Epidemiology and classification for canine and feline mammary gland tumors: a histopathological survey of 437 mammary gland tumor biopsies performed in a secondary care hospital in Chiang Mai, Thailand from 2012 to 2019.

Background: Metastatic disease resulting from mammary gland tumors (MGTs) is a known cause of death among dogs and cats. Keys to successful prevention and management strategies involve the accurate recording of diagnostic data. Methods: This retrospective study reviewed the epidemiology and classification of canine mammary gland tumors (CMTs) and feline mammary gland tumors (FMTs), as well as the factors including sex, age, and breed related to the occurrence of these tumors. Accordingly, 1,736 tumor biopsy cases were reported from 2012 to 2019 at Chiang Mai University Small Animal Hospital, Thailand, with 1,639 canine tumor biopsy cases and 97 feline tumor biopsy cases. Results: The proportion of CMTs was reported at 24.5% (401/1,639) for all canine tumor biopsy cases. Benign and malignant tumors were reported at 14.5% (58/401) and 85.5% (343/401) for all CMT cases, respectively. The mean age of dogs affected by benign CMTs was 9.0 ± 3.0 years, which was significantly lower than for malignant CMTs at 9.9 ± 2.8 years (P = 0.0239). According to histopathological classification, benign mixed tumors and simple carcinoma types were highest among benign and malignant CMT cases, respectively. Moreover, female dogs were at significantly higher risk of developing mammary gland tumors (OR = 45.8, 95% CI [3.9-86.0], P < 0.0001) than male dogs, as well as older dogs (>8 years) (OR = 1.7, 95% CI [1.2-2.2], P = 0.0001) compared to young ones (≤8 years). The proportion of FMTs was 37.1% (36/97) for all feline tumor biopsy cases. Benign and malignant tumors for all FMTs were reported at 16.7% (6/36) and 83.3% (30/36), respectively. According to histopathological classifications, adenoma and simple carcinoma were present in the highest proportion among benign and malignant FMTs, respectively. Female cats were at a significantly higher risk of developing mammary gland tumors than male cats (OR = 25.7, 95% CI [3.9-272.8], P < 0.0001). Conclusions and clinical importance: There was a high proportion of MGT cases compared with other tumor cases reported in a secondary care hospital in Chiang Mai, Thailand from 2012 to 2019, and malignant tumor biopsies have been more frequently observed than benign tumor biopsies in both CMT and FMT cases. The resulting data originating from this study can be an aid for veterinary oncologists in better educating clients and planning treatment and prevention strategies and it can be used as a basis for further experimental studies in the oncology section.

Immune response enhancement by dietary supplementation with Caesalpinia sappan extract in weaned pigs challenged with porcine reproductive and respiratory syndrome virus.

BACKGROUND: At present, porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe epidemics impacting pig farming globally. Despite the fact that a number of studies have been conducted on potential solutions to this problem, none have proven effective. The focus of problem solving is the use of natural ingredients such as plant extracts. Popular throughout Asia, Caesalpinia sappan (CS) is a therapeutic plant that inhibits PRRSV in vitro. Therefore, this study was performed to determine the efficacy of CS extract dietary supplementation on the productive performance, antibody levels, immunological indicators, and lung pathology of PRRSV-challenged weaned pigs. A total of 32 weaned piglets (28 days old) were randomized into 4 groups and kept separately for 14 days. The treatments were organized in a 2 × 2 factorial design involving two factors: PRRSV challenge and supplementation with 1 mg/kg CS extract. The pigs in the PRRSV-challenged groups were intranasally inoculated with 2 mL of PRRSV (VR2332) containing 104 TCID50/mL, while those in the groups not challenged with PRRSV were inoculated with 2 mL of normal saline. RESULTS: In the PRRSV-challenged group (CS + PRRSV), supplementation with CS extract led to an increase in white blood cells (WBCs) on Day 7 post infection (p < 0.05) and particularly in lymphocytes on Days 7 and 14. The antibody titer was significantly greater in the CS + PRRSV group than in the PRRSV-challenged group not administered CS (PRRSV group) on Day 14 postinfection (S/P = 1.19 vs. 0.78). In addition, CS extract administration decreased the prevalence of pulmonary lesions, which were more prevalent in the PRRSV-challenged pigs that did not receive the CS extract. CONCLUSION: The findings of this study suggest that supplementation with CS extract is beneficial for increasing WBC counts, especially lymphocytes, increasing the levels of antibodies and reducing the prevalence of lung lesions in PRRSV-infected pigs.

Decreased circulating transforming growth factor-beta (TGF-ß) and kidney TGF-ß immunoreactivity predict renal disease in cats with naturally occurring chronic kidney disease.

OBJECTIVES: The aim of the present study was to compare the circulating transforming growth factor-beta (TGF-ß) of clinically normal age-matched and naturally occurring chronic kidney disease (CKD) cats and to determine the correlation between the TGF-ß expression and histopathological changes in cats with CKD. METHODS: A total of 11 clinically normal age-matched and 27 cats with naturally occurring CKD were included in this study. Circulating TGF-ß was quantified by immunoassays. Kaplan-Meier analysis was used to calculate the association between survival time and the concentration of circulating TGF-ß. A general linear model was used to compare the circulating TGF-ß between groups. Immunohistochemical analyses revealed TGF-ß expression in renal tissues from cats with CKD that died during the study (n = 7) and in available archived renal tissue specimens taken at necropsy from cats that had previous CKD with renal lesions (n = 10). Correlations of the TGF-ß expression and clinical parameters (n = 7) and histopathological changes (n = 17) were analysed using Spearman's rank correlation. RESULTS: The median survival time of cats with a lower concentration of circulating TGF-ß was shorter than that of cats with a higher concentration. The area under the curve of circulating TGF-ß for predicting CKD was 0.781, indicating good differentiation. The study indicated a significant difference in circulating TGF-ß concentrations between clinically normal cats and those with CKD and demonstrated that TGF-ß expression is correlated with tubular atrophy. CONCLUSIONS AND RELEVANCE: The study findings suggest that decreased serum TGF-ß and tubular atrophy with TGF-ß immunoreactivity may be significant in cats with CKD.

Identifying the patterns and sizes of the first lumpy skin disease outbreak clusters in Northern Thailand with a high degree of dairy farm aggregation using spatio-temporal models.

Lumpy skin disease (LSD) is one of the most important notifiable transboundary diseases affecting cattle in many parts of the world. In Thailand, LSD outbreaks in cattle farming areas have been reported in 69 out of 77 provinces, indicating a serious nationwide situation. Understanding the dynamics of spatial and temporal LSD epidemic patterns can provide important information on disease transmission and control. This study aims to identify spatial and temporal clusters in the first LSD outbreaks in dairy farming areas with a high degree of aggregation in Northern Thailand using spatio-temporal models. The data were obtained from an official LSD outbreak investigation conducted between June and August 2021 on dairy farms (n = 202). The outbreak of LSD was confirmed by employing clinical observations and laboratory analysis. The spatio-temporal models including space-time permutation (STP), Poisson, and Bernoulli were applied to the outbreak data with the settings of 10%, 25%, and 50%, respectively, for the maximum reported cluster size (MRCS). Overall, the number of most likely and secondary clusters varied depending on the model and MRCS settings. All MRCS settings in the STP model detected the most likely clusters in the same area and the Poisson models in different areas, with the largest being defined by a 50% MRCS. Although the sizes of the most likely clusters identified by the Bernoulli models were different, they all had the same cluster period. Based on the sizes of the detected clusters, strict LSD insect-vector control should be undertaken within one kilometer of the outbreak farm in areas where no LSD vaccination has been administered. This study determines the sizes and patterns of LSD outbreak clusters in the dairy farming area with a high degree of farm aggregation. The spatio-temporal study models used in this study, along with multiple adjusted MRCS, provide critical epidemiological information. These models also expand the options for assisting livestock authorities in facilitating effective LSD prevention and control programs. By prioritizing areas for resource allocation, these models can help improve the efficiency of such programs.

Seasonal patterns of oxidative stress markers in captive Asian elephants in Thailand and relationships to elephant endotheliotropic herpesvirus shedding.

Introduction: Oxidative stress refers to an imbalance between oxidant and antioxidant activity and accumulation of reactive oxygen species, which can have detrimental effects on animal health. Annual fluctuations in oxidative stress status can occur, increasing disease susceptibility during certain time periods. However, a full understanding of factors related to oxidative stress in Asian elephants and how to mitigate the negative consequences is lacking. Methods: This study measured six serum oxidative stress markers [reactive oxygen species (ROS), malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), albumin, glutathione peroxidase (GPx), and catalase] and two stress markers [serum cortisol and fecal glucocorticoid metabolites (fGCM)] in 23 captive Asian elephants in Thailand over a 12 months period to examine relationships with age and season. Results: Seasonal variations were observed, with several markers exhibiting significantly higher concentrations in the summer (ROS, MDA, 8-OHdG, albumin) and lower values during the rainy/winter seasons (MDA, 8-OHdG, albumin, catalase). By contrast, GPx was the only marker to be highest during the rainy season. For the stress markers, higher fGCM concentrations were noted during the rainy season, which contrasts with earlier studies showing more activity in the winter (tourist season). Positive correlations were found between the temperature-humidity index and ROS, GPx, and fGCM, while a negative correlation was observed with serum albumin. Elephant endotheliotropic herpesvirus (EEHV) shedding events were associated with higher concentrations of ROS and MDA. A moderate negative correlation was observed between 8-OHdG and the PCR threshold cycle of EEHV shedding (Ct), indicating DNA damage may be involved in EEHV shedding in elephants. Discussion: Results revealed significant age and seasonal effects on several oxidative stress markers, indicating those factors should be considered in study design and data interpretation. There also may be physiological adaptations in oxidative stress conditions in relation to environmental changes that could impact health outcomes.

Measures of Oxidative Status Markers in Relation to Age, Sex, and Season in Sick and Healthy Captive Asian Elephants in Thailand.

Oxidative stress is a pathological condition that can have adverse effects on animal health, although little research has been conducted on wildlife species. In this study, blood was collected from captive Asian elephants for the assessment of five serum oxidative status markers (reactive oxygen species (ROS) concentrations; malondialdehyde, MDA; albumin; glutathione peroxidase, GPx; and catalase) in healthy (n = 137) and sick (n = 20) animals. Health problems consisted of weakness, puncture wounds, gastrointestinal distress, eye and musculoskeletal problems, and elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD). Fecal samples were also collected to assess glucocorticoid metabolites (fGCMs) as a measure of stress. All data were analyzed in relation to age, sex, sampling season, and their interactions using generalized linear models, and a correlation matrix was constructed. ROS and serum albumin concentrations exhibited the highest concentrations in aged elephants (>45 years). No sex differences were found for any biomarker. Interactions were observed for age groups and seasons for ROS and catalase, while GPx displayed a significant interaction between sex and season. In pairwise comparisons, significant increases in ROS and catalase were observed in summer, with higher ROS concentrations observed only in the adult female group. Lower catalase activity was exhibited in juvenile males, subadult males, adult females, and aged females compared to subadult and adult elephants (males and females) in winter and the rainy season. There was a positive association between catalase activity and fGCMs (r = 0.23, p < 0.05), and a number of red blood cell parameters were positively associated with several of these biomarkers, suggesting high oxidative and antioxidative activity covary in red cells (p < 0.05). According to health status, elephants with EEHV-HD showed the most significant changes in oxidative stress markers, with MDA, GPx, and catalase being higher and albumin being lower than in healthy elephants. This study provides an analysis of understudied health biomarkers in Asian elephants, which can be used as additional tools for assessing the health condition of this species and suggests age and season may be important factors in data interpretation.

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.

Development of in-house ELISA for detection of antibodies against lumpy skin disease virus in cattle and assessment of its performance using a bayesian approach.

Lumpy skin disease (LSD) is a contagious disease among cattle and buffalo worldwide. Currently, an enzyme-linked immunosorbent assay (ELISA) has been recognized as an efficient diagnostic tool that is less time-consuming and easier than the viral neutralization test to measure the antibody levels. In the present study, an in-house method of indirect ELISA was developed to detect the bovine antibodies against Lumpy skin disease virus (LSDV) and its performance was assessed using field samples. This in-house method has been compared with the commercial ELISA test kit for detection of bovine antibodies against LSDV. The sensitivity (Se) and the specificity (Sp) of the test were estimated using a Bayesian latent class model. Checkerboard titration was performed using the naturally LSDV-infected bovine sera and colostrum-deprived calf sera. The LSDV antigen concentrations (1 TCID50/mL), the sample serum (1:500), and goat anti-bovine immunoglobulin G (IgG) labeled with horseradish peroxidase (HRP) (1:10,000) were determined to be optimal for this assay. The calculated cut-off value was 0.067, and there were no differences in the results of tests that utilized positive and negative sera (p < 0.05). The characteristics of two diagnostic tests were evaluated using a conditional dependent and one-population Bayesian model. The Se value of an in-house indirect ELISA were almost similar to ELISA test kit. On the other hand, the Sp value of the in-house ELISA test was lower than that of the commercial ELISA test with the median values of 89% (95% PPI = 75.9-99.3%) and 91.4% (95% PPI = 85.3-95.5%), respectively. A posterior estimate for the prevalence was 66.9% (95% PPI = 60.8-83.3%) and higher than initially expected.

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.

Brazilin from Caesalpinia sappan inhibits viral infection against PRRSV via CD163ΔSRCR5 MARC-145 cells: an in silico and in vitro studies.

This research aimed to identify bioactive compounds from Caesalpinia sappan extract that function as novel porcine reproductive and respiratory syndrome virus (PRRSV) infection inhibitors by computational molecular screening. We obtained a set of small-molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. In addition, the functions of positive hits were assessed and verified utilizing an in vitro antiviral activity assay with PRRSV-infected MARC-145 cells. Combining molecular docking with the results of binding affinity and ligand conformation, it was found that brazilin had the highest binding energy with the SRCR5 receptor compared to catechin and epicatechin (- 5.8, - 5.5, and - 5.1 kcal/mol, respectively). In terms of molecular mechanics, the binding free energy between the SRCR5 receptor was - 15.71 kcal/mol based on the Poisson-Boltzmann surface area of brazilin. In addition, PRRSV infection in MARC-145 cells was significantly inhibited by brazilin compared to the control (virus titer, 4.10 vs. 9.25 TCID50/mL, respectively). Moreover, brazilin successfully limited the number of PRRSV RNA copies in MARC-145 cells as determined by RT-qPCR. By inhibiting the PRRSV-CD163 interaction with brazilin from Caesalpinia sappan, it may be possible to prevent PRRSV infection in pigs, as suggested by this research.

Development of Nonstructural Protein-Based Indirect ELISA to Identify Elephant Endotheliotropic Herpesvirus (EEHV) Infection in Asian Elephants (Elephas maximus).

Disease caused by elephant endotheliotropic herpesvirus (EEHV) infection is the most highly fatal hemorrhagic disease in Asian elephant calves worldwide. To date, adult elephants that have been infected with EEHV have predominantly displayed mild clinical signs, while they are believed to serve as EEHV shedders to other elephants. Hence, the diagnostic tools employed for monitoring EEHV-active infection are considered vitally important. In this study, partial EEHV-DNA polymerase (DNApol) nonstructural proteins (NSPs) were used to detect anti-EEHV antibodies through the use of an in-house indirect enzyme-linked immunosorbent assay (ELISA). The results were then compared to those obtained from a PCR test. In this study, a total of 175 serum samples were collected from Asian elephants living in elephant camps located in Chiang Mai and Lampang Provinces, Thailand. The elephants were aged between 2 and 80 years old. The overall percentages of positive samples by the PCR and EEHV-DNApol ELISA tests were 4% (21/175) and 12% (21/175), respectively. The ELISAs demonstrated values of 77.9% (95% posterior probability interval (PPI) = 52.5-95%) sensitivity and 87.7% (PPI = 82.5-91.9%) specificity, respectively. Accordingly, the sera obtained from the elephants exhibiting no clinical signs of EEHV infection, and those who were negative according to PCR tests, revealed a value of 14% seropositivity for EEHV-DNApol. Our results indicate that these asymptomatic, active EEHV-infected elephants could likely serve as a source of EEHV shedding within elephant herds. Consequently, the developed EEHV-DNApol NSPs-based ELISA test employed in the present study may be of use for routine monitoring and identification of EEHV shedders in elephant herds, and could be an alternative diagnostic tool for EEHV detection in Asian elephants.

Molecular Characterization and Phylogenetic Analysis of Lumpy Skin Disease Virus Collected from Outbreaks in Northern Thailand in 2021.

Understanding molecular epidemiology is essential for the improvement of lumpy skin disease (LSD) eradication and control strategies. The objective of this study was to perform a molecular characterization and phylogenetic analysis of lumpy skin disease virus (LSDV) isolated from dairy cows presenting LSD-like clinical signs in northern Thailand. The skin nodules were collected from 26 LSD-suspected cows involved in six outbreaks during the period from July to September of 2021. LSDVs were confirmed from clinical samples using the polymerase chain reaction (PCR). The PCR-positive samples were subsequently amplified and sequenced using a G-protein-coupled chemokine receptor (GPCR) gene for molecular characterization and phylogenetic analyses. All 26 samples were positive for LSDV by PCR. A phylogenetic analysis indicated that the 24 LSDV isolates obtained from cattle in northern Thailand were closely related to other LSDV sequences acquired from Asia (China, Hong Kong, and Vietnam). On the other hand, two LSDV isolates of the cows presenting LSD-like clinical signs after vaccination were clustered along with LSDV Neethling-derived vaccines. The outcomes of this research will be beneficial in developing effective control strategies for LSDV.

Transcriptome analysis of infected Crandell Rees Feline Kidney (CRFK) cells by canine parvovirus type 2c Laotian isolates.

The advent of RNA sequencing technology provides insight into the dynamic nature of tremendous transcripts within Crandell-Reese feline kidney (CRFK) cells in response to canine parvovirus (CPV-2c) infection. A total of 1,603 genes displayed differentially expressed genes (DEGs), including 789 up-regulated genes and 814 downregulated genes in the infected cells. Gene expression profiles have shown a subtle pattern of defense mechanism and immune response to CPV through significant DEGs when extensively examined via Gene Ontology (GO) and pathway analysis. Prospective GO analysis was performed and identified several enriched GO biological process terms with significant participating roles in the immune system process and defense response to virus pathway. A Gene network was constructed using the 22 most significantly enriched genes of particular interests in defense response to virus pathways to illustrate the key pathways. Eleven genes (C1QBP, CD40, HYAL2, IFNB1, IFNG, IL12B, IL6, IRF3, LSM14A, MAVS, NLRC5) were identified, which are directly related to the defense response to the virus. Results of transcriptome profiling permit us to understand the heterogeneity of DEGs during in vitro experimental study of CPV infection, reflecting a unique transcriptome signature for the CPV virus. Our findings also demonstrate a distinct scenario of enhanced CPV responses in CRFK cells for viral clearance that involved multistep and perplexity of biological processes. Collectively, our data have given a fundamental role in anti-viral immunity as our highlights of this study, thus providing outlooks on future research priorities to be important in studying CPV.

Evaluation of Bcl-2 as a marker for chronic kidney disease prediction in cats.

Chronic kidney disease (CKD) is a frequent condition in elderly cats. Bcl-2 is linked to kidney disease through the processes of apoptosis and fibrosis. The purpose of this study is to examine Bcl-2 levels in CKD and clinically healthy age-matched cats in order to evaluate the relationship between Bcl-2 levels, signalment, and blood parameters in cats with CKD. The circulating levels of Bcl-2 were determined using an immunoassay in twenty-four CKD cats and eleven clinically healthy age-matched cats by the utilization of the general linear model (GLM), Pearson correlation, principal component analysis (PCA), ROC curves, the Cox hazard model, and Kaplan-Meier survival analysis. These were all conducted in order to explore Bcl-2 levels and their connection with other variables. The Bcl-2 immunohistochemical intensity was graded in each glomerulus and tubulointerstitium. McNemar's test was performed in order to compare the expression of Bcl-2 in the two renal tissue sites. The circulating Bcl-2 of CKD cats was significantly lower than those of clinically healthy age-matched cats (P = 0.034). The presence of circulating Bcl-2 (P < 0.01) and the severity of CKD (P = 0.02) were both linked with the survival time of cats with CKD. The area under the curve (AUC) of Bcl-2 for detection of CKD was 0.723. In cats, decreased circulating Bcl-2 was associated with increased blood BUN, creatinine levels, and CKD severity. Bcl-2 protein expression was reduced in the renal tissues of CKD cats as the disease progressed, resulting in a decrease in their survival time. This study demonstrated that Bcl-2 may be effective in diagnosing feline CKD.

Potentiality of Melittin-Loaded Niosomal Vesicles Against Vancomycin-Intermediate Staphylococcus aureus and Staphylococcal Skin Infection.

BACKGROUND: Staphylococcus aureus is an important human pathogen, especially causing skin and soft tissue infections (SSTIs). Over the decades, the infections caused by antibiotic-resistant strains have often become life-threatening. Consequently, exploration and development of competent approaches to combat these serious circumstances are urgently required. METHODS: The antibacterial activity of melittin (Mel) on S. aureus, methicillin-resistant S. aureus (MRSA) and clinical isolates of vancomycin-intermediate S. aureus (VISA) was investigated by minimum inhibitory concentration (MIC) and time-killing assays. The localization of Mel on the bacterial cell was visualized by confocal laser scanning microscopy and its effect on the membrane was indicated based on propidium iodide uptake. The non-ionic surfactant vesicle (NISV) or niosome nanocarrier was established for Mel loading (Mel-loaded NISV) by the thin-film hydration method. Physicochemical and in vitro biological properties of Mel-loaded NISVs were characterized. The cellular uptake of Mel-loaded NISVs was evaluated by holotomography analysis. In addition, an ex vivo study was conducted on a porcine ear skin model to assess the permeation ability of Mel-loaded NISVs and their potential to inhibit bacterial skin infection. RESULTS: The effective inhibitory activity of Mel on skin pathogens was demonstrated. Among the tested strains, VISA was most susceptible to Mel. Regarding to its function, Mel targeted the bacterial cell envelope and disrupted cell membrane integrity. Mel-loaded NISVs were successfully fabricated with a nano-size of 120-200 nm and entrapment efficiency of greater than 90%. Moreover, Mel-loaded NISVs were taken up and accumulated in the intracellular space. Meanwhile, Mel was released and distributed throughout the cytosol and nucleus. Mel-loaded NISVs efficiently inhibited the growth of bacteria, particularly MRSA and VISA. Importantly, they not only penetrated epidermal and dermal skin layers, but also reduced the bacterial growth in infected skin. CONCLUSION: Mel-loaded NISVs have a great potential to exhibit antibacterial activity. Therapeutic application of Mel-loaded NISVs could be further developed as an alternative platform for the treatment of skin infection via dermal and transdermal delivery.

Clinical characteristics of elephant endotheliotropic herpesvirus (EEHV) cases in Asian elephants (Elephas maximus) in Thailand during 2006-2019.

BACKGROUND: Elephant endotheliotropic herpesvirus causes a hemorrhagic disease (EEHV-HD) that is a major cause of death in juvenile Asian elephants with EEHV1 and EEHV4 being the most prevalent. AIM: To perform a retrospective clinical data analysis. METHODS: Records of a total of 103 cases in Thailand confirmed by polymerase chain reaction (PCR) on blood and/or tissue samples. RESULTS: The severity of clinical signs varied among EEHV subtypes. EEHV1A was the most prevalent with 58%, followed by EEHV4 with 34%, EEHV1B with 5.8% and EEHV1&4 co-infection with 1.9%. Overall case fatality rate was 66%. When compared among subtypes, 100% case fatality rate was associated with EEHV1&4 co-infection, 83% with EEHV1B, 75% with EEHV1A, and the lowest at 40% for EEHV4. Calves 2- to 4-year old were in the highest age risk group and exhibited more severe clinical signs with the highest mortality. Majority of cases were found in weaned or trained claves and higher number of cases were observed in rainy season. A gender predilection could not be demonstrated. Severely affected elephants presented with thrombocytopenia, depletion of monocytes, lymphocytes and heterophils, a monocyte:heterophil (M:H) ratio lower than 2.37, hypoproteinemia (both albumin and globulin), severe grade of heterophil toxicity, and low red blood cell counts and pack cell volumes. Survival was not affected by antiviral drug treatment in the severely compromised animals. CONCLUSION: Early detection by laboratory testing and aggressive application of therapies comprising of supportive and anti-viral treatment can improve survival outcomes of this disease.

Effect of Ethanolic Caesalpinia sappan Fraction on In Vitro Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major epidemic in pig production, leading to economic losses in the pig industry worldwide. The use of medicinal plants with antiviral properties might be useful help to prevent and control PRRSV outbreaks. Caesalpinia sappan (CS) heartwood is an important herbal ingredient used in Thai folk medicine, possessing various biological activities, including antiviral activity. The present study focuses on the in vitro antiviral activity against PRRSV of a semi-purified fraction of ethanolic CS crude extract using preparative high-performance liquid chromatography. Qualification of the fractions illustrating positive antiviral activity was carried out with liquid chromatography-quadrupole time-of-flight mass spectrometry. The preparative chromatography separated the crude extract into six consecutive fractions, among which the first fraction showed potential antiviral activity by inhibiting PRRSV replication in a MARC-145 monolayer (virus titer 2.75 median tissue culture infective dose (TCID50)/mL (log10) vs. 9.50 median log10 TCID50/mL of the control) at 72 h post-infection, and this fraction included byakangelicin, brazilin, naringenin, and brazilein. These results provide useful information for further study to effectively develop the CS bioactive antiviral compounds against PRRSV as a feed additive or veterinary drug in the pig industry.

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.

Prevalence of autoantibodies that bind to kidney tissues in cats and association risk with antibodies to feline viral rhinotracheitis, calicivirus, and panleukopenia.

BACKGROUND: The feline viral rhinotracheitis, calicivirus, and panleukopenia (FVRCP) vaccine, prepared from viruses grown in the Crandell-Rees feline kidney cell line, can induce antibodies to cross-react with feline kidney tissues. OBJECTIVES: This study surveyed the prevalence of autoantibodies to feline kidney tissues and their association with the frequency of FVRCP vaccination. METHODS: Serum samples and kidneys were collected from 156 live and 26 cadaveric cats. Antibodies that bind to kidney tissues and antibodies to the FVRCP antigen were determined by enzyme-linked immunosorbent assay (ELISA), and kidney-bound antibody patterns were investigated by examining immunofluorescence. Proteins recognized by antibodies were identified by Western blot analysis. RESULTS: The prevalences of autoantibodies that bind to kidney tissues in cats were 41% and 13% by ELISA and immunofluorescence, respectively. Kidney-bound antibodies were observed at interstitial cells, apical border, and cytoplasm of proximal and distal tubules; the antibodies were bound to proteins with molecular weights of 40, 47, 38, and 20 kDa. There was no direct link between vaccination and anti-kidney antibodies, but positive antibodies to kidney tissues were significantly associated with the anti-FVRCP antibody. The odds ratio or association in finding the autoantibody in cats with the antibody to FVRCP was 2.8 times higher than that in cats without the antibody to FVRCP. CONCLUSIONS: These preliminary results demonstrate an association between anti-FVRCP and anti-cat kidney tissues. However, an increase in the risk of inducing kidney-bound antibodies by repeat vaccinations could not be shown directly. It will be interesting to expand the sample size and follow-up on whether these autoantibodies can lead to kidney function impairment.