Sex-dependent host-microbiome dynamics in zebrafish: Implications for toxicology and gastrointestinal physiology.

The physiology of males and females can be vastly different, complicating interpretation of toxicological and physiological data. The objectives of this study were to elucidate the sex differences in the microbiome-gastrointestinal (GI) transcriptome of adult zebrafish. We compared microbial composition and diversity in both males and females fed the same diet and housed in the same environment. There were no sex-specific differences in weight gain nor gastrointestinal morphology based on histopathology. There was no difference in gut microbial diversity, richness (Shannon and Chao1 index) nor predicted functional composition of the microbiome between males and females. Prior to post-hoc correction, male zebrafish showed higher abundance for the bacterial families Erythrobacteraceae and Lamiaceae, both belonging to the phyla Actinobacteria and Proteobacteria. At the genus level, Lamia and Altererythrobacter were more dominant in males and an unidentified genus in Bacteroidetes was more abundant in females. There were 16 unique differentially expressed transcripts in the gastrointestinal tissue between male and female zebrafish (FDR corrected, p < 0.05). Relative to males, the mRNA expression for trim35-9, slc25a48, chchd3b, csad, and hsd17b3 were lower in female GI while cyp2k6, adra2c, and bckdk were higher in the female GI. Immune and lipid-related gene network expression differed between the sexes (i.e., cholesterol export and metabolism) as well as networks related to gastric motility, gastrointestinal system absorption and digestion. Such data provide clues as to putative differences in gastrointestinal physiology between male and female zebrafish. This study identifies host-transcriptome differences that can be considered when interpreting the microgenderome of zebrafish in studies investigating GI physiology and toxicology of fishes.

Microbiome analysis and predicted relative metabolomic turnover suggest bacterial heme and selenium metabolism are altered in the gastrointestinal system of zebrafish (Danio rerio) exposed to the organochlorine dieldrin.

Dietary exposure to chemicals alters the diversity of microbiome communities and can lead to pathophysiological changes in the gastrointestinal system. The organochlorine pesticide dieldrin is a persistent environmental contaminant that bioaccumulates in fatty tissue of aquatic organisms. The objectives of this study were to determine whether environmentally-relevant doses of dieldrin altered gastrointestinal morphology and the microbiome of zebrafish. Adult zebrafish at ∼4 months of age were fed a measured amount of feed containing either a solvent control or one of two doses of dieldrin (measured at 16, and 163.5 ng/g dry weight) for 4 months. Dieldrin body burden levels in zebrafish after four-month exposure were 0 (control), 11.47 ± 1.13 ng/g (low dose) and 18.32 ± 1.32 ng/g (high dose) wet weight [mean ± std]. Extensive histopathology at the whole organism level revealed that dieldrin exposure did not induce notable tissue pathology, including the gastrointestinal tract. A repeated measure mixed model analysis revealed that, while fish gained weight over time, there were no dieldrin-specific effects on body weight. Fecal content was collected from the gastrointestinal tract of males and 16S rRNA gene sequencing conducted. Dieldrin at a measured feed dose of 16 ng/g reduced the abundance of Firmicutes, a phylum involved in energy resorption. At the level of class, there was a decrease in abundance of Clostridia and Betaproteobacteria, and an increase in Verrucomicrobiae species. We used a computational approach called predicted relative metabolomic turnover (PRMT) to predict how a shift in microbial community composition affects exchange of metabolites. Dieldrin was predicted to affect metabolic turnover of uroporphyrinogen I and coproporphyrinogen I [enzyme]-cysteine, hydrogen selenide, selenite, and methyl-selenic acid in the fish gastrointestinal system. These pathways are related to bacterial heme biosynthesis and selenium metabolism. Our study demonstrates that dietary exposures to dieldrin can alter microbiota composition over 4 months, however the long-term consequences of such impacts are not well understood.

Evaluation of Microbiome-Host Relationships in the Zebrafish Gastrointestinal System Reveals Adaptive Immunity Is a Target of Bis(2-ethylhexyl) Phthalate (DEHP) Exposure.

