Background and Aim: Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite- dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats. Materials and Methods: T. gondii-GRA7 was cloned and expressed in the Expresso® small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test. Results: A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite- GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94). Conclusion: In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

Rhipicephalus (Boophilus) microplus is one of the most widespread ticks causing a massive loss to livestock production. The long-term use of acaracides rapidly develops acaracide resistance. In R. microplus, enhancing the metabolic activity of glutathione S-transferase (RmGST) is one of the mechanisms underlying acaracide resistance. RmGST catalyzes the conjugation of glutathione (GSH) to insecticides causing an easy-to-excrete conjugate. The active RmGST dimer contains two active sites (hydrophobic co-substrate binding site (H-site) and GSH binding site (G-site)) in each monomer. To preserve the insecticide efficacy, s-hexyl glutathione (GTX), a GST inhibitor, has been used as a synergist. To date, no molecular information on the RmGST-GSH/GTX complex is available. The insight is important for developing a novel RmGST inhibitor. Therefore, in this work, molecular dynamics simulations (MD) were performed to explore the binding of GTX and GSH to RmGST. GSH binds tighter and sits rigidly inside the G-site, while flexible GTX occupies both active sites. In GSH, the backbone mainly interacts with W8, R43, W46, K50, N59, L60, Q72, and S73, while its thiol group directs to Y7. In contrast, the aliphatic hexyl of GTX protrudes into the H-site and allows a flexible peptide core to form various interactions. Such high GTX flexibility and the protrusion of its hexyl moiety to the H-site suggest the dual role of GTX in preventing the conjugation reaction and the binding of acaracide. This insight can provide a better understanding of an important insecticide-resistance mechanism, which may in turn facilitate the development of novel approaches to tick control.

Inhibition of proteases shows therapeutic potential. Our previous studies demonstrated the cardioprotection by the Secretory Leukocyte Protease Inhibitor (SLPI) against myocardial ischaemia/reperfusion (I/R) injury. However, it is unclear whether the cardioprotective effect of SLPI seen in our previous works is due to the inhibition of protease enzymes. Several studies demonstrate that the anti-protease independent activity of SLPI could provide therapeutic benefits. Here, we show for the first time that recombinant protein of anti-protease deficient mutant SLPI (L72K, M73G, L74G) (mt-SLPI) could significantly reduce cell death and intracellular reactive oxygen species (ROS) production against an in vitro simulated I/R injury. Furthermore, post-ischaemic treatment of mt-SLPI is found to significantly reduce infarct size and cardiac biomarkers lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity, improve cardiac functions, attenuate I/R induced-p38 MAPK phosphorylation, and reduce apoptotic regulatory protein levels, including Bax, cleaved-Caspase-3 and total Capase-8, in rats subjected to an in vivo I/R injury. Additionally, the beneficial effect of mt-SLPI was not significantly different from the wildtype (wt-SLPI). In summary, SLPI could provide cardioprotection without anti-protease activity, which could be more clinically beneficial in terms of providing cardioprotection without interfering with basal serine protease activity.

Background and Aim: Canine parvovirus (CPV) is one of the most common viral infections in dogs, causing acute hemorrhagic gastroenteritis and high mortality. Vaccination effectively prevents CPV infection. However, the currently available CPV vaccines have concerns such as maternal immunity interference, shedding of virus vaccine, and false-positive result based on polymerase chain reaction after vaccination. A subunit vaccine can overcome these problems. This study aimed to express the recombinant 35 kDa fragment of the VP2 protein (consisting of epitopes 1-7) and the recombinant full-length VP2 protein (consisting of epitopes 1-10) and to study the ability of these two recombinant proteins to react with rabbit anti-CPV polyclonal antibodies. Materials and Methods: The full length and 35 kDa fragment of VP2 gene of CPV were cloned into the pBAD202 Directional TOPOTM expression vector and expressed in E. coli. The recombinant full-length and the recombinant 35 kDa fragment proteins of VP2 were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Results: The recombinant full-length and the recombinant 35 kDa fragment VP2 genes were successfully cloned and expressed. The optimum concentrations of arabinose and induction time for the recombinant full-length and the recombinant 35 kDa fragment VP2 proteins were 0.2% for 6 h and 0.02% for 6 h, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 protein molecular weights were approximately 81 and 51 kDa, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 proteins specifically interacted with rabbit anti-CPV polyclonal antibodies. Conclusion: These results suggest that the recombinant 35 kDa fragment and the recombinant full-length VP2 proteins may be useful in developing a CPV diagnostic test or vaccine.

Background: Blood parasites cause serious drawbacks in the livestock industry. The detection methods rely on microscopic examination and polymerase chain reaction (PCR), which requires an expert person, labor, time, and cost. Aim: In this study, multiplex polymerase chain reaction (PCR) has been developed for hemoparasite consisting of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi detections. Methods: Blood parasite infections in cattle were investigated using 3 different methods: microscopic examination, single PCR, and multiplex PCR. The Anaplasma 16s rRNA gene, Babesia 18s rRNA gene, Theileria MPSP gene, and Trypanosome ITS1 gene have been used to detect Anaplasma spp., Babesia spp., Theileria spp., and T. evansi, respectively, using molecular methods. The sensitivity and specificity of multiplex PCR were evaluated. Multiplex PCR results were compared with microscopic examination and single PCR. Results: Multiplex PCR assay revealed a limit of detection of 0.01–10 pg of parasite DNA. According to the evaluation of 60 bovine blood samples, blood smear, single PCR, and multiplex PCR revealed 50.0%, 18.3%, and 26.7% of single infections, and 40.0%, 81.7%, and 50.0% of co-infections, respectively. The comparative analysis between multiplex PCR with microscopic examination and single PCR revealed that triple infection, Babesia spp., Theileria spp., and T. evansi, showed 50.0% sensitivity, 100% specificity and PPV, 98% NPV, and substantial agreement indicated by a Cohen’s Kappa value of 0.659. Conclusion: The multiplex PCR assay developed in this study may be helpful for improved hemoparasite prevention and control when combined with farmer education, proper hygiene practices, and effective environmental management.

Neosporosis, caused by N. caninum, is an emerging protozoan disease responsible for significant economic losses in the global dairy and meat industries, primarily due to abortion in cattle. Dogs serve as both definitive and intermediate hosts and play a key role in the parasite transmission cycle. Currently, effective control strategies remain limited, partly due to insufficient information on infection status. In Vietnam, data on N. caninum infection are scarce and mainly limited to cattle and buffalo. In this study, an indirect enzyme-linked immunosorbent assay (iELISA) based on recombinant NcGRA4 protein was applied and evaluated for the detection of anti-N. caninum antibodies in dogs. A total of 142 shelter dogs from Hanoi, northern Vietnam, were tested to determine seroprevalence. The NcGRA4-based iELISA detected an overall seroprevalence of 28.87% (41/142), whereas the indirect fluorescent antibody test (iFAT) showed a lower prevalence of 14.08% (20/142), indicating substantial exposure to N. caninum among shelter dogs in this region. Using iFAT as the reference method, the NcGRA4-based iELISA demonstrated a sensitivity of 90.00%, a specificity of 81.15%, and an overall accuracy of 82.39%. These findings indicate that the NcGRA4-based iELISA is a suitable screening tool for seroepidemiological surveillance of N. caninum infections in dogs. Univariable and multivariable logistic regression analyses showed no significant associations between N. caninum seropositivity and the investigated variables, including age, sex, breed, and housing conditions. This study also provides the first serological evidence of canine exposure to N. caninum in Vietnam.