Effects of Mangosteen (Garcinia mangostana) Peel Extract Loaded in Nanoemulsion on Growth Performance, Immune Response, and Disease Resistance of Nile Tilapia (Oreochromis niloticus) against Aeromonas veronii Infection.

Nanotechnology can enhance nutrient delivery and bioavailability; hence, it has recently been considered the most practical alternative technology for nutritional supplements and disease control in fish farming. The present study was designed to evaluate the effects of mangosteen peel extract loaded in nanoemulsion (MSNE) on the inhibition of A. veronii (in vitro) and in vivo growth performance, serum biochemical parameters, the immune response, and the disease resistance of Nile tilapia (Oreochromis niloticus) against A. veronii challenge. The particle size, polydispersity index, and particle surface charge of MSNE were 151.9 ± 1.4 nm, >0.3, and -30 mV, respectively. Furthermore, MSNE, mangosteen peel extract (MPE), and nanoemulsion (NE) improved the antimicrobial activity against A. veronii. Fish fed MSNE, MPE, and NE-supplemented diets had a significantly lower (p < 0.05) feed conversion ratio (FCR) and higher specific growth rate (SGR) than fish fed the control diet. Furthermore, the MSNE had significantly higher serum glucose and protein levels than the control group in Nile tilapia. Total immunoglobulin, serum lysozyme, alternative complement activity, and survival of Nile tilapia fed with MSNE were significantly higher (p < 0.05) than the control diet. Therefore, MSNE has the potential to be employed as a supplement in sustainable Nile tilapia farming.

Masculinization of Red Tilapia (Oreochromis spp.) Using 17α-Methyltestosterone-Loaded Alkyl Polyglucosides Integrated into Nanostructured Lipid Carriers.

The aim of the present study was to optimize a masculinization platform for the production of all-male red tilapia fry by oral administration of 30 and 60 ppm of MT and alkyl polyglucoside nanostructured lipid carriers (APG-NLC) loaded with MT, respectively, for 14 and 21 days. The characterization, encapsulation efficiency and release kinetics of MT in lipid-based nanoparticles were assessed in vitro. The results showed that the MT-loaded nanoparticles were spherical, ranging from 80 to 125 nm in size, and had a negative charge with a narrow particle distribution. The APG-NLC loaded with MT provided higher physical stability and encapsulation efficacy than the NLC. The release rate constants of MT from MT-NLC and MT-APG-NLC were higher than those of free MT, which is insoluble in aqueous media. There was no significant difference in survival between the fish administered MT or the those fed orally with MT-APG-NLC fish. According to the logistic regression analysis, the sex reversal efficacy of MT-APG-NLC (30 ppm) and MT (60 ppm), resulted in significantly higher numbers of males after 21 days of treatment compared with the controls. The production cost of MT-APG-NLC (30 ppm) after 21 days of treatment was reduced by 32.9% compared with the conventional MT treatment group (60 ppm). In all the treatments, the length-weight relationship (LWR) showed negatively allomeric growth behavior (b < 3), with a relative condition factor (Kn) of more than 1. Therefore, MT-APG-NLC (30 ppm) would seem to be a promising, cost-effective way to reduce the dose of MT used for the masculinization of farmed red tilapia.

Domestic cat hepadnavirus associated with hepatopathy in cats: A retrospective study.

BACKGROUND: Whether domestic cat hepadnavirus (DCH) infection is associated with clinical disease remains to be determined. OBJECTIVES: To determine the relationship between DCH detection, hematology, serum bichemistry and liver histology in DCH-positive cats. ANIMALS: One thousand twenty-two cats in Thailand without concurrent diseases and not undergoing treatments adversely affecting the liver. METHODS: Retrospective cross-sectional study. Samples derived from cats with concurrent virus detection were excluded. DCH detection was determined in blood and fresh-frozen liver by quantitative polymerase chain reaction (qPCR) and further investigated in liver sections showing histological parenchymal disorders (HPD) and normal liver (HNL) using in situ hybridization (ISH). Proliferative/apoptotic activities were determined using immunohistochemistry and ISH panels. Biochemical variables and risk factors for DCH infection were investigated. RESULTS: Six hundred sixty-one (557 blood and 119 liver samples) cats were included. DCH was detected in 18.50% (103/557), 13.85% (9/65), and 3.70% (2/54) of the blood, HPD, and HNL groups, respectively. Cats with DCH revealed abnormally high activity of aspartate aminotransferase (AST) (P = .001) and alanine aminotransferase (ALT) (P < .001). Among DCH-positive HPD case 2/9 an 7/9 were acute and chronic hepatitis, of which 4/7 had hepatitis. Log viral copy number (LVCN) was positively correlated with ALT (P < .001), triglyceride (P < .001), and gamma-glutamyl transpeptidase (GGT) (P = .022). The LVCN also had a positive association with degree of hepatitis (P < .05). There was hepatocyte proliferation activity in DHC positive cats. CONCLUSION AND CLINICAL IMPORTANCE: Domestic cat hepadnavirus infection was associated with high serum activity of liver enzymes and chronic lymphoplasmacytic hepatitis (LPH).