To improve physical characteristics of plastics such as flexibility and durability, producers enrich materials with phthalates such as di-2-(ethylhexyl) phthalate (DEHP). DEHP is a high production volume chemical associated with metabolic and immune disruption in animals and humans. To reveal mechanisms implicated in phthalate-related disruption in the gastrointestinal system, male and female zebrafish were fed DEHP (3 ppm) daily for two months. At the transcriptome level, DEHP significantly upregulated gene networks in the intestine associated with helper T cells' (Th1, Th2, and Th17) specific pathways. The activation of gene networks associated with adaptive immunity was linked to the suppression of networks for tight junction, gap junctional intercellular communication, and transmembrane transporters, all of which are precursors for impaired gut integrity and performance. On a class level, DEHP exposure increased Bacteroidia and Gammaproteobacteria and decreased Verrucomicrobiae in both the male and female gastrointestinal system. Further, in males there was a relative increase in Fusobacteriia and Betaproteobacteria and a relative decrease in Saccharibacteria. Predictive algorithms revealed that the functional shift in the microbiome community, and the metabolites they produce, act to modulate intestinal adaptive immunity. This finding suggests that the gut microbiota may contribute to the adverse effects of DEHP on the host by altering metabolites sensed by both intestinal and immune Th cells. Our results suggest that the microbiome-gut-immune axis can be modified by DEHP and emphasize the value of multiomics approaches to study microbiome-host interactions following chemical perturbations.

Dermatitis caused by autochthonous Cercopithifilaria bainae from a dog in Florida, USA: clinical, histological and parasitological diagnosis and treatment.

BACKGROUND: Cercopithifilaria bainae is a tick-vectored filarioid nematode associated with erythematous dermatitis in dogs. It has not been reported previously in the United States. HYPOTHESIS/OBJECTIVE: To describe clinical, histological and parasitological diagnosis and treatment of C. bainae in a dog. ANIMALS: An 11-month-old golden retriever/standard poodle mixed breed dog from Florida (USA). METHODS AND MATERIALS: The dog had no travel history within or outside the United States, was presented with a one month history of annular erythematous plaques on the head and ulcers on the medial canthi. Lesions were unresponsive to antibiotic treatment. RESULTS: Histopathological evaluation of skin biopsies revealed an eosinophilic to lymphohistiocytic perivascular dermatitis with multiple microgranulomas and rare 5-10 µm diameter microfilariae within microgranulomas. Microfilarial morphology was consistent with C. bainae. PCR and sequencing of 18S rRNA and mitochondrial cytochrome oxidase subunit I genes confirmed the nematodes as C. bainae. The dog was treated with a commercial spot-on containing imidacloprid and moxidectin, and clinical resolution occurred. CONCLUSIONS AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first report of C. bainae in a dog in the United States and the first description of dermatological lesions caused primarily by C. bainae.

Rhabdomyosarcoma associated with the lead wire of a pacemaker generator implant.

An 11-year-old female spayed Labrador Retriever was presented for a draining, painful subcutaneous mass palpated over a previously implanted pacemaker generator. Infection was suspected and the mass was removed surgically. On cut surface, the mass was friable and mottled tan to brown with firm pale tan nodules, surrounding the pacemaker lead wire adjacent to the pacemaker generator. Cytologic interpretation of impression smears was consistent with a sarcoma, and suggestive of a rhabdomyosarcoma due to the presence of strap-like cells. On histopathologic examination, a highly invasive nodular mass surrounded the pacemaker lead, composed of pleomorphic round, spindle and strap cells, and multinucleated giant cells. The population exhibited microscopic invasion into the deep portion of the fibrous capsule surrounding the pacemaker generator. There were tumor emboli within small to medium subcutaneous veins adjacent to the mass. Immunohistochemically, the neoplastic cells stained positive for α-sarcomeric actin and vimentin, and negative for α-smooth muscle actin, consistent with a rhabdomyosarcoma arising at the site of the pacemaker generator. To our knowledge, this is the first report of a rhabdomyosarcoma associated with the lead wire of a pacemaker generator in a dog.

Laryngeal rhabdomyoma in a dog.

