Wen-Ta Li

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

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-AgNP20) induce different cytomorphological alterations and intracellular distributions in cetacean polymorphonuclear cells (cPMNs) and peripheral blood mononuclear cells (cPBMCs). At high concentrations of C-AgNP20 (10 and 50 μg/ml), the time- and dose-dependent cytotoxicity in cPMNs and cPBMCs involving apoptosis is demonstrated. C-AgNP20 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.

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

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.

Th2 cytokine bias induced by silver nanoparticles in peripheral blood mononuclear cells of common bottlenose dolphins (Tursiops truncatus)

Background 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 in vitro cytokine responses of cetacean peripheral blood mononuclear cells (cPBMCs) to 20 nm citrate-AgNPs (C-AgNP20) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Methods Blood samples were collected from six captive common bottlenose dolphins (Tursiops truncatus). The cPBMCs were isolated and utilized for evaluating the in vitro cytokine responses. The cytokines evaluated included IL-2, IL-4, IL-10, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. The geometric means of two housekeeping genes (HKGs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M), of each sample were determined and used to normalize the mRNA expression levels of target genes. Results The ratio of late apoptotic/necrotic cells of cPBMCs significantly increased with or without concanavalin A (ConA) stimulation after 24 h of 10 µg/ml C-AgNP20 treatment. At 4 h of culture, the mRNA expression level of IL-10 was significantly decreased with 1 µg/ml C-AgNP20 treatment. At 24 h of culture with 1 µg/ml C-AgNP20, the mRNA expression levels of all cytokines were significantly decreased, with the exceptions of IL-4 and IL-10. The IFN-γ/IL-4 ratio was significantly decreased at 24 h of culture with 1 µg/ml C-AgNP20 treatment, and the IL-12/IL-4 ratio was significantly decreased at 4 or 24 h of culture with 0.1 or 1 µg/ml C-AgNP20 treatment, respectively. Furthermore, the mRNA expression level of TNF-α was significantly decreased by 1 µg/ml C-AgNP20 after 24 h of culture. Discussion The present study demonstrated that the sublethal dose of C-AgNP20 (≤1 µg/ml) had an inhibitory effect on the cytokine mRNA expression levels of cPBMCs with the evidence of Th2 cytokine bias and significantly decreased the mRNA expression level of TNF-α. Th2 cytokine bias is associated with enhanced immunity against parasites but decreased immunity to intracellular microorganisms. TNF-α is a contributing factor for the inflammatory response against the infection of intracellular pathogens. In summary, our data indicate that C-AgNP20 suppresses the cellular immune response and thereby increases the susceptibility of cetaceans to infection by intracellular microorganisms.

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 antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

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

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.

Diffuse-type Gastric Mucinous and Signet Ring Cell Adenocarcinoma in a Captive California King Snake (Lampropeltis getula californiae)

An adult female California king snake (Lampropeltis getula californiae) housed in Taipei Zoo was presented with a 2-week history of anorexia, fatigue and abdominal swelling. Exploratory laparotomy revealed a gastric mass with two circular perforations and multiple mottled white to beige protuberances along the mucosal surface. Histologically, the gastric mass showed an invasive, transmural growth of epithelial cells arranged in nests, lobules, acini and sheets in the mucosa and submucosa that progressively transformed into signet ring cells in the muscularis externa and subserosa. All of the neoplastic cells expressed pan-cytokeratin immunohistochemically. Based on the World Health Organization histological criteria, a diagnosis of diffuse-type gastric mucinous and signet ring cell adenocarcinoma was made.

A PENILE SQUAMOUS CELL CARCINOMA WITH SYSTEMIC METASTASIS IN A THOROUGHBRED HORSE

A 22-year-old gelding Thoroughbred horse had been in poor physical condition with a prolapsed penis. On necropsy, numerous white nodules/plaques were found on the lungs, omentum, and multiple lymph nodes, as well as several ulcerations and cauliflower-like growths throughout the glans penis to the internal fold. Histologically, the white nodules/plaques were composed of similar neoplastic squamous cells, which were immunostained positive for cytokeratin (CK), CK 5/6, and P63. The cauliflower-like growths were diagnosed as squamous papilloma, inverted papilloma, and penile squamous cell carcinoma (PSCC). It is speculated that the lung metastasis and peritoneal carcinomatosis in this case are derived from the PSCC. The lesion tissues from the penis and omentum were characterized negative, by polymerase chain reaction, for Equus caballus papillomavirus 2 (EcPV2). This finding suggests that the penile lesions may have been irrelevant to the EcPV2, further implying that multiple pathogeneses exist for the development of an equine PSCC.

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

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.

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

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.

MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF A RHABDOMYOSARCOMA WITH SYSTEMIC METASTASIS IN AN AVIAN LEUKOSIS VIRUS (ALV) INFECTED CHICKEN

Immunohistochemistry features and cross-reactivity of commercially available antibodies are poorly understood in avian species. Here, we present a confirmative diagnosis of a rare systemically metastatic rhabdomyosarcoma in a seven-month-old male domestic chicken (Gallus domesticus). The animal had multiple grey to dark-red masses in the pectoral muscle, coelomic cavity, and multiple visceral organs. Microscopically, these masses were composed of spindloid neoplastic cells arranged into interwoven and patternless arrays with the presence of binucleated to multinucleated and karyomegalic neoplastic cells. A systemically metastatic rhabdomyosarcoma associated with avian leukosis viruses (ALV) infection was diagnosed by immunohistochemically demonstrating neoplastic cells dispersedly positive for desmin and p27 major capsid protein of ALVs, but diffusely negative for cytokeratin, vimentin, α-smooth muscle actin, MyoD1, S-100, and neuron-specific enolase. These tumors and internal organs were further characterized positive for subgroups A, B, C, D, or E ALVs, but negative for subgroup J ALV, by polymerase chain reactions. The report suggests the potential of using IHC for a confirmative diagnosis of rhabdomyosarcoma and ALV infection in chickens. The role of endogenous and exogenous ALVs in the rhabdomyosarcoma is also discussed in the present report.