Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

National Taiwan University

An ~21-year-old female Barbary sheep (Ammotragus lervia) died spontaneously following a lengthy episode of difficulty in walking. An ~6 × 3 × 3 cm, unilocular cystic growth was found in the cranioventral thorax. The fibrotic cystic wall, lined by a single layer of flattened to cuboidal epithelial cells, was invaginated and partially encircled solid masses of fusiform neoplastic cells with multiple intratumoral cystic structures. The fusiform neoplastic cells were intensely positive for cytokeratin (CK) and partially positive for α-smooth muscle actin and vimentin, but negative for thyroid transcription factor-1 (TTF-1) and neuron-specific enolase (NSE). The intratumoral cysts were lined by CK-positive but TTF-1- negative, NSE-negative, flattened, cuboidal to columnar epithelial cells, suggestive of cystically dilated medullary duct epithelium-derived structures. Based on the location and histopathologic findings of the growth, concurrent spindle-cell thymoma and thymic cysts was diagnosed. We also discuss the correlation between thymic cysts and thymoma and review the literature of thymomas in ovine and wildlife species.

Concurrent spindle-cell thymoma and thymic cysts in a Barbary sheep (Ammotragus lervia): case report and review of the literature.

A mass mortality event of captive Hong Kong warty newts Paramesotriton hongkongensis with non-granulomatous necrotic lesions occurred in Taipei Zoo, Taiwan, in 2014. Clinically, the sick newts were lethargic and often covered with water mold Saprolegnia sp. on the skin of the body trunk or extremities. Predominant pathological findings were multifocal non-granulomatous necrotic lesions in the liver, spleen, and kidneys and severe skin infection with Saprolegnia sp., with deep invasion and involvement of underlying muscles. The possibility of ranavirus infection was ruled out by negative PCR results. Unexpectedly, abundant intralesional acid-fast positive bacilli were found in the necrotic lesions of the liver, spleen, and kidney in all 14 sick newts. PCR targeting the hsp65, ITS region, and partial 16S rRNA genes was performed, and the sequence identity from amplified amplicons of hsp65 and partial 16S rRNA genes was 100% identical to that of the corresponding gene fragment of Mycobacterium marinum. Further molecular investigations demonstrated that the current M. marinum was a mycolactone-producing mycobacterium with the presence of esxA/esxB genes. Mycolactone is a plasmid-encoded, immunosuppressive, and cytotoxic toxin. The possible immunosuppression phenomenon characterized by systemic non-granulomatous necrotic lesions caused by M. marinum and the unusual deep invasive infection caused by water mold might be associated with the immunosuppressive effect of mycolactone. Therefore, it should be noted that non-granulomatous necrotic lesions in amphibians can be caused not only by ranavirus infection but also by mycobacteriosis.

Mycolactone-producing Mycobacterium marinum infection in captive Hong Kong warty newts and pathological evidence of impaired host immune function.

Scuticociliatosis, caused by ciliated protozoa in the subclass Scuticociliatia of the phylum Ciliophora, can cause fatal disease in teleost fish species. However, information on scuticociliatosis in elasmobranchs is still scarce. In this report, we describe a case of locally extensive meningoencephalitis caused by Miamiensis avidus (syn. Philasterides dicentrarchi) in a 2 yr old captive zebra shark Stegostoma fasciatum. Granulocytic meningoencephalitis was observed through histological assessment. Inflammation was confined to the ventral aspect of the brain with a large number of ciliated protozoa, transforming into non-suppurative meningitis in the lateral aspect, and gradually vanished in the dorsal aspect. No histopathological and polymerase chain reaction (PCR) evidence of systemic dissemination of M. avidus was found. PCR targeting the gene coding the small-subunit ribosomal RNA (SSUrRNA) of M. avidus was performed on the brain, liver, and gill tissues, and only brain tissue yielded a positive result. The DNA sequences from amplicons of the protozoal SSUrRNA gene were completely matched to that of M. avidus. The distribution of protozoa in the current case was mainly located in the brain and suggests the possibility of a direct neural invasive pathway of M. avidus through the nasal cavity/ampullary system and/or a unique tissue tropism of M. avidus specific to the brain in zebra sharks. Further investigations on the pathogenesis of M. avidus in elasmobranchs, especially zebra sharks, are needed.

Locally extensive meningoencephalitis caused by Miamiensis avidus (syn. Philasterides dicentrarchi) in a zebra shark.

Silver, such as silver nanoparticles (AgNPs), has been widely used in commercial products and may be released into the environment. The interaction between Ag deposition and biological systems is raising serious concerns because of one health consideration. Cetaceans, as the top predators of the oceans, may be exposed to Ag/Ag compounds and suffer negative health impacts from the deposition of these compounds in their bodies. In the present study, we utilized autometallography (AMG) to localize the Ag in the liver and kidney tissues of cetaceans and developed a model called the cetacean histological Ag assay (CHAA) to estimate the Ag concentrations in the liver and kidney tissues of cetaceans. Our results revealed that Ag was mainly located in hepatocytes, Kupffer cells and the epithelial cells of some proximal renal tubules. The tissue pattern of Ag/Ag compounds deposition in cetaceans was different from those in previous studies conducted on laboratory rats. This difference may suggest that cetaceans have a different metabolic profile of Ag, so a presumptive metabolic pathway of Ag in cetaceans is advanced. Furthermore, our results suggest that the Ag contamination in cetaceans living in the North-western Pacific Ocean is more severe than that in cetaceans living in other marine regions of the world. The level of Ag deposition in cetaceans living in the former area may have caused negative impacts on their health condition. Further investigations are warranted to study the systemic Ag distribution, the cause of death/stranding, and the infectious diseases in stranded cetaceans with different Ag concentrations for comprehensively evaluating the negative health effects caused by Ag in cetaceans.

Investigation of silver (Ag) deposition in tissues from stranded cetaceans by autometallography (AMG).

Nephroblastomas are uncommon embryonal tumors in dogs. We report herein a blastema-predominant nephroblastoma with gingival metastasis in an 8-y-old Miniature Pinscher dog. Histologically, the mass was composed mainly of blastemal elements with minor epithelial and mesenchymal differentiation. Metastatic masses in the gingiva had histologic and immunohistochemical features similar to those of the primary renal nephroblastoma. Neoplastic cells were extensively positive for both vimentin and PAX8, and scattered positive for cytokeratin. Using the clinical staging of human Wilms tumor, we staged our case as stage IV with <4 mo of survival time. We summarized previous studies of canine renal and spinal nephroblastomas, and analyzed the correlations among clinical staging, histologic classification, and mean survival time of dogs with renal nephroblastomas. Clinical staging was significantly correlated with survival time, as shown in humans. In dogs, however, additional factors can potentially influence the outcome of treatment and disease development.

A blastema-predominant canine renal nephroblastoma with gingival metastasis: case report and literature review.

Silver nanoparticles (AgNPs) have been extensively used and are considered as an emerging contaminant in the ocean. The environmental contamination of AgNPs is expected to increase greatly over time, and cetaceans, as the top ocean predators, will suffer the negative impacts of AgNPs. In the present study, we investigate the immunotoxicity of AgNPs on the leukocytes of cetaceans using several methods, including cytomorphology, cytotoxicity, and functional activity assays. The results reveal that 20 nm Citrate-AgNPs (C-AgNP) induce different cytomorphological alterations and intracellular distributions in cetacean polymorphonuclear cells (cPMNs) and peripheral blood mononuclear cells (cPBMCs). At high concentrations of C-AgNP (10 and 50 μg/ml), the time- and dose-dependent cytotoxicity in cPMNs and cPBMCs involving apoptosis is demonstrated. C-AgNP at sub-lethal doses (0.1 and 1 μg/ml) negatively affect the functional activities of cPMNs (phagocytosis and respiratory burst) and cPBMCs (proliferative activity). The current study presents the first evidence of the cytotoxicity and immunotoxicity of AgNPs on the leukocytes of cetaceans and improves our understanding of environmental safety concerning AgNPs. The dose-response data of AgNPs on the leukocytes of cetaceans are invaluable for evaluating the adverse health effects in cetaceans and for proposing a conservation plan for marine mammals.

Immunotoxicity of Silver Nanoparticles (AgNPs) on the Leukocytes of Common Bottlenose Dolphins (Tursiops truncatus).

Silver nanoparticles (AgNPs) have been widely used in many commercial products due to their excellent antibacterial ability. The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect animals at high trophic levels, such as cetaceans and humans, via the food chain. Hence, the negative health impacts caused by AgNPs in cetaceans are of concern. Cytokines play a major role in the modulation of immune system and can be classified into two types: Th1 and Th2. Th1/Th2 balance can be evaluated by the ratios of their polarizing cytokines (i.e., interferon [IFN]-γ/Interleukin [IL]-4), and animals with imbalanced Th1/Th2 response may become more susceptible to certain kinds of infection. Therefore, the present study evaluated the  cytokine responses of cetacean peripheral blood mononuclear cells (cPBMCs) to 20 nm citrate-AgNPs (C-AgNP) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Wen-Ta Li

Graduate Institute of Molecular and Comparative Pathobiology

National Taiwan University

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Wen-Ta Li

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Graduate Institute of Molecular and Comparative Pathobiology

National Taiwan University

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Graduate Institute of Molecular and Comparative Pathobiology