Toxoplasma gondii (T. gondii) a widespread zoonotic parasite infecting various host species. Since cattle serve as major intermediate hosts for T. gondii, giving a significant public health concern, developing effective diagnostic tools is essential. This study evaluated the serodiagnostic potential of an indirect enzyme linked immunosorbent assay (iELISA) using recombinant T. gondii dense granule 14 protein (TgGRA14) in cattle population. The recombinant TgGRA14 used was synthesized and evaluated as previously described. The microplate was coated with the purified TgGRA14 protein at 1 µg/mL and incubated overnight. The assay utilized a 1:200 dilution of bovine sera and a 1:5000 dilution of HRP-conjugated anti-bovine IgG. The reaction was developed with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate and the absorbance was measured at 450 nm. A sensitivity of 93.30

Toxoplasma gondii is a widespread zoonotic protozoan parasite, with domestic cats as definitive hosts. To improve serodiagnosis surveillance and control, this study aimed to develop and evaluate an indirect enzyme-linked immunosorbent assay (iELISA) using recombinant TgGRA14 protein for the serological detection of T. gondii in domestic cats. The TgGRA14 gene (nucleotides 114–858 encoding amino acids 38–286) was synthesized and cloned into the pET-28a vector containing an N-terminal FLAG tag. The recombinant plasmid was transformed into E. coli BL21, and protein expression was purified via anti-FLAG affinity chromatography. Protein integrity, antigenicity and identity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blotting, while mass spectrometry verified protein identity. The purified TgGRA14 protein was used as a coating antigen in iELISA, which was applied to 149 feline serum samples and compared with results from the indirect fluorescent antibody test (IFAT). The recombinant TgGRA14 protein (approximately 29 kDa) was successfully expressed and purified, with confirmed antigenicity and 99.6–100% identity by mass spectrometry. The iELISA detected  38.3% seroprevalence in feline samples, compared to 32.9% by IFAT, and showed high diagnostic performance with 95.9% sensitivity, 90.0% specificity, and a kappa value of 0.824. These results indicate that the iELISA based on recombinant TgGRA14 is a highly sensitive and specific diagnostic tool for detecting T. gondii infection in domestic cats, which has strong potential for application in epidemiological surveillance and serological screening programs.

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.

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, a multiplex PCR has been developed for hemoparasites consisting of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi detections. Methods:Blood parasite infections in cattle were investigated using three different methods: microscopic examination, single PCR, and multiplex PCR. The Anaplasma 16s rRNA gene, Babesia 18s rRNA gene, Theileria major piroplasm surface protein 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 positive predictive value, 98% negative predictive value, 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.

Phylogenetic and population genetic analyses were conducted on tick specimens collected from cattle in northern, northeastern, central, and southern regions of Thailand. Morphological identification indicated these ticks consisted of three species, Rhipicephalus microplus from all four regions, R. sanguineus from the northern and northeastern regions, and a Haemaphysalis species only collected from the northeastern region. Analysis of cytochrome c oxidase subunit I gene (COI) sequences identified R. microplus clades A and C, while clade B was not detected in this study. The same analysis indicated specimens morphologically identified as Haemaphysalis were H. bispinosa, confirming previous reports of their prevalence in northeastern Thailand. H. bispinosa showed low haplotype and nucleotide diversity, suggesting either a bottleneck or founder effect. Both R. microplus clades displayed high haplotype diversity and low nucleotide diversity, a pattern associated with population expansion. Genetic structural analysis revealed significant genetic differences in R. microplus clade A, especially between mainland (northern, northeastern, and central regions) and peninsular (southern region) populations, which indicated limited gene flow between these areas while suggesting movement of these ticks across the mainland. The sequence analyses described in this report enhance understanding of the natural history of ticks in Thailand and are expected to guide and strengthen tick control strategies across Southeast Asia.

Toxoplasma gondii, a pathogen of significant concern in animal production, companion animal health, and public health, particularly affects immunocompromised individuals and pregnant women. Current diagnostic techniques employ both direct and indirect methods, with serological assays widely used for detecting T. gondii infections in humans and animals. In this study, the TIM-barrel structure of Br2 β-glucosidase was engineered to create 10 chimeric multi-epitope proteins for T. gondii serological detection. Indirect ELISA screening identified three promising candidate proteins, V4Z, SFF, and S7V-V4Z-SFF, with sensitivities ranging from 71–86% and specificities ranging from 68–76%. Among these, ELISA-V4Z achieved the highest concordance with the reference IFAT method (Kappa = 0.58, 95% CI = 0.32–0.84) and demonstrated a moderate positive predictive value (PPV, 67%) and strong negative predictive value (NPV, 90%). These results suggest that the V4Z chimeric protein demonstrated the strongest performance among the tested candidates for T. gondii detection, exhibiting the highest sensitivity and specificity along with moderate agreement with the reference IFAT. However, its overall diagnostic performance remains limited. These findings highlight the need for further refinement and validation to enhance its diagnostic potential and assess its applicability for broader serological testing.

