Utilizing liposomal encapsulation approach to address nephrotoxic challenges of colistimethate sodium through a preclinical study.

The use of Colistin, a last-resort antimicrobial drug, carries the risk of acute kidney injury. The objective of the study was to assess the effectiveness of colistin-encapsulated liposomes (CL) in reducing nephrotoxicity. Additionally, a liposomal preparation of colistimethate sodium was formulated using the reverse phase evaporation method with a 3:1 ratio of phospholipids to cholesterol. The liposomal properties were evaluated using scanning electron microscopy, photon correlation spectroscopy, and release kinetic assay. The killing kinetics of the formulations on embryonic kidney cells were assessed using in vitro MTT reduction assay. The nephrotoxicity of CL and colistimethate sodium solution (CS) was evaluated in vivo by administering a dose of 20 mg/kg to rats every 12 h for 3 days, with a negative control group receiving a 0.9% saline solution (NSS). The study results revealed that monodisperses of CL showed a smooth surface and distinct boundaries, with an average size of 151.50 ± 0.46 nm and a narrow size distribution of 0.25 ± 0.01. The liposomal particles showed high entrapment efficiency of 96.45% ± 0.41%, with a ζ-potential of -60.80 ± 1.01 mV and a release rate of 50% of colistimethate sodium within the first 480 min. The CL induced nephrocytotoxicity in a concentration- and time-dependent manner. However, CS had notably lower IC50 values compared to its liposome preparations at 48 and 72 h (p < 0.05). In vivo study results show that serum levels of symmetric dimethylarginine (SDMA) and total white blood cell count (WBC) were significantly lower in the CL group (SDMA = 8.33 ± 1.70 µg/dL; WBC = 7.29 ± 0.99 log10 cells/mL) compared to the CS group (SDMA = 15.00 ± 1.63 µg/dL; WBC = 9.73 ± 0.51 log10 cells/mL). Our study findings enhance the understanding of the safety profile of CL and its potential to improve patient outcomes through the use of liposomal colistin medication. Additional clinical studies are necessary to establish the optimal safety regiment in humans.

Effects of Nelumbonucifera Gaertn. Petal Tea Extract on Hepatotoxicity and Oxidative Stress Induced by Mancozeb in Rat Model.

Mancozeb (Mz) is one of the most widely used pesticides that has been reported to cause adverse human health risks. White Nelumbo nucifera (N. nucifera) petals have therapeutic properties to prevent toxicity. Hence, this study attempted to determine the effects of N. nucifera extract on hepatotoxicity and oxidative stress in mancozeb-treated rats. Seventy-two male rats were divided into nine groups and designed with a control; N. nucifera extract was administered at the doses of 0.55, 1.1, and 2.2 mg/kg bw/day, Mz was administered at 500 mg/kg bw/day, and the co-treatment groups (N. nucifera and Mz) were administered 0.55, 1.1, and 2.2 mg/kg bw/day of N. nucifera followed by administering Mz 500 mg/kg bw/day daily for 30 days. The results showed that all doses of N. nucifera extract did not induce hepatic toxicity and could suppress the toxicity of mancozeb by increasing body weight gain and decreasing relative liver weight, lobular inflammation, and total injury score. The combination treatment also decreased the molecular markers of oxidative stress (2-hydroxybutyric acid, 4-hydroxynonenal, l-tyrosine, pentosidine, and N6-carboxymethyllysine). Furthermore, the reduced glutathione and oxidized glutathione contents were adjusted close to the normal level. Therefore, N. nucifera extract is a natural antioxidant supplement that could decrease the toxicity of mancozeb and can be safely consumed.

Comparative Susceptibility of Pathogenic Methicillin-Resistant and Methicillin-Susceptible Staphylococcus pseudintermedius to Empirical Cotrimazole for Canine Pyoderma.

The prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) that causes pyoderma has been gradually shifting, according to many surveillance studies, with annual changes. The empirical cotrimazole regimen remains interesting, but research on cotrimazole susceptibility to MRSP is limited. The objective of this study was to evaluate the susceptibility of cotrimazole to canine pyoderma MRSP isolates. Sixty isolates of S. pseudintermedius were identified as 16 MRSP and 44 methicillin-susceptible S. pseudintermedius (MSSP) using an oxacillin disk diffusion test and VITEK 2 system with VITEK GP card. Using the VITEK 2 system with a VITEK AST-GP81 card, the susceptibility rates of MRSP (15.00%) and MSSP (35.00%) to cotrimazole was observed. The median MIC of cotrimazole on MSSP (median, ≤10; IQR, 10-320) was lower than that of MRSP (median, ≥320; IQR, 10-320) (p = 0.5889, Mann-Whitney test). Percent attainment of PK/PD targets in MRSP (q 12 h, 43.75; q 8 h, 43.75) were lower than that of MSSP (q 12 h, 52.27; q 8 h, 52.27) (p = 0.7710). These findings show the moderately phenotypic cotrimazole susceptibilities of both MRSP and MSSP. Further study is required to develop clinical trials examining the use of cotrimazole in dogs with pyoderma.

Response of elephant peripheral blood mononuclear cells when stimulated with elephant endotheliotropic herpesvirus glycoprotein B (EEHV-gB).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the most highly fatal infectious disease among young Asian elephants. Despite the fact that antiviral therapy has been widely used, its therapeutic outcomes remain uncertain. Additionally, the virus has yet to be successfully cultivated in vitro in the process of develop viral envelope glycoproteins for vaccine design. The present study aims to investigate and evaluate EEHV1A glycoprotein B (gB) antigenic epitopes as potential candidates for further vaccine development. Epitopes of EEHV1A-gB were employed in in silico predictions and designed by using online antigenic predicting tools. Candidate genes were then constructed, transformed and expressed in the E. coli vectors prior to examine their potential for acceleration elephant immune responses in vitro. Elephant peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy juvenile Asian elephants were investigated for their proliferative capability and cytokine responses after being stimulated with EEHV1A-gB epitopes. Exposure of elephant PBMCs to 20 µg/mL of gB for 72 h resulted in a significant proliferation of CD3 + cells when compared with the control group. Furthermore, proliferation of CD3 + cells was associated with a marked up-regulation of cytokine mRNA expression, involving IL-1ß, IL-8, IL-12 and IFN-γ. It remains to be determined whether these candidate EEHV1A-gB epitopes could activate immune responses in animal models or elephants in vivo. Our potentially promising results demonstrate a degree of feasibility for the use of these gB epitopes in expanding EEHV vaccine development.

Comparative Efficacy of Chimeric Porcine Circovirus (PCV) Vaccines against Experimental Heterologous PCV2d Challenges.

The objective of this study was to evaluate the efficacy of two multivalent commercial porcine circovirus (PCV) vaccines against heterologous PCV2d challenges. A total of 24 crossbred male pigs aged 26 days selected from a specific pathogen-free herd were randomly divided into four groups (six pigs per group) and assigned as follows: negative control (unvaccinated/sham-challenge), vaccinated with chimeric PCV1-2a vaccine (PCV1-2a/PCV2d-challenge), vaccinated with chimeric PCV1-2a-2b vaccine (PCV1-2a-2b/PCV2d-challenge) and positive control (unvaccinated/PCV2d-challenge). At 21 days after vaccination, the pigs were intranasally and intramuscularly inoculated with either sham or field isolates of PCV2d (PCV2d/149/TH/2020). After being challenged, blood samples were obtained weekly and analyzed for levels of PCV2d viremia, neutralizing antibodies, and IgG against PCV2. At 30 days post-challenge (DPC), the pigs were euthanized and then subjected to pathological evaluations and molecular analysis. The results indicated that pigs in the PCV1-2a-2b/PCV2d-challenge and the PCV1-2a/PCV2d-challenge groups possessed significantly greater levels of PCV2d-neutralizing antibody titer when compared with the positive control group. Moreover, pigs in the PCV1-2a-2b/PCV2d-challenge group exhibited a lower degree of severity in terms of gross lesion scores and lower levels of PCV2 viremia when compared with the positive control group. This study demonstrated that vaccinating pigs with either the PCV1-2a or PCV1-2a-2b chimeric vaccines elicits a potent immune response against PCV2d infection and reduces viremia after PCV2d inoculation in pigs.

Development of an immunochromatographic strip test for antigen detection of elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus (EEHV) is the causative agent of EEHV-hemorrhagic disease (EEHV-HD) in elephants worldwide. This disease is highly virulent and a predominant cause of fatalities in young Asian elephants. Rapid diagnosis and aggressive therapies have been determined to be a key strategy in the successful treatment of this disease. Herein, we have developed the immunochromatographic strip test for EEHV detection. Accordingly, 31.2 kDa of partial EEHV DNA polymerase (DNApol) protein was expressed in Escherichia coli and used to generate rabbit polyclonal anti-EEHV DNApol antibodies. These were then used to develop an ICS test for EEHV antigen detection using the double-antibody sandwich colloidal gold method. Anti-EEHV DNApol antibodies conjugated with 40 nm colloidal gold solution were used as a detector, while rabbit anti-EEHV DNApol and goat anti-rabbit IgG antibodies immobilized on the nitrocellulose membrane were used as the test and control lines, respectively. The test had a detection limit of 1.25 × 105 viral genome copies (vgc)/mL of EEHV obtained from blood samples. Moreover, no specialized equipment or laboratory infrastructure was required in the administration of this test. This developed ICS test for EEHV antigen detection can be used in field application for the rapid detection of EEHV in resource-limited environments.

Pathogenesis of hemorrhagic disease caused by elephant endotheliotropic herpesvirus (EEHV) in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is an acute fatal disease in elephants. Despite the fact that the underlying pathogenesis of EEHV-HD has been proposed, it remains undetermined as to what mechanisms drive these hemorrhagic and edematous lesions. In the present study, we have investigated and explained the pathogenesis of acute EEHV-HD using blood profiles of EEHV-HD and EEHV-infected cases, hematoxylin and eosin (H&E) stain, special stains, immunohistochemistry, quantitative polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR). It was found that EEHV genomes were predominantly detected in various internal organs of EEHV-HD cases. Damage to endothelial cells, vasculitis and vascular thrombosis of the small blood vessels were also predominantly observed. Increases in platelet endothelial cell adhesion molecules-1 (PECAM-1)- and von Willebrand factor (vWF)-immunolabeling positive cells were significantly noticed in injured blood vessels. The expression of pro-inflammatory cytokine mRNA was significantly up-regulated in EEHV-HD cases when compared to EEHV-negative controls. We have hypothesized that this could be attributed to the systemic inflammation and disruption of small blood vessels, followed by the disseminated intravascular coagulopathy that enhanced hemorrhagic and edematous lesions in EEHV-HD cases. Our findings have brought attention to the potential application of effective preventive and therapeutic protocols to treat EEHV infection in Asian elephants.

Disseminated histiocytic sarcoma in Asian palm civet (Paradoxurus hermaphroditus).

This case study had focused on a male, 7-year-old Asian palm civet (Paradoxurus hermaphroditus) with a history of biting its tail and the development of skin masses around its inguinal area, prior to its death. Macroscopically, multiple firm white nodular masses of 0.5-5 cm in diameter were found in the subcutis of the inguinal area, and in the lungs, spleen and liver. Microscopically, masses in the skin, lungs and spleen were composed of neoplastic spindle cells admixed with mononuclear cells and multinucleated giant cells. The neoplastic cells were arranged in a sheet pattern. Immunohistochemically, the neoplastic cells were immunohistochemically positive for vimentin, Iba-1, CD 204 and Human leukocyte antigen (HLA)-DR, while the cells were negative for cytokeratin and smooth muscle actin. Based on the histopathological and immunohistochemical results, disseminated histiocytic sarcoma was diagnosed.

Attempt to Isolate Elephant Endotheliotropic Herpesvirus (EEHV) Using a Continuous Cell Culture System.

Elephant endotheliotropic herpesvirus (EEHV) infection is known to cause acute fatal hemorrhagic disease, which has killed many young Asian elephants (Elephas maximus). Until recently, in vitro isolation and propagation of the virus have not been successful. This study aimed to isolate and propagate EEHV using continuous cell lines derived from human and/or animal origins. Human cell lines, including EA. hy926, A549, U937, RKO, SW620, HCT-116 and HT-29, and animal cell lines, including CT26.CL25 and sp2/0-Ag14, were investigated in this study. Mixed frozen tissue samples of the heart, lung, liver, spleen and kidney obtained from fatal EEHV1A- or EEHV4-infected cases were homogenized and used for cell inoculation. At 6, 24, 48 and 72 h post infection (hpi), EEHV-inoculated cells were observed for cytopathic effects (CPEs) or were assessed for EEHV infection by immunoperoxidase monolayer assay (IPMA) or quantitative PCR. The results were then compared to those of the mock-infected controls. Replication of EEHV in the tested cells was further determined by immunohistochemistry of cell pellets using anti-EEHV DNA polymerase antibodies or re-inoculated cells with supernatants obtained from passages 2 or 3 of the culture medium. The results reveal that no CPEs were observed in the tested cells, while immunolabeling for EEHV gB was observed in only U937 human myeloid leukemia cells. However, quantitation values of the EEHV terminase gene, as well as those of the EEHV gB or EEHV DNA polymerase proteins in U937 cells, gradually declined from passage 1 to passage 3. The findings of this study indicate that despite poor adaptation in U937 cells, this cell line displays promise and potential to be used for the isolation of EEHV1 and EEHV4 in vitro.

In vivo characterization of target cells for acute elephant endotheliotropic herpesvirus (EEHV) infection in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

Possible roles of monocytes/macrophages in response to elephant endotheliotropic herpesvirus (EEHV) infections in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the primary cause of acute, highly fatal, hemorrhagic diseases in young Asian elephants. Although monocytopenia is frequently observed in EEHV-HD cases, the role monocytes play in EEHV-disease pathogenesis is unknown. This study seeks to explain the responses of monocytes/macrophages in the pathogenesis of EEHV-HD. Samples of blood, frozen tissues, and formalin-fixed, paraffin-embedded (FFPE) tissues from EEHV1A-HD, EEHV4-HD, co-infected EEHV1A and 4-HD, and EEHV-negative calves were analyzed. Peripheral blood mononuclear cells (PBMCs) from the persistent EEHV4-infected and EEHV-negative calves were also studied. The results showed increased infiltration of Iba-1-positive macrophages in the inflamed tissues of the internal organs of elephant calves with EEHV-HD. In addition, cellular apoptosis also increased in the tissues of elephants with EEHV-HD, especially in the PBMCs, compared to the EEHV-negative control. In the PBMCs of persistent EEHV4-infected elephants, cytokine mRNA expression was high, particularly up-regulation of TNF-α and IFN-γ. Moreover, viral particles were observed in the cytoplasm of the persistent EEHV4-infected elephant monocytes. Our study demonstrated for the first time that apoptosis of the PBMCs increased in cases of EEHV-HD. Furthermore, this study showed that monocytes may serve as a vehicle for viral dissemination during EEHV infection in Asian elephants.

Quercetin manipulates the expression of genes involved in the reactive oxygen species (ROS) process in chicken heterophils.

Chicken heterophils generate reactive oxygen species (ROS) molecules to defend against invading pathogens. The present study examined effects of quercetin on chicken heterophils. Heterophils were stimulated with PBS, 50 µM quercetin (QH), PMA or Escherichia coli (EC) and the resulting intracellular ROS molecules were determined. Flow cytometry results showed that cells stimulated with QH, PMA and EC had a higher ROS production. Increases in intracellular ROS molecules were identified in all treatment groups by fluorescence microscopy. Determination of the ability of quercetin to manipulate mRNA expression of ROS subunits was assessed using real-time RT-PCR. Quercetin and other stimulants up-regulated the majority of genes involved in ROS production: CYBB (NOX2), NCF1 (p47phox), NCF2 (p67phox), NOX1 and RAC2. The antioxidant property of QH was explored by measuring mRNA expression of CAT and SOD1. The data indicate increased levels of CAT with all treatments; however, only QH attenuated the expression of the SOD1 gene. To further investigate the effects of ROS-driven inflammation or cell death, IL6, CASP8 and MCL1 genes were preferentially tested. The inflammatory gene (IL6) was profoundly down-regulated in the QH- and PMA-treated groups while EC induced a strikingly high IL6 expression level. Investigation of the known apoptotic (CASP8) and anti-apoptotic (MCL1) genes found down-regulation of CASP8 in the QH- and PMA-treated groups which were contradicted to the MCL1 gene. In conclusion, quercetin can enhance ROS production by regulating the expression of genes involved in ROS production as well as in subsequent processes.