Clove Oil-Nanostructured Lipid Carriers: A Platform of Herbal Anesthetics in Whiteleg Shrimp (Penaeus vannamei).

Whiteleg shrimp (Penaeus vannamei) have been vulnerable to the stress induced by different aquaculture operations such as capture, handling, and transportation. In this study, we developed a novel clove oil-nanostructured lipid carrier (CO-NLC) to enhance the water-soluble capability and improve its anesthetic potential in whiteleg shrimp. The physicochemical characteristics, stability, and drug release capacity were assessed in vitro. The anesthetic effect and biodistribution were fully investigated in the shrimp body as well as the acute multiple-dose toxicity study. The average particle size, polydispersity index, and zeta potential value of the CO-NLCs were 175 nm, 0.12, and -48.37 mV, respectively, with a spherical shape that was stable for up to 3 months of storage. The average encapsulation efficiency of the CO-NLCs was 88.55%. In addition, the CO-NLCs were able to release 20% of eugenol after 2 h, which was lower than the standard (STD)-CO. The CO-NLC at 50 ppm observed the lowest anesthesia (2.2 min), the fastest recovery time (3.3 min), and the most rapid clearance (30 min) in shrimp body biodistribution. The results suggest that the CO-NLC could be a potent alternative nanodelivery platform for increasing the anesthetic activity of clove oil in whiteleg shrimp (P. vannamei).

Renal epitheliotropism of feline morbillivirus in two cats.

The association of feline morbillivirus (FeMV) with kidney disease in cats is controversial. Two cats with a history of severe hematuria had eosinophilic inclusion-like bodies in the renal tubular epithelial cells, without any inflammatory cellular reaction. Ultrastructurally, aggregations of electron-dense viral-like particles were found where the inclusion-like bodies were located. Immunohistochemistry (IHC) using antibodies against FeMV matrix protein labeled these inclusion-like bodies, and also labeled the cytoplasm of tracheal and bronchiolar epithelial cells, and lymphocytes and macrophages in spleen and mesenteric lymph node. Using double IHC, FeMV antigen was detected in astroglia and oligodendroglia but not in microglia. Phylogenetic characterization of the fusion and hemagglutinin gene sequences revealed FeMV-1A genotypes in both cats. These findings indicated an active viral infection with FeMV. We propose that FeMV is a renal epitheliotropic virus and also localizes in various other tissues.

Japanese encephalitis virus infection in meerkats (Suricata suricatta).

Japanese encephalitis virus (JEV) infection has been recognized as a serious disease in humans. Wildlife animal infections due to JEV have not been well described. This study identified JEV infection in two deceased meerkats in Thailand, with clinical signs of neurological disease. Histopathology of brains revealed severe lymphoplasmacytic necrotizing meningoencephalitis, while similar inflammation was observed in the lung and liver. Partial JEV sequences were identified from the formalin-fixed paraffin-embedded-derived brain sections of two meerkats and were found to be genetically similar to a JEV strain detected in China but not from a local strain. Using immunohistochemistry, the virus was identified in neurons and glial cells, and also found in bronchial glands, Kupffer's cells in liver, lymphocytes in the spleen and pancreatic acini, which suggests extraneural infection. Transmission electron microscopy confirmed the presence of spheroid viral particles in the lungs. These findings may suggest that infection of extraneural organs in meerkats is similar to that described in JEV-infected humans. In conclusion, this study identified the first JEV infection in meerkats as an interesting case study. The JEV should be considered as an important differential diagnosis in meerkats with encephalitis. Further surveillance on JEV infection in meerkats and other wildlife species in a large cohort is needed in the future study.

Feline morbillivirus-1 in dogs with respiratory diseases.

Feline morbillivirus-1 (FeMV-1) is a viral pathogen associated with kidney disease in domestic cats and wild felids. We initially identified the FeMV-1 from the lung of a necropsied dog with severe pulmonary disease by the reverse transcription polymerase chain reaction (RT-PCR). Thereafter, we investigated FeMV-1 in nasal and oral swab samples from 73 healthy and 113 dogs with respiratory illnesses. We found polymerase chain reaction (PCR)-positive FeMV-1 from only 14/113 (12.39%) dogs with respiratory disease (p = .001). Of these 14 dogs, six were co-infected with other canine respiratory viruses (6/14; 42.86%). Two independent immunohistochemistry procedures, using antibodies against matrix and phosphoprotein of FeMV-1, confirmed the presence of FeMV-1 in lung tissues of two necropsied dogs (out of a total of 22 dogs, 9.09%) that died from respiratory disease. This finding corresponded to transmission electron microscopy findings that paramyxoviral particles exist in lung epithelia. FeMV-1 antigen localization was also evident in the kidney, lymphoid and brain tissues of two deceased dogs. FeMV-1 was successfully isolated from a necropsied dog and from two living dogs, all with respiratory illnesses, which supports FeMV infection in dogs. The detection of FeMV-1 in dog tissues expands the known tropism of this virus to a non-felid host. Our findings indicate that FeMV-1, alone or in co-infection with other viral pathogens, might contribute to respiratory illness and death in dogs.

Canine bocavirus-2 infection and its possible association with encephalopathy in domestic dogs.

Canine bocaviruses (CBoVs) have been recognized as pathogens associated with intestinal diseases. Hematogenous spreading caused by CBoV has been documented and may potentiate the virus entry across the blood-brain barrier to initiate a brain infection. This study focused attention on CBoV detection in cases of encepahlopathy and attempted to determine its viral localization. A total of 107 dog brains that histologically exhibited encephalopathy (ED) were investigated for the presence of CBoVs using polymerase chain reaction (PCR). Thirty-three histologically normal brain samples from dogs were used as a control group (CD). CBoV-2 was detected in 15 ED dogs (14.02%) but not in CD dogs (p = 0.02), while no CBoV-1 and -3 were detected. Among the CBoV-2 positive dogs, brain histological changes were characterized by nonsuppurative encephalitis, with inclusion body-like materials in some brains. In situ hybridization (ISH) and transmission electron microscopy (TEM) confirmed the presence of CBoV-2 viral particles in glial cells, supporting neurotropism of this virus. ISH signals were also detected in the intestines, lymphoid organs, and the heart, suggesting both enteral and parenteral infections of this virus. Whole genome characterization and evolutionary analysis revealed genetic diversity of CBoV-2 sequences and it was varying among the different countries where the virus was detected. This study points to a possible association of CBoV-2 with encephalopathy in dogs. It also highlights the genetic diversity and cellular tropism of this virus.

Epizootic reptilian ferlavirus infection in individual and multiple snake colonies with additional evidence of the virus in the male genital tract.

Reptilian ferlavirus, a pathogen of serious concern in snakes, has been reported in Western countries, but little is known about its prevalence in Thailand, where many snake breeding farms are located. In this study, we investigated the reptilian ferlavirus via swab samples derived from 49 diseased snakes and 77 healthy snakes as well as tissue samples taken from nine dead snakes from five independent snake farms. Using molecular detection, we found the ferlavirus in 8.16% of diseased snakes, but not in healthy snakes. Out of nine farmed snakes, eight snakes derived from four farms were found to be positive. Four complete genome sequences of the ferlavirus were successfully obtained and phylogenetically clustered to the highly pathogenic ferlavirus. Tissue tropism of the ferlavirus was identified in various epithelial cell types using the in situ hybridization technique. Interestingly, the hybridization signals were strongly labeled in the male genital tract. Transmission electron microscopy was used to support the ferlaviral localization in the male genital tract. This study provides the first evidence of ferlavirus localization in the male genital tract and contributes to the knowledge about ferlavirus epidemiology, indicating that there needs to be further awareness and elucidation regarding vertical transmission of reptilian ferlavirus.

Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys.

