Longitudinal humoral immune response and maternal immunity in horses after a single live-attenuated vaccination against African horse sickness during the disease outbreak in Thailand.

Background and Aim: African horse sickness (AHS) has become a newly emerging disease after an outbreak in northeastern Thailand in March 2020. Mass vaccination in horses with live-attenuated AHS virus (AHSV) vaccine is essential for AHS control and prevention. This study aimed to monitor the longitudinal humoral immune response before and after a single vaccination using a live-attenuated vaccine against AHS in stallions, mares, and pregnant mares, including maternal immunity in foals born from pregnant mares during the outbreak in Thailand. Materials and Methods: A total of 13 stallions and 23 non-pregnant and 21 pregnant mares were vaccinated with live-attenuated AHSV vaccines. Serum samples from selected horses were collected on the day of vaccination and 1, 6, 8, 9, 10, and 12-months post-vaccination. Furthermore, seven serum samples of foals born from vaccinated pregnant mares were collected on parturition date and 1, 3, and 6-months old. The antibody titer against AHS in all collected serum samples was evaluated using a commercial enzyme-linked immunosorbent assay kit. All data were analyzed for mean and standard deviation for each group of samples using a spreadsheet program. Antibody titers between times were analyzed using a one-way analysis of variance as repeated measurement, and antibody titers between horse groups were analyzed using a general linear model for statistically significant differences when p < 0.05. Results: In stallion and non-pregnant mare groups, there were no statistically significant differences in antibody titers in all 6 time periods after vaccination. The antibody titer in the pregnant mare group showed a non-statistically significant difference between each gestation stage, except at 8 months post-vaccination. Furthermore, increasing antibody titers on days 1 and 3 after receiving colostrum in foals indicate the major role of transcolostral antibody transfer for AHS. Conclusion: This study demonstrated that a single AHS vaccination using a live-attenuated vaccine could stimulate high antibody titers sufficient for AHS control and prevention during the outbreak in Thailand. Similarly, the antibody response of vaccinated horses of both genders, including various stages of pregnant mares, was statistically not different.

First study on the immunohistochemical expression of cyclooxygenase-2 and clinicopathological association in canine hepatoid gland neoplasms.

Background and Aim: Hepatoid gland neoplasms (HGNs) constitute one of the most common cutaneous tumors that arise from perianal glands in dogs and are clinically characterized by rapid growth. Cyclooxygenase-2 (COX-2), the inducible form of the enzyme, is associated with several hallmarks of tumorigenesis. Its expression has been confirmed in several human and animal neoplastic tissues, but there are no reports in hepatoid gland tissues. Therefore, this study aimed to investigate COX-2 immunoexpression in canine HGNs, compare the expression among groups of normal hepatoid glands, hepatoid gland adenomas (HGAs), hepatoid gland epitheliomas (HGEs), and hepatoid gland carcinomas (HGCs), and assess the association of the COX-2 expression with clinicopathological features. Materials and Methods: Sixty-one formalin-fixed paraffin-embedded canine hepatoid gland tissues (20 samples of HGAs, 16 of HGEs, 15 of HGCs, and 10 of normal hepatoid glands) were analyzed for COX-2 expression using immunohistochemistry with scoring for percentage positivity and intensity. Multiple comparisons of COX-2 expression among normal and neoplastic hepatoid glands and the associations between COX-2 expression and clinicopathological features were analyzed. Results: Cyclooxygenase-2 expression was not detected in 60% of normal hepatoid glands and 25% of HGAs. Seventy-five percent of HGAs had a weak expression, while 43.7% and 56.3% of HGEs showed weak and moderate expression, respectively. The expression of HGCs ranged from weak (13.3%) to moderate (33.3%) and strong (53.3%). The immunoreactivity score of COX-2 labeling was significantly different among the normal and neoplastic hepatoid glands (p < 0.0001). The highest score was observed in the HGCs. Only in HGCs, the strong COX-2 expression was significantly associated with some clinicopathological features, including tissue invasion (p = 0.007) and necrosis (p = 0.029). Conclusion: These results suggest that COX-2 may play a role in the modulation of neoplastic cell growth. These preliminary data lead to further investigation on the potential of COX-2 expression as a prognostic indicator and COX-2 inhibitors for canine HGCs treatment.

Sensitivity and specificity for African horse sickness antibodies detection using monovalent and polyvalent vaccine antigen-based dot blotting.

Background and Aim: The immune responses of animals infected with African horse sickness (AHS) virus are determined by enzyme-linked immunosorbent assay (ELISA), complement fixation, and virus neutralization test. During the outbreaks of AHS in Thailand, the immune response after vaccination has been monitored using commercial test kits such as blocking ELISA, which are expensive imported products unavailable commercially in Thailand. This study aimed to assess the sensitivity and specificity of anti-AHS virus antibodies using dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. Materials and Methods: A total of 186 horse sera, namely, 93 AHS-unvaccinated samples and 93 AHS-vaccinated samples, were used in this study. All sera underwent antibodies detection using commercial blocking ELISA and in-house dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. The numbers of true positive, false positive, true negative, and false negative results in the dot blotting were compared with those in blocking ELISA and the sensitivity and specificity of dot blotting were assessed. Results: For the monovalent antigen, there were 78, 19, 74, and 15 true positive, false positive, true negative, and false negative results, respectively, while for the polyvalent antigen, the corresponding numbers were 84, 34, 58, and 9. Meanwhile, the diagnostic sensitivity and specificity for monovalent antigen were 83.87% and 79.57%, respectively, but 90.32% and 62.37% for polyvalent antigen. Conclusion: Dot blotting for AHS antibodies detection using vaccine antigen showed high sensitivity and rather a high specificity compared with the findings with the commercial ELISA test kit. In countries where commercial ELISA test kits are not available and when the size of a serum sample is small, dot blotting could become a good alternative test given its advantages, including its simplicity, rapidity, and convenience. To the best of our knowledge, these findings are the first report on the use of dot blotting for detecting AHS antibodies in horses. In conclusion, monovalent antigen-based dot blotting could be used as a reliable alternative serodiagnostic test for monitoring AHS humoral immune response, especially in vaccinated horses.

Sensitivity of RNA viral nucleic acid-based detection of avian influenza virus, Newcastle disease virus, and African horse sickness virus on flinders technology associates card using conventional reverse-transcription polymerase chain reaction.

Background and Aim: The flinders technology associates (FTA) card is a cotton-based cellulose membrane impregnated with a chaotropic agent that inactivates infectious microorganisms, lyses cellular material, and fixes DNA and/or RNA within the fiber matrix. However, little is known about the effectiveness of these cards for detecting RNA viruses in animals. This study aimed to evaluate the sensitivity of RNA virus detection using conventional reverse-transcription polymerase chain reaction (RT-PCR) on FTA cards. Materials and Methods: A highly virulent Newcastle disease virus (NDV) and an avian influenza virus (AIV) with low pathogenicity were propagated using chicken embryonic eggs. Three days after inoculation, the allantoic fluid was harvested, stored at -80°C, and the stock virus was tested for virus titration. African horse sickness virus (AHSV) was obtained from a live attenuated vaccine that was dissolved and stored at -80°C. For sample preparation, each stock virus was 10-fold serially diluted and each dilution was inoculated onto an FTA card, followed by drying in a Class II safety cabinet. Both the stock virus and infected FTA card were genomically isolated using an extraction kit, FTA purification kit, and extraction kit with Tris-EDTA (TE) buffer. The target genome was then detected by one-step RT-PCR for NDV and AIV, and two-step RT-PCR for African horse sickness, including gel electrophoresis for the detection of specific nucleic acids. Results: The detection limit of stock AIV was compared on FTA cards, using the FTA purification kit, and with TE buffer with an extraction kit. The corresponding results were 1.47, 1.17, and 2.18 log10 EID50, respectively, while for NDV the results were 4.13, 4.83, and 4.84 log10 ELD50. Finally, detection limit of stock AHSV and AHSV on the FTA card extracted using TE buffer with an extraction kit were 4.30 and 4.01 log10 plaque-forming units, respectively. Conclusion: This study demonstrated that the detection limit or sensitivity of all tested RNA viruses on FTA cards did not differ when compared with those of the stock virus and in both methods for RNA isolation on FTA cards. These cards are suitable for collecting and transporting samples infected with RNA viruses, particularly AIV, NDV, and AHSV. Flinders technology associates cards also provide hazard-free samples, a reliable source of RNA for molecular characterization, and sufficient quantity for diagnostic applications based on nucleic acid-based detection.

In vitro primary porcine alveolar macrophage cell toxicity and African swine fever virus inactivation using five commercially supply compound disinfectants under various condition.

Efficacy of African swine fever virus (ASFV) inactivation using five commercially supply compound disinfectants was evaluated under various condition. Virucidal efficacy demonstrated that products A and E could inactivate at 1:800 within 1 min for both temperatures, while products B, C and D inactivated at 1:400. However, product D could inactivate at 1:800 when the exposure time was extended to 30 min and effected only 20°C. In addition, the cytotoxicity demonstrated that products A, B, C, D and E did not significantly affect to cell at 1:51,200, 1:12,800, 1:12,800, 1:25,600 and 1:12,800 dilution, respectively. In conclusion, these disinfectants could inactivate ASFV, however, the application of these products should be performed under safety precautions to prevent cytotoxicity in humans and animals.

In vitro cytotoxicity and virucidal efficacy of potassium hydrogen peroxymonosulfate compared to quaternary ammonium compound under various concentrations, exposure times and temperatures against African swine fever virus.

BACKGROUND AND AIM: The selection and proper application of disinfectants are crucial to the prevention of many diseases, so disinfectants must be evaluated before being used for the prevention of African swine fever (ASF). Three disinfectant products belonging to the group of potassium hydrogen peroxymonosulfates, product A and product B, and a quaternary ammonium compound called product C, were examined in vitro for host cell cytotoxicity and the efficacy of ASF virus inactivation. The study parameters included various concentrations, exposure times, temperatures, and degrees of cytotoxicity. MATERIALS AND METHODS: Three disinfectant products were evaluated for cytotoxicity using primary porcine alveolar macrophage (PAM) cells at dilutions from 1:200 to 1:51,200. Disinfectants in concentrations of 1:200, 1:400, and 1:800 were prepared, the pH and the virucidal activity were tested. An equal volume of each dilution was mixed with the ASF virus and incubated at room temperature (20°C) or on ice (4°C) for 1 min, 5 min, or 30 min. Hemadsorption (HAD) or rosette formation was observed using an inverted microscope for 5 days after inoculation, and the virus titer was calculated as HAD50/mL. Each treatment and virus control were tested in triplicate, and the titers were reported as means and standard deviations. The reduction factor was used to measure inactivation. RESULTS: Products A, B, and C at 1:400, 1:800, and 1:25,600 of dilution, respectively, did not show significant cytotoxic effects on PAM cells. Products A and B could inactivate ASF virus at 1:200 dilution within 5 min after exposure at 4°C. However, at 20°C, the exposure time had to be extended to 30 min to inactivate the virus. Product C could inactivate the virus at 1:400 dilution within 5 min under both temperature conditions, whereas at 1:800 dilution, the exposure time had to be extended to 30 min to completely inactivate the virus at 20°C. CONCLUSION: All disinfectants could inactivate ASF virus in various concentrations, under appropriate exposure times and reaction temperatures, and there was no evidence of host cell cytotoxicity. For the control of ASF in pig farms, the appropriate concentration, ambient temperature, and contact time of these disinfectants should be taken into account.

Intrastromal refractive surgery with ultrashort laser pulses: in vivo study on the rabbit eye.

BACKGROUND: Femtosecond (fs) laser pulses may offer new possibilities in the field of refractive surgery, especially when using the laser as a microkeratome. By induction of nonlinear absorption processes the laser can be used to perform intrastromal cuts. The conventional microkeratome, associated with numerous potential side effects, can possibly be replaced. Furthermore, refractive lenticules can be prepared within the stroma and removed in a single-step operation. METHODS: In 10 rabbits, cuts were made to create both a lamellar flap and an intrastromal refractive lenticule. The flap was lifted, the lenticule was extracted and, finally, the flap was repositioned (intrastromal laser keratomileusis, ILK). The corneal samples were collected up to 120 days after treatment and processed for histopathological analysis. RESULTS: All flaps could be opened and prepared lenticules could be extracted in one piece by the surgeon. The treated corneas developed a mild wound healing reaction, comparable to that known from excimer laser in situ keratomileusis (LASIK) studies. The wound healing was restricted to the flap-stroma interface, most pronounced at the periphery of the flaps. CONCLUSIONS: The use of the fs-laser offers new possibilities in preparation of corneal flaps, possibly providing advantages over conventional microkeratomes. Furthermore, the fs-laser has the potential to create intrastromal refractive lenticules for complete refractive procedures (ILK).

Effects of epithelial growth factor receptor (EGFR) kinase inhibitors on genetically reconstituted mouse mammary glands.

UNLABELLED: The aim of the study was to determine the effects of a specific epithelial growth factor Receptor kinase inhibitor (EGFR-KI) and Taxol on tumor growth in a novel tumor model. MATERIAL & METHODS: A genetically engineered tumor model which uses "transgenic" organs in immune competent mice was used. NeuT-transfected immortalized HC11 epithelial cells and primary mouse mammary epithelial cells have been transplanted into the gland-free mammary fat pad of female BALB/c mice. Mammary tumors developed after a latency period of three to four weeks. The mice were thereafter daily orally treated over a 19 or 22-day period with 0, 38, 75, 2 x 75 mg/kg body weight (b.w.) EGFR-KI (n: 7-9 per group) or intravenously with 10 mg/kg b.w. Taxol. After necropsy the histopathological evaluation of the tumors was performed in a coded manner. The proliferation activity of tumor cells was analyzed by laser scanning cytometry (LSC) using anti-Ki67-antibodies. RESULTS: Oral Treatment with EGFR-KI in this transgenic organ model showed clear antitumor efficacy in a dose-dependent manner in the range between 38 and 75 mg/kg b.w. This antiproliferative effect appears to be minimally increased at 75 mg/kg/day twice per day. For all treatments a strong correlation between the biological behavior of the tumor, histopathology and cell proliferation could be established. In contrast, treatment with Taxol showed no significant reduction of tumor growth or cell proliferation in this model. This new transgenic organ model comprising histopathological evaluation and cell proliferation analysis appears to be a suitable test system for drug candidates that affect specific biochemical pathways. It may have greater predictive nature for clinical effects in humans as compared to conventional tumor models because of its c-erb B2 gene overexpression.