Bovine sperm HSP-70 molecules: a potential cryo-tolerance marker associated with semen quality and fertility rate.

Introduction: Freezability is the ability of sperm to maintain its vitality and quality from various stress during the cryopreservation process, which is very important for the success of fertilization in AI programs. Heat shock proteins (HSPs) are unique proteins induced in response to various stress, including excess reactive oxygen species (ROS) and oxidative damage to intracellular enzymes that can harm cells. This study aimed to analyze the potential of HSP-70 molecules in bovine sperm as a marker of freezability or cryo-tolerance, as well as its association with semen quality and fertility rate. Methods: The classification of bulls is based on freezability (good freezability/GF and poor freezability/PF), which is obtained from the value of post-thaw viability using the SYBR-14/PI-flow cytometry. Semen quality assessed included sperm motility and kinetics (computer-assisted sperm analyses), plasma membrane integrity (HOS test), acrosome integrity (FITC-PNA), mitochondrial membrane (JC-1), and DNA damage (Halomax kit). The bull fertility rate assessment was analyzed based on the first service conception rate of each bull derived from data on the success of artificial insemination contained in the Indonesian-integrated National Animal Health Information System (iSIKHNAS). Gene expression levels of HSP-70 bovine sperm were performed using the RT-qPCR method. The protein abundance of HSP-70 bovine sperm was determined using the enzyme immunoassay (EIA) method. Results: Bovine sperm HSP-70 molecules, at the gene and protein level, showed a higher abundance in GF (p < 0.05) than in PF bulls. The percentage of each parameter of frozen-thawed sperm quality was significantly higher in GF (p < 0.05) than in PF bulls. The HSP-70 molecules at the gene and protein levels were significantly positively correlated (p < 0.01) with the fertility rate. Furthermore, HSP-70 molecules were negatively associated (p < 0.01) with low mitochondrial membrane potential and sperm DNA damage and positively correlated (p < 0.01) with other frozen-thawed sperm quality parameters. The overall quality of frozen-thawed sperm was closely related (p < 0.01) to the fertility rate. Conclusion: We may conclude that HSP-70 molecules in bovine sperm at the gene and protein level have the potential to be developed as a marker for cryo-tolerance or freezability, which may be utilized as a predictor of fertility and frozen-thawed sperm quality in bulls.

Sperm Head Morphology Alterations Associated with Chromatin Instability and Lack of Protamine Abundance in Frozen-Thawed Sperm of Indonesian Local Bulls.

This study aimed to analyze various alterations in the morphology of the sperm head and its association with nucleus instability and insufficient sperm protamine. Frozen-thawed semen from twenty local Indonesian bulls was used for all stages in this study. The results of sperm head defect assessments are used for bull grouping, high (HD) and low (LD). Sperm DNA damage was assessed using Acridine Orange and Halomax. The PRM1 protein abundance was carried out using an enzyme immunoassay, while PRM1 gene expression was carried out using the RT-qPCR. PRM deficiency was performed using CMA3. Several kinds of sperm head defects in the HD were significantly higher (p < 0.05) than in the LD bulls. Sperm DNA damage showed a significant (p < 0.05) difference between the HD and LD bulls. PRM1 abundance was significantly (p < 0.05) decreased in HD bulls. PRM deficiency was significantly (p < 0.05) higher in HD bulls than in LD bulls. PRM deficiency in bulls correlated significantly (p < 0.01) with sperm head defects, DNA damage, and PRM1 abundance. The lack of sperm protamine might affect the sperm nucleus's stability and induce morphological alterations in the sperm head.

Severe acute respiratory syndrome coronavirus-2 detection in domestic animals as a reservoir for the virus transmission to humans in Yogyakarta, Indonesia.

Background and Aim: The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that attacks the respiratory and digestive tract. The SARS-CoV-2 showed systemic characteristics with various clinical symptoms from subclinical to fatal (causing death). Transmission of SARS-CoV-2 has been reported to occur from humans to pets (cats, dogs, tigers, ferrets, and poultry). Knowledge about the role of domestic animals in the transmission of SARS-CoV-2 to humans, and as reservoirs of this virus needs to be investigated further. This study aimed to detect the presence of SARS-CoV-2 in domestic animals such as dogs, cats, pigs, cows, birds, and bats that are often in contact with humans. Materials and Methods: A total of 157 samples, which included nasopharyngeal and oropharyngeal swabs, along with sera samples from domestic animals such as cats, pigs, cows, birds, and bats, were taken from Veterinary Hospitals, Veterinary Clinics, and farms around the Yogyakarta region. Detection of the virus was done using rapid detection of viral antigens, antibodies, and reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Results: The results showed that 5/157 (3.1%) samples found positive against the COVID-19 virus using a rapid antibody test; however, the results were negative on the rapid antigen and RT-PCR tests. Antibody-positive samples came from animals that had a history of household COVID-19 human infection. Conclusion: Thus, findings of the present study conclude that there is a potential for transmission of the COVID-19 virus between animals and humans.

Expression and epitope prediction of MPT64 recombinant proteins from clinical isolates of Mycobacterium tuberculosis as immunoserodiagnostic candidates.

Background and Aim: The success in the handling and prevention of tuberculosis (TB) cases is highly dependent on their rapid detection, monitoring, and treatment. The efficacy of the Bacille Calmette-Guerin (BCG) vaccine is inconclusive in eastern Indonesia. The RV1980c gene of Mycobacterium tuberculosis encodes an antigenic protein that is considered to be a virulence factor, as it can stimulate the immune response in patients with TB. This study aimed to study the expression and epitope indicator of MPT64 recombinant proteins from clinical isolates of M. tuberculosis as immunoserodiagnostic candidates for pET SUMO plasmids from clinical isolates as candidates for serodiagnostic tests and recombinant vaccines. Materials and Methods: The polymerase chain reaction (PCR) product of the RV1980c gene was inserted into the SUMO pET plasmid, which was then transformed into Escherichia coli BL21 (DE3) cells and expressed in Luria Bertani media induced by 1.0 M IPTG. Subsequently, sequencing was performed and the results were analyzed using the ClustalW and National Center for Biotechnology Information BLAST software. The T-cell epitope prognosis was then explained by GENETYX version 8.0., for the prediction of B-cell epitope, as assessed using an Immune Epitope Database analysis. Results: The PCR product of the RV1980c gene had a length of 619 bp. Moreover, SDS- polyacrylamide gel electrophoresis and Western blotting revealed that the protein encoded by the Rv1980c gene weighed 36 kDa. We gained nine specific T-cell epitopes according to Iad Pattern position and eight epitopes according to Rothbard/Taylor Pattern Position; furthermore, we detected five B-cell epitopes in the RV1980c gene. Conclusion: The MPT64 protein encoded by the RV1980c gene carries epitopes that are realized by lymphocytes and represent potential immunoserodiagnostic candidates in diagnostic immunology.

Potential of polylactic-co-glycolic acid (PLGA) for delivery Jembrana disease DNA vaccine Model (pEGFP-C1-tat).

BACKGROUND: The development of a vaccine for Jembrana disease is needed to prevent losses in Indonesia's Bali cattle industry. A DNA vaccine model (pEGFP-C1-tat) that requires a functional delivery system will be developed. Polylactic-co-glycolic acid (PLGA) may have potential as a delivery system for the vaccine model. OBJECTIVES: This study aims to evaluate the in vitro potential of PLGA as a delivery system for pEGFP-C1-tat. METHODS: Consensus and codon optimization for the tat gene was completed using a bioinformatic method, and the product was inserted into a pEGFP-C1 vector. Cloning of the pEGFP-C1-tat was successfully performed, and polymerase chain reaction (PCR) and restriction analysis confirmed DNA isolation. PLGA-pEGFP-C1-tat solutions were prepared for encapsulated formulation testing, physicochemical characterization, stability testing with DNase I, and cytotoxicity testing. The PLGA-pEGFP-C1-tat solutions were transfected in HeLa cells, and gene expression was observed by fluorescent microscopy and real-time PCR. RESULTS: The successful acquisition of transformant bacteria was confirmed by PCR. The PLGA:DNA:polyvinyl alcohol ratio formulation with optimal encapsulation was 4%:0.5%:2%, physicochemical characterization of PLGA revealed a polydispersity index value of 0.246, a particle size of 925 nm, and a zeta potential value of -2.31 mV. PLGA succeeded in protecting pEGFP-C1-tat from enzymatic degradation, and the percentage viability from the cytotoxicity test of PLGA-pEGFP-C1-tat was 98.03%. The PLGA-pEGFP-C1-tat demonstrated luminescence of the EGFP-tat fusion protein and mRNA transcription was detected. CONCLUSIONS: PLGA has good potential as a delivery system for pEGFP-C1-tat.

In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate.

INTRODUCTION: The development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex. MATERIAL AND METHODS: Cloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection. RESULTS: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent. CONCLUSION: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

Use of reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick for an easy and rapid detection of Jembrana disease virus.

Jembrana disease virus (JDV) is a viral pathogen that causes Jembrana disease in Bali cattle (Bos javanicus) with high mortality rate. An easy and rapid diagnostic method is essential for further control this disease. We used a reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with lateral flow dipstick (LFD), based on conserved tm subunit of Jembrana disease virus env gene. The RT-LAMP conditions were optimized by varying the concentration of MgSO4, betaine, dNTP, and temperature as well as the time and duration of reaction. The primers sensitivity for JDV was confirmed. The method was able to detect env-tm gene dilution which contained 2 × 10(-15) g of template. Comparatively, the sensitivity of RT-LAMP/LFD was 100-fold more sensitive than reverse transcription-polymerase chain reaction. The primers specificity for JDV was also confirmed using positive and negative controls. This work also showed that virus detection could be done not only on total RNA extracted from blood but various organs could also be analyzed for the presence of JDV using RT-LAMP/LFD method. The whole process, including the LAMP reaction and the LFD hybridization step only lasts approximately 75 min. Results of analysis can be easily observed with naked eyes without addition of any chemical or further analysis. The combination of RT-LAMP with LFD makes the method a more suitable diagnostic tool in conditions where sophisticated and expensive equipments are not available for field investigations on Jembrana disease in Bali cattle.

Development of loop-mediated isothermal amplification (LAMP) as a diagnostic tool of toxoplasmosis.

Infection with Toxoplasma gondii is one of the most common parasitic infections in humans and other warm-blooded animals. This paper describes the development of loop-mediated isothermal amplification (LAMP) specific to the single-copy gene SAG1 as a diagnostic tool of toxoplasmosis. A set of primers, composed of outer primers, inner primers and loop primers was designed from a published sequence data (GeneBank Acc. no. AY651825). Experiments showed that when LAMP was applied to sample organs, amplification absolutely required the loop primers to complete. SAG1-based LAMP turned out to be very sensitive, exhibiting a degree of sensitivity higher than the conventional PCR. LAMP is a convenient and sensitive diagnostic tool for routine health control of toxoplasmosis.

Primary structure of mature SAG1 gene of an Indonesian Toxoplasma gondii and comparison with other strains.

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.