Carnivore protoparvovirus-1 (CPPV-1), a viral species containing feline panleukopenia virus (FPV) and canine parvovirus (CPV) variants, are widely spread among domestic and wild carnivores causing systemic fatal diseases. Wild fishing cats (Prionailurus viverrinus), a globally vulnerable species, have been found dead. Postmortem examination of the carcasses revealed lesions in intestine, spleen and kidney. CPPV-1 antigen identification in these tissues, using polymerase chain reaction (PCR) and immunohistochemistry (IHC), supported the infection by the virus. PCR- and IHC-positivity in kidney tissues revealed atypical localization of the virus while in situ hybridization (ISH) and transmission electron microscopy (TEM) with the pop-off technique confirmed the first description of viral localization in kidneys. Complete genome characterization and deduced amino acid analysis of the obtained CPPV-1 from the fishing cats revealed FPV as a causative agent. The detected FPV sequences showed amino acid mutations at I566M and M569R in the capsid protein. Phylogenetic and evolutionary analyses of complete coding genome sequences revealed that the fishing cat CPPV-1 genomes are genetically clustered to the FPV genomes isolated from domestic cats in Thailand. Since the 1970s, these genomes have also been shown to share a genetic evolution with Chinese FPV strains. This study is the first evidence of CPPV-1 infection in fishing cats and it is the first to show its localization in the kidneys. These findings support the multi-host range of this parvovirus and suggest fatal CPPV-1 infections may result in other vulnerable wild carnivores.

Improvement of the multi-performance biocharacteristics of cordycepin using BiloNiosome-core/chitosan-shell hybrid nanocarriers.

Cordycepin, a derivative of the nucleotide adenosine, has displayed several pharmacological activities including enhanced apoptosis and cancer cells inhibition. However, oral administration of cordycepin has limited practical use due to its poor bioavailability in the intestine. Herein, we developed and demonstrated a hybrid nanocarrier system in the form of biloniosome-core/chitosan-shell hybrid nanocarriers (HNCs) in order to improve the bio-characteristics of cordycepin. In this study, HNCs were prepared by using a solvent (ethanol) injection method involving cordycepin as the biloniosome core and mucoadhesive chitosan biopolymer as a coating shell. Our results showed that the cordycepin-loaded HNCs were positively charged with enhanced mucoadhesive characteristics and highly stable in gastric fluid. The increased permeability of cordycepin-loaded HNCs compared with standard cordycepin was confirmed by in vitro intestinal permeation study across the human intestinal barrier. In addition, we demonstrated that the cordycepin-loaded HNCs are able to release their components in an active form resulting in enhanced anti-cancer activity in two-dimensional (2D) cell cultures as well as in three-dimensional (3D) multi-cellular spheroids of colon cancer cells. Further, quantitative real time PCR analysis of apoptotic gene expression revealed that cordycepin HNCs can induce apoptosis in cancer cells by negatively regulating the expression of B-cell lymphoma-extra large (BCL-XL). I Overall our results showed that the hybrid nanocarrier systems represent a promising strategy for improving the bio-characteristics of cordycepin which can be considered as a potential anti-cancer agent for colorectal cancer chemotherapy.

Insights into the genetic diversity, recombination, and systemic infections with evidence of intracellular maturation of hepadnavirus in cats.

Hepatitis B virus (HBV) is a human pathogen of global concern, while a high diversity of viruses related to HBV have been discovered in other animals during the last decade. Recently, the novel mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), was detected in an immunocompromised cat. Herein, a collection of 209 cat sera and 15 hepato-diseased cats were screened for DCH using PCR, resulting in 12.4% and 20% positivity in the tested sera and necropsied cats, respectively. Among the DCH-positive sera, a significantly high level of co-detection with retroviral infection was found, with the highest proportion being co-detection with feline immunodeficiency virus (FIV). Full-length genome characterization of DCH revealed the genetic diversity between the nine Thai DCH sequences obtained, and that they phylogenetically formed three distinct monophyletic clades. A putative DCH recombinant strain was found, suggesting a possible role of recombination in DCH evolution. Additionally, quantitative PCR was used to determine the viral copy number in various organs of the DCH-moribund cats, while the pathological findings were compared to the viral localization in hepatocytes, adjacent to areas of hepatic fibrosis, by immunohistochemical (IHC) and western blot analysis. In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, providing evidence of systemic infection. Ultrastructure of infected cells revealed electron-dense particles in the nucleus and cytoplasm of hepatocytes, bronchial epithelial cells, and fibroblasts. We propose the intracellular development mechanism of this virus. Although the definitive roles of pathogenicity of DCH remains undetermined, a contributory role of the virus associated with systemic diseases is possible.

