nitric oxide production in HKDM was not significantly different between controls and experimental groups. This indicates that HKDM have limited capacity to produce NO. Levels of urea produced in infected and control HKDM were at the lowest limit of assay detection and were not significantly different from one another. Together, these data show that E. ictaluri uses its urease and AdiA to neutralize phagosome pH, and that it uses urea derived from HKDM arginase to do so

Quantitative analysis of photodynamic therapy effects in rat mammary tumor vascular density using image-pro plus software

Photodynamic therapy (PDT) is a treatment modality that has advanced rapidly in recent years. It causes tissue and vascular damage with the interaction of a photosensitizing agent (PS), light of a proper wavelength, and molecular oxygen. Evaluation of vessel damage usually relies on histopathology evaluation. Results are often qualitative or at best semi-quantitative based on a subjective system. The aim of this study was to evaluate, using CD31 immunohistochem- istry and image analysis software, the vascular damage after PDT in a well-established rodent model of chemically induced mammary tumor. Fourteen Sprague-Dawley rats received a single dose of 7,12-dimethylbenz(a)anthraxcene (80 mg/kg by gavage), treatment efficacy was evaluated by comparing the vascular density of tumors after treatment with Photogem® as a PS, intraperitoneally, followed by interstitial fiber optic lighting, from a diode laser, at 200 mW/cm and light dose of 100 J/cm directed against his tumor (7 animals), with a control group (6 animals, no PDT). The animals were euthanized 30 hours after the lighting and mammary tumors were removed and samples from each lesion were formalin-fixed. Immunostained blood vessels were quantified by Image Pro-Plus version 7.0. The control group had an average of 3368.6 ± 4027.1 pixels per picture and the treated group had an average of 779 ± 1242.6 pixels per area (P < 0.01), indicating that PDT caused a significant decrease in vascular density of mammary tumors. The combined immu- nohistochemistry using CD31, with selection of representative areas by a trained pathology, followed by quantification of staining using Image Pro-Plus version 7.0 system was a practical and robust methodology for vessel damage evalua- tion, which probably could be used to assess other antiangiogenic treatments.FAPESPCePO

\u3ci\u3eClinostomum poteae\u3c/i\u3e n. sp. (Digenea: Clinostomidae), in the trachea of a double-crested cormorant \u3ci\u3ePhalacrocorax auritus\u3c/i\u3e Lesson, 1831 and molecular data linking the life-cycle stages of \u3ci\u3eClinostomum album\u3c/i\u3e 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 farmraised 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

Assessment of the Live Attenuated and Wild-Type Edwardsiella ictaluri-Induced Immune Gene Expression and Langerhans-Like Cell Profiles in the Immune-Related Organs of Catfish

Edwardsiella ictaluri is a Gram-negative intracellular pathogen that causes enteric septicemia of catfish (ESC). Successful vaccination against intracellular pathogens requires T cell priming by antigen presenting cells (APCs) that bridge innate and adaptive immunity. However, the evidence on immunological mechanisms that underscore E. ictaluri pathogenesis and the protective role of live attenuated vaccines (LAVs) is scarce. We assessed the expression of immune genes related to antigen presentation by real-time PCR and the distribution patterns of Langerhans-like (L/CD207+) cells by immunohistochemistry in the immune-related tissues of channel catfish challenged with two novel E. ictaluri LAVs, EiΔevpB, and ESC-NDKL1 and wild type (WT) strain. Our results indicated significantly elevated expression of IFN-γ gene in the anterior kidney (AK) and spleen of vaccinated catfish at the early stages of exposure, which correlated with increased numbers of L/CD207+ cells. In general, the ESC-NDKL1-induced IFN-γ gene expression patterns in the AK resembled that of the patterns induced by EiΔevpB. However the MHCII gene expression patterns differed between the strains with significant increases at 6 h post-challenge (pc) with the EiΔevpB and at 7 d pc with the ESC-NDKL1 strains, respectively. Significant increases in activity of T helper type polarization genes such as IFN-γ and T cell co-receptors after exposure to ESC-NDKL1, in combination with elevated numbers of L/CD207+ cells at 7 d pc with both LAVs compared to uninfected and the WT-exposed counterparts, were documented in the spleen. The dominant pro-inflammatory environment with dramatically overexpressed inflammatory genes in the AK and 7 d pc in the spleen in response to E. ictaluri was found in exposed catfish. In general, the pro-inflammatory gene expression profiles in the ESC-NDKL1 pc showed more similarities to the WT strain-induced gene profiles compared to the EiΔevpB counterpart. In addition, E. ictaluri WT significantly decreased the numbers of Langerhans-like L/CD207+ cells in the AK and spleen at 3 and 7 days pc. In conclusion, we report the differential framework of initiation of innate and adaptive immune responses between E. ictaluri strains with both LAVs having a potential of satisfying the stringent requirements for successful vaccines

Experimental Elucidation of the Life Cycle of \u3ci\u3eDrepanocephalus Spathans\u3c/i\u3e (Digenea: Echinostomatidae) with Notes on the Morphological Plasticity of \u3ci\u3eD. Spathans\u3c/i\u3e in the United States

The echinostomatid Drepanocephalus spathans (syn. Drepanocephalus auritus) parasitizes the doublecrested 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 naı¨ve 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

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.Keywords: misdiagnosis, fish histopathology, diagnostic accuracy, tissue fixation, artifacts, nonlesion

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