The bovine rumen contains a large consortium of residential microbes that release a variety of digestive enzymes for feed degradation. However, the utilization of these microbial enzymes is still limited because these rumen microorganisms are mostly anaerobes and are thus unculturable. Therefore, we applied a sequence-based metagenomic approach to identify a novel 2,445-bp glycoside hydrolase family 3 β-glucosidase gene known as BrGH3A from the metagenome of bovine ruminal fluid. BrGH3A β-glucosidase is a 92-kDa polypeptide composed of 814 amino acid residues. Unlike most glycoside hydrolases in the same family, BrGH3A exhibited a permuted domain arrangement consisting of an (α/β)6 sandwich domain, a fibronectin type III domain and a (β/α)8 barrel domain. BrGH3A exhibited greater catalytic efficiency toward laminaribiose than cellobiose. We proposed that BrGH3A is an exo-acting β-glucosidase from Spirochaetales bacteria that is possibly involved in the intracellular degradation of β-1,3-/1,4-mixed linkage glucans that are present in grass cell walls. BrGH3A exhibits rich diversity in rumen hydrolytic enzymes and may represent a member of a new clan with a permuted domain topology within the large family.

Ruminant animals rely on the activities of β-glucosidases from residential microbes to convert feed fibers into glucose for further metabolic uses. In this report, we determined the structures of Br2, which is a glycoside hydrolase family 1 β-glucosidase from the bovine rumen metagenome. Br2 folds into a classical (β/α)8-TIM barrel domain but displays unique structural features at loop β5→α5 and α-helix 5, resulting in different positive subsites from those of other GH1 enzymes. Br2 exhibited the highest specificity toward laminaritriose, suggesting its involvement in β-glucan hydrolysis in digested feed. We then substituted the residues at subsites +1 and + 2 of Br2 with those of Halothermothrix orenii β-glucosidase. The C170E and C221T mutations provided favorable interactions with glucooligosaccharide substrates at subsite +2, while the A219N mutation probably improved the substrate preference for cellobiose and gentiobiose relative to laminaribiose at subsite +1. The N407Y mutation increased the affinity toward cellooligosaccharides. These results give further insights into the molecular determinants responsible for substrate specificity in GH1 β-glucosidases and may provide a basis for future enzyme engineering applications.

Rhipicephalus (Boophilus) microplus is tick parasite that affects the cattle industry worldwide. In R. (B.) microplus, acaricide resistance develops rapidly against many commercial acaricides. One of main resistance strategies is to enhance the metabolic detoxification mediated by R. (B.) microplus glutathione-S-transferase (RmGST). RmGST detoxifies acaricides by catalyzing the conjugation of glutathione to acaricides. Although structural and dynamic details of RmGST are expected to elucidate the biologic activity of this molecule, these data have not been available to date. Thus, Molecular Dynamics simulations were employed to study ligand-free RmGST at an atomic level. Like other m-class GSTs, the flexible m loop (m1) of RmGST was observed. M1 seems to shield the active sites from the bulk. A RmGST dimer is stabilized by the lock-and-key motif (F57 as "key") and hydrogen bonds of R82-E91 and R82-D98 at the dimer interface. Without substrates, conserved catalytic Y116 and N209 can interact with V112, G210 (for Y116) and F215 (for N209). Overall, most residues involving in RmGST function and stability are similar to other m-class GSTs. This implies similar structural stability and catalytic activity of RmGST to other GSTs. An insight obtained here will be useful for management of acaricide resistance and tick control.Communicated by Ramaswamy H. Sarma.