Formulation, physical, in vitro and ex vivo evaluation of nanomedicine-based chemosterilant for non-surgical castration of male animals.

The overpopulation of free-roaming companion animals has become the global crisis. The development and application of a suitable, effective, non-surgical approach for animal sterilization would have an enormous advantage over the current surgical method. The main purpose of this study was to develop and evaluate a novel nanomedicine-based chemosterilant for non-surgical castration of male companion animals. In this study, we first sought to investigate the testicular toxicity of different apoptosis-inducing agents. We next synthesized and characterized nano-sized particles which encapsulated the most potent testicular toxicants and evaluated in vitro sterilant properties. Our result showed that doxorubicin exhibited the highest cytotoxic activity against mouse spermatogenic cells. We therefore synthesized and characterized doxorubicin-encapsulated nanoemulsion. The negatively charged particle of doxorubicin-encapsulated nanoemulsion exhibited the anti-proliferative activity towards spermatogetic cells. Apoptosis studies revealed activation of Caspases 3 and 7 as well as annexin V expression. In addition, doxorubicin-encapsulated nanoemulsion exhibited anti-inflammatory activity in lipopolysaccharide-stimulated macrophages. Cell death was observed following treatment of isolated and cultured rat seminiferous tubules with doxorubicin-encapsulated nanoemulsion. In conclusion, nanoemulsion can be a potential carrier for prolonged release and to enhance activity of doxorubicin that may have utility in non-surgical castration of male animals.

Nanocarrier-mediated delivery of α-mangostin for non-surgical castration of male animals.

The overpopulation of abandoned and stray companion animals has become a global crisis. The main purpose of this study was to develop a novel nanomedicine-based antifertility compound for non-surgical castration of male animals. Mangosteen (Garcinia mangostana L) pericarp extract has been shown to exhibit anti-fertility property. α-mangostin (AM)-loaded nanostructured lipid carrier (AM-NLC) was developed to improve male germ cell apoptosis. This study was conducted to investigate physicochemical properties of AM-NLC and determine the biological effects of AM-NLC on spermatogonia cells and testicular explants obtained from castrated testes. AM-NLC was produced through a hot homogenization technique. The negatively charged particle of AM-NLC was nano-sized with a narrow dispersity. AM-NLC exhibited antiproliferative activity towards spermatogonium cells. It induced apoptosis in the cells. In addition, AM-NLC exhibited anti-inflammatory activities in lipopolysaccharide-activated macrophages. Abnormal anatomy of seminiferous tubule was noted following treatment of testicular explant with AM-NLC. This nanomedicine-based sterilant would be a promising platform that may have utility in non-surgical castration of male animals by intra-testicular injection.

Surface modification of nanostructure lipid carrier (NLC) by oleoyl-quaternized-chitosan as a mucoadhesive nanocarrier.

A nanostructure lipid carrier (NLC) composed of solid, and liquid lipid as a core has been developed as a delivery system for hydrophobic drug molecules. The aim of this research was to fabricate an oleoyl-quaternized-chitosan (CS)-coated NLC, where the mucoadhesive property of nanoparticles is enhanced for more efficient drug delivery. NLC loaded with alpha-mangostin (AP), a model hydrophobic drug, were fabricated using a high pressure homogenization process and subsequently coated with CS. The fabricated nanoparticles showed particle sizes in the range of 200-400nm, with low polydispersity, high physical stability and excellent encapsulation efficiency (EE>90%). Additionally, in vitro viability, cytotoxicity and ability of NLC and CS-NLC to affect apoptosis in carcinoma Caco-2 cells were determined using the Triplex assay. Gene expressiom analysis were performed using quantitative reverse transcription Polymerase Chain Reaction (RT-qPCR). Moreover, in vivo toxicological testing of NLCs was conducted in zebrafish embryos. Results indicated that CS-NLC provieded high cytotoxicity than NLC itself. In the case of AP loaded nanoparticles, NLC loaded with AP (AP-NLC), and CS-NLC loaded with AP (CS-AP-NLC) exhibited higher cytotoxicity to Caco-2 over Hela cells. These results indicate that CS-NLC shows enhanced cellular uptake but increased cytotoxicity characteristics over NLC and therefore careful optimization of dosage and loading levels in CS-NLC is needed to allow cancer cell targeting, and for exploiting the potential of these systems in cancer therapy.