A 3-year-old, female spayed Australian Shepherd dog was presented to Veterinary Healthcare Associates in Winter Haven, FL with a history of respiratory stridor, difficulty swallowing, and a change in bark for approximately 3 months. Radiographs revealed a soft tissue mass caudal to the epiglottis. Oral and computerized tomographic (CT) examinations were performed under general anesthesia and revealed a small firm mass in the caudal larynx on the right side. Cytologic evaluation of a fine-needle aspirate of the mass revealed cells consistent with a rhabdomyoma, oncocytoma, or granular cell tumor. Histopathologic interpretation was rhabdomyoma or oncocytoma. Histochemical staining with periodic acid-Schiff was mostly negative with a low number of weakly positive cells and with phosphotungstic acid-hematoxylin was strongly positive with visible cross striations. Tumor cells did not express pancytokeratin AE1/AE3, were strongly immunoreactive for myoglobin and muscle-specific actin, and did not express vimentin except for a low number of weakly immunoreactive cells. These findings confirmed a diagnosis of rhabdomyoma. Primary neoplasia of the larynx is rare, and widely varying types of neoplasms have been documented in this location. Due to the cytologic and histologic similarities between rhabdomyoma, oncocytoma, and granular cell tumor, these neoplasms should be differentiated using histochemical staining and immunohistochemical analysis.

Serum amyloid A (SAA) as a biomarker of chronic infection due to boat strike trauma in a free-ranging Florida manatee (Trichechus manatus latirostris) with incidental polycystic kidneys.

Watercraft-related trauma is the predominant cause of human-induced mortality in manatees (Trichechus manatus latirostris), a federal- and state-listed endangered species. Pyothorax (documented in this case report) and other secondary infections are common sequelae of inhalation of water and the open wounds caused by boat propellers. These secondary infections can lead to the demise of the animal weeks to months after the traumatic incident when external wounds have healed. Diagnosis of underlying disease on physical examination during capture and restraint can be difficult. Acute phase proteins, including serum amyloid A, fibrinogen, and albumin can be used to diagnose inflammatory disease in manatees and improve quality of medical care and husbandry. We also provide the first report of polycystic kidneys in Sirenians.

B-cell lymphoma with Mott cell differentiation in two young adult dogs.

Two young adult dogs with gastrointestinal signs were each found to have an intra-abdominal mass based on physical examination and diagnostic imaging. On exploratory laparotomy, small intestinal masses and mesenteric lymphadenopathy were found in both dogs; a liver mass was also found in dog 1. Cytologic and histologic examination of intestinal and liver masses and mesenteric lymph nodes revealed 2 distinct lymphoid cell populations: lymphoblasts and atypical Mott cells. With Romanowsky stains, the atypical Mott cells contained many discrete, clear to pale blue cytoplasmic inclusions consistent with Russell bodies that were positive by immunohistochemistry for IgM and CD79a in both dogs and for IgG in dog 2. The Mott cells and occasional lymphoblasts stained strongly positive with periodic acid-Schiff. Using flow cytometric immunophenotyping in dog 1, 60% of peripheral blood mononuclear cells and 85% of cells in an affected lymph node were positive for CD21, CD79a, IgM, and MCH II, indicative of B-cells. With electron microscopy, disorganized and dilated endoplasmic reticulum was seen in Mott cells in tumors from both dogs. Antigen receptor gene rearrangement analysis of lymph node and intestinal masses indicated a clonal B-cell population. Based on cell morphology, tissue involvement, and evidence for clonal B-cell proliferation, we diagnosed neoplasms involving Mott cells. To the authors' knowledge, this is the second report of Mott cell tumors or, more appropriately, B-cell lymphoma with Mott cell differentiation, in dogs. More complete characterization of this neoplasm requires further investigation of additional cases. This lymphoproliferative disease should be considered as a differential diagnosis for canine gastrointestinal tumors.

Endobronchial polyp derived from a myxosarcoma in the lung of a dog.

An endobronchial polyp was visible radiographically and bronchoscopically in an 11-year-old, mixed-breed dog with a persistent cough. The polyp was removed by traction. Initial histological examination suggested it was a myxomatous fibroma. The cough resolved but recurred with polyp regrowth. Two additional lung masses became visible radiographically. The polyp was removed twice more at 6-month intervals. Euthanasia was performed 15 months after first presentation when coughing recurred soon after the final bronchoscopy. Histological examination revealed that the mass was a myxomatous sarcoma. The lung contained two other unrelated tumors: a bronchioloalveolar carcinoma and a carcinoma of unknown origin.

West Nile virus infection in farmed American alligators (Alligator mississippiensis) in Florida.

In September and October 2002, an epizootic of neurologic disease occurred at an alligator farm in Florida (USA). Three affected American alligators (Alligator mississippiensis) were euthanatized and necropsied, and results confirmed infection with West Nile virus (WNV). The most significant microscopic lesions were a moderate heterophilic to lymphoplasmacytic meningoencephalomyelitis, necrotizing hepatitis and splenitis, pancreatic necrosis, myocardial degeneration with necrosis, mild interstitial pneumonia, heterophilic necrotizing stomatitis, and glossitis. Immunohistochemistry identified WNV antigen, with the most intense staining in liver, pancreas, spleen, and brain. Virus isolation and RNA detection by reverse transcription-polymerase chain reaction confirmed WNV infection in plasma and tissue samples. Of the tissues, liver had the highest viral loads (maximum 10(8.9) plaque-forming units [PFU]/0.5 cm3), whereas brain and spinal cord had the lowest viral loads (maximum 10(6.6) PFU/0.5 cm3 each). Virus titers in plasma ranged from 10(3.6) to 10(6.5) PFU/ml, exceeding the threshold needed to infect Culex quinquefasciatus mosquitoes (10(5) PFU/ml). Thus, alligators may serve as a vertebrate amplifying host for WNV.

Evaluation of a percutaneously controlled hydraulic occluder in a rat model of gradual venous occlusion.

OBJECTIVE: To evaluate the efficacy of a percutaneously controlled hydraulic occluder (HO) in a rat model of gradual venous occlusion. STUDY DESIGN: Experimental study. ANIMALS: Ten male Sprague-Dawley rats. METHODS: HOs and perivascular transit time ultrasound flow probes were applied to the caudal vena cava (CVC). Occluders in group I rats (n=6) were gradually attenuated over 8 weeks, whereas occluders in group II rats (n=4) were not attenuated. Vena caval blood flow measurements were performed weekly in all rats for 8 weeks. After euthanasia, the CVC was evaluated grossly and by histopathology. RESULTS: Premature occlusion of the vena cava occurred during the 1st week in 3 rats (1 in group I, 2 in group II) because of kinking of the vena cava between the HO and the flow probe. For the remaining rats, mean blood flow in group I decreased significantly from 40.71 mL/min/kg at baseline to 4.68 mL/min/kg at 8 weeks (P=.0094, power=0.91). Group II rats maintained vena caval blood flow at all times during the study. Complete occlusion of the vena cava was confirmed at necropsy in all group I rats. CONCLUSIONS: Gradual occlusion of the CVC of rats was achieved with HOs over 8 weeks. This model is limited by the size of the experimental animals and comparatively large implants. CLINICAL RELEVANCE: HOs may provide a method for gradual occlusion of congenital portosystemic shunts.

Intrapelvic granuloma formation six years after total hip arthroplasty in a dog.

A 9-year-old Golden Retriever that had undergone left total hip arthroplasty 6 years previously was evaluated because of constipation and tenesmus. Abdominal radiography and ultrasonography revealed a large intrapelvic mass that was contiguous with a mass of polymethyl methacrylate that had been extruded through a defect in the medial wall of the acetabulum at the time of total hip arthroplasty. Clinical signs resolved following resection of the mass and associated polymethyl methacrylate from the pelvic canal. Results of histologic examination of the mass were consistent with a diagnosis of foreign body granuloma, most likely secondary to particulate debris. There was no clinical or radiographic evidence of aseptic loosening of the acetabular or femoral components, and the mass may have represented a response to wear debris.

Effect of a single dose of ponazuril on neural infection and clinical disease in Sarcocystis neurona-challenged interferon-gamma knockout mice.

Interferon gamma-knockout mice were challenged with 5000 Sarcocystis neurona sporocysts acquired from a naturally infected opossum. Ponazuril was administered once, by gavage, at day 1, 3, 7, 10, or 14 post-infection (pi). Ponazuril was given at either 20 or 200mg/kg. Mice that survived to day 30 pi were euthanized. Severity of CNS infection was quantified as schizont density in the cerebellum. Unchallenged mice in treatment and non-treatment groups remained free of disease and gained weight throughout the experiment. All challenged mice, regardless of treatment, developed histologic evidence of CNS infection even though clinical signs were prevented in some groups. The greatest treatment benefits were seen in mice given 200mg/kg ponazuril between days 4 and 14 pi. Weight gain over the course of the experiment occurred only in mice that were given 200mg/kg ponazuril on day 7 or 10 pi. With the exception of groups given 200mg/kg ponazuril on day 7 or 14 pi, mice in groups that got sporocysts developed abnormal neurologic signs. No deaths before day 30 pi occurred in mice given ponazuril at 20mg/kg on day 7 pi or 200mg/kg on day 1, 7, 10, or 14 pi. This effect was not significant. Mice given 200mg/kg on day 7 pi had significantly fewer cerebellar schizonts than did those of the control group that was not given ponazuril. These results indicate that single-dose administration of ponazuril for prevention of CNS infection is partially protective when given between days 4 and 14 pi.

Dermal malignant melanoma in a horse with multifocal pancytokeratin expression.

The current report describes a malignant melanoma in the dermis of a 13-year-old bay Thoroughbred mare. Microscopic examination revealed that tumor cells were arranged in cords and packets within an abundant collagenous stroma containing scattered myxomatous foci. Tumor cells stained positively for S-100, neuron-specific enolase, and vimentin and some contained melanin granules. Some clusters of tumor cells were also positive for pancytokeratin. Expression of epithelial cell markers has been described in small numbers of human melanomas but has not been reported previously in equine melanomas.

Safety of long-term administration of a 0.01% fluocinolone shampoo in allergic dogs.

Nine dogs with allergic dermatitis were bathed twice weekly with a 0.01% fluocinolone shampoo for 6 months to evaluate the shampoo's effects on the adrenal-pituitary axis, serum chemistries, complete blood counts and cutaneous histology. A group of 5 normal control dogs were bathed twice weekly for 6 months with the shampoo vehicle to determine whether or not the stress of bathing would have any effects on the monitored indices. Skin biopsies were evaluated at the beginning and end of the study, and serum chemistries and serum cortisols postadrenocorticotrophic hormone administration were evaluated every 8 weeks. There was no effect of the drug over time on serum chemistries or cortisol. Individual dogs in the treatment group had decreased epidermal and dermal thickness as well as decrease in size or number of sebaceous glands after treatment. None of the dogs exhibited the combination of morphologic changes characteristic of an atrophic dermatosis due to hyperadrenocorticism.

Canine Subcutaneous Zygomycosis Caused by Conidiobolus sp.: A Case Report and Review of Conidiobolus Infections in other Species.

Résumé- Un chien, jeune adulte, a été présenté pour une masse sous-cutanée évoluant depuis un mois sur la partie droite du thorax et une masse ulcérée sous-cutanée sur la cuisse droite. L'examen histo-pathologique a révélé une inflammation pyogranulomateuse du derme et du tissu sous-cutané avec de larges hyphes fongiques. De nouvelles lésions sont apparues. Un traitement à base d'itraconazole pendant 2 semaines a été inefficace. L'euthanasie a été pratiquée 10 semaines après la première consultation. La culture fongique faite à partir de prélèvements chirurgicaux a permis d'isoler un Conidiobolus sp. Des infections àConidiobolus sp. ont été rapportées chez l'homme, le cheval, le mouton et le lama. Histologiquement, les hyphes sont difficiles à différencier de ceux d'autres zygomycètes ou de Pythium insidiosum. La culture est nécessaire pour une identification définitive. Une discussion sur les aspects comparés des infections cutanées àConidiobolus sp. est développée. [Hiller, A., Kunkle, G. A., Ginn, P. E., Padhye, A. A. Canine subcutaneous zygomycosis caused by Conidiobolus sp.: a case report and review of Conidibolus infections in other species. (Zygomycose sous-cutanée àConidiobolus sp. chez un chien: cas clinique et revue des infections àConidiobolus dans les autres espèces). Resumen- Un perro adulto joven presentaba una masa subcutánea en la región torácica derecha y otra ulcerada en la parte proximal de la extremidad posterior derecha, ambas de un mes de duración. El exámen histopatológico mostró una inflamación piogranulomatosa de la dermis y del tejido subcutáneo con grandes hifas füngicas. A pesar de que la enfermedad cutánea presentó un curso oscilante, aparecieron nuevas lesiones. Un tratamiento de dos semanas con itraconazol no alteró el curso de la enfermedad. El animal fue eutanasiado a las 10 semanas de la primera visita. Se ais lóConidiobolus sp. a partir de las muestras quirúrjicas. Las infecciones por Conidiobolus sp. estan descritas en la especie humana, la equina, la ovina y en las llamas. Las hifas no se diferencian fácilmente a nivel histológico de otros cigomicetales o de Pythium insidiosum y es necesaria la identificatión mediante un cultivo fúngico para establecer un diagnóstico definitive Se discuten las caracteristicas de la infección cutánea por Conidiobolus sp. y se describen las caracteristicas de la pitiosis cutánea. [Hillier, A., Kunkle, G. A., Ginn, P. E. and Padhye, A. A. Canine subcutaneous zygomycosis caused by Conidiobolus sp.: a case report and review of Conidibolus infections in other species (Cigomcosis subcutánea canina causada por Conidibolus sp.: un caso clinico y revisién de las infecciones por Conidibolus sp. en otras especies). Zusammenfassung- Ein junger ausgewachsener Hund wurde vorgestellt, der seit einem Monat an der rechten Thoraxwand ein subkutanes Gebilde und proximal an der rechten Hintergliedmaße eine ulzer-ierende, subkutane Masse aufwies. Bei der histopathologischen Untersuchung wurde eine pyogranulomatöse Entzündung der Dermis und Subkutis mit breiten Pilzhyphen entdeckt. Trotz des "Auf und Ab" der Hautkrankheit erschionon laufend neue Hautveränderungen. Eine zweiwöchige Behandlung mit Itrakonazol veränderte den Verlauf der Krankheit nicht. 10 Wochen nach der Erstvorstellung des Patienten wurde en euthenesiert. In der Kultur von chrirugischen Proben wurde Conidiobolus sp. isoliert. Conidiobolus sp.-Infektionen sind bei Menschen, Pferden, Schafen und Lamas beschrieben. Die Hyphen können histologisch schlecht von denen anderen Zygomyzeten oder von Pythium insidiosum unterschieden werden. Für die endgültige Diagnose ist eine Identifizierung mittels Pilzkultur notwendig. Die Erscheinungsbilder von kutanen Conidiobolus sp.-Infektionen werdon diskutiert und mit denen kutaner Phytiosis verglichen. [Canine subcutaneous zygomycosis caused by Conidiobolus sp.: a case report and reviews of Conidiobolus infections in other species (Subkutane Zygomykose des Hundes durch Conidiobolus sp.: Fallbericht und übersicht über Conidioblus-Infektionen bie anderen Tierarten). Abstract- A young adult dog was presented with a 1-month history of a subcutaneous mass on the right thoracic wall and an ulcerative, subcutaneous mass of the proximal right hindlimb. Pyogranulomatous inflammation of the dermis and subcutis with broad fungal hyphae were found on histopathologic examination. Despite some waxing and waning of the skin disease, new lesions continued to appear. A 2-week course of itraconazole did not alter the course of the disease. Euthanasia was performed 10 weeks after initial presentation. Conidiobolus sp. was isolated in culture from surgical specimens. Conidiobolus sp. infection has been reported in humans, horses, sheep and llamas. The hyphae cannot be easily distinguished histologically from those of some other zygomycetes or Pythium insidiosum and fungal culture identification of the organism is required for definitive diagnosis. A discussion of the features of cutaneous Conidiobolus sp. infection is given and comparative features of cutaneous pythiosis are presented.