Importance of the work: Trypanosoma evansi is a vector-borne pathogen, responsible for T. evansi infection (surra) which has a major impact on animal health in Southeast Asia, including Thailand.Objectives: To update the current situation regarding trypanosome infections of cattle and buffaloes in Sakon Nakhon province, Thailand.Materials & Methods: Blood samples were collected from buffaloes (455) and beef (1,077) and dairy (227) cattle in eight districts of Sakon Nakhon province and tested for trypanosome infections using T. evansi indirect-enzyme-linked immunosorbent assay (ELISA) and TRYP1 polymerase chain reaction (PCR).Results: Among the 1,759 samples, there were 77 positives (4.38%; 77/1,759) from T. evansi indirect-ELISA and 8 positives (0.45%; 8/1,759) from TRYP1 PCR, of which two samples were positives for both tests.The ITS1 sequencing analysis revealed high T. evansi sequence identities (97.22-100%).Factors associated with T. evansi infections were age, species, farm size and district.The highest prevalence was in buffaloes (9.67%; 44/455), followed by beef cattle (3.25%; 35/1,077) and dairy cattle (1.76%; 4/227).The significant associations with T. evansi infection were an animal age of 1-3 yr (6.82%; 53/777; p = 0.00), a medium herd size (5.99%;13/217; p = 0.03) and farm located in Phanna Nikhom district (21.33%; 32/150; p = 0.00).Main finding: Cattle and buffaloes in Sakon Nakhon province had a low prevalence (less than 5%) for current infection by T. evansi.However, the prevalence of trypanosome infections might be increasing due to the high densities of blood-sucking flies.

Tilapia lake virus (TiLV) now affects Nile tilapia culture worldwide, with no available commercial vaccine for disease prevention. DNA and recombinant protein-based vaccines were developed and tested following viral isolation and characterization. The viral strain isolated from diseased hybrid red tilapia (Oreochromis sp.) shared high levels of morphological and genomic similarity (95.49-99.52%) with other TiLV isolates in the GenBank database. TiLV segment 9 (Tis9) and segment 10 (Tis10) DNA vaccines (pcDNA-Tis9 and pcDNA-Tis10) and recombinant protein vaccines (Tis9 and Tis10) were prepared and tested for their efficacy in juvenile hybrid red tilapia. Fish were immunized with either single vaccines (pcDNA-Tis9, pcDNA-Tis10, Tis9 and Tis10) or combined vaccines (pcDNA-Tis9 + pcDNA-Tis10 and Tis9 + Tis10) by intramuscular injection and intraperitoneal injection for DNA and protein vaccines, respectively. Negative controls were injected with PBS or a naked pcDNA3.1 vector in the same manner. An experimental challenge with TiLV was carried out at 4 weeks post-vaccination (wpv) by intraperitoneal injection with a dose of 1 × 105 TCID50 per fish. Relative percent survival (RPS) ranged from 16.67 ± 00.00 to 61.11 ± 9.62%. The Tis10 and pcDNA-Tis10 vaccines conferred better protection compared to Tis9 and pcDNA-Tis9. Highest levels of protection were observed in pcDNA-Tis9 + pcDNA-Tis10 (61.11 ± 9.62%) and Tis9 + Tis10 (55.56 ± 9.62%) groups. Specific antibody was detected in all vaccinated groups at 1-4 wpv by Dot Blot method, with the highest integrated density at 2 and 3 wpv. In silico analysis of Tis9 and Tis10 revealed a number of B-cell epitopes in their coil structure, possibly reflecting their immunogenicity. Findings suggested that the combination of Tis9 and Tis10 in DNA and recombinant protein vaccine showed high efficacy for the prevention of TiLV disease in hybrid red tilapia.

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, χ 2 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.

ABSTRACT Cattle ticks, Rhipicephalus (Boophilus) microplus, have a serious impact on livestock worldwide. For tick control, host vaccination is more sustainable than chemical. Salivary serpin (serine protease inhibitor) from R. microplus (RmS-3) is a potential anti-tick vaccine candidate antigen. Like other serpins, RmS-3 has a suicide inhibition mechanism, where the reactive centre loop (RCL) is cleaved and then inserted into the main β-pleated sheet. This causes the breakdown of serpin into two regions (core domain and cleaved polypeptide). A small sequence variation of RmS-3 was observed among specimens in Thailand included three extra amino acids (T147, K180, N193). Molecular Dynamics simulations of the previously reported (Micro) and the novel Thai RmS-3 (Thai) were conducted in comparison. Overall, Micro is more rigid than Thai. Having additional T147 and K180 enhance the structural flexibility in Thai. Both residues are located close to areas that perform a distinct conformational change, so T147 and K180 may impact inhibitory activity of Thai RmS-3. In contrast, N193 seems to have a minor effect. A presence of T147 and K180 in Thai seems to cause significant differences in structural dynamics which may lead to different degrees of inhibition activity. Further experimental studies are required.

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 TOPO (TM) 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.

Ixodid ticks have a crucial impact on people and domestic animals worldwide. These parasites also pose a serious threat to livestock. To date, vaccination of hosts against ticks is a safer, more sustainable alternative to chemical control of ticks and the disease agents they transmit. Because of their roles in tick physiology, serpins (serine protease inhibitors) from tick saliva are among the candidates for anti-tick vaccines. Inhibitory serpins employ a suicide inhibition mechanism to inhibit proteases, where the serpin reactive centre loop (RCL) is cleaved, by the targeted protease, and then inserted into the main beta-sheet of the serpin. This causes a massive conformational change called the 'stressed to relaxed' (S -> R) transition, leading to the breakdown of serpin into two regions (core domain and cleaved polypeptide). Recently, the first tick serpin crystal structure from Ixodes ricinus in R-state was reported. We thus employed molecular dynamics simulations to better understand serpin structure and dynamics in atomic detail. Overall, R-state serpin showed high rigidity, especially the core domain. The most flexible region is the terminal of the cleaved polypeptide, due to its high-water exposure, while the rest of the cleaved polypeptide is stably trapped behind the core domain. T363, D367 and N375 are found to play a vital role in protein-protein attachment. This finding can be used to explain the high stability of the R-state serpin at the atomic level and provides insight into this tick serpin which will be useful for rational anti-tick vaccine development.

The efficacy of antibody detection tools for all stages of Ehrlichia canis infections and for various genotypes remains unclear. We produced recombinant gp36 (rgp36) antigens from different isolates of Thai E. canis to confirm the immunoreactivities to these recombinant proteins from naturally infected dogs. Sera and blood samples were taken from 21 dogs naturally infected with E. canis and in the clinical stages of acute phase ehrlichiosis. The expression vectors and competent E. coli produced two isolates of rgp36. These two major rgp36s were recognized by the dogs' sera in Western blotting, with both anti-dog IgM and IgG used as secondary antibodies. The two different genotypes of these local recombinant immunoreactive proteins were gp36 subgroup A (isolate 1055) and subgroup B (isolate 533). The Western blot analyses successfully identified both specific IgM and IgG from the dogs' sera. Of all 21 cases, five dogs presented specific IgM, twenty dogs presented specific IgG, and the commercial test used found fifteen seropositive dogs. There were four dogs that presented both specific IgM and IgG. Only one dog presented specific IgM only. This report is the first identification of a specific IgM in dogs in response to acute infections with E. canis. The recombinant gp36 isolates may be useful as potential antigenic material for subsequent serological tests that have a high possibility for differentiating between acute, chronic, primary, and nonprimary infections with E. canis.

A novel β-glucosidase from higher termite Microcerotermes annandalei (MaBG) was obtained via a screening method targeting β-glucosidases with increased activities in the presence of glucose. The purified natural MaBG showed a subunit molecular weight of 55 kDa and existed in a native form as a dimer without any glycosylation. Gene-specific primers designed from its partial amino acid sequences were used to amplify the corresponding 1,419-bp coding sequence of MaBG which encodes a 472-amino acid glycoside hydrolase family 1 (GH1) β-glucosidase. When expressed in Komagataella pastoris, the recombinant MaBG appeared as a ~ 55-kDa protein without glycosylation modifications. Kinetic parameters as well as the lack of secretion signal suggested that MaBG is an intracellular enzyme and not involved in cellulolysis. The hydrolytic activities of MaBG were enhanced in the presence of up to 3.5-4.5 M glucose, partly due to its strong transglucosylation activity, which suggests its applicability in biosynthetic processes. The potential synthetic activities of the recombinant MaBG were demonstrated in the synthesis of para-nitrophenyl-β-D-gentiobioside via transglucosylation and octyl glucoside via reverse hydrolysis. The information obtained from this study has broadened our insight into the functional characteristics of this variant of termite GH1 β-glucosidase and its applications in bioconversion and biotechnology.

Abstract β-Glucosidases play an important role in biomass degradation as they hydrolyze cellobiose to glucose in a final step of cellulolysis. In particular, ruminant animals rely on β-glucosidases from rumen microorganisms for conversion of plant cellulosic materials into glucose. In this study, we are interested in characterization of a novel β-glucosidase from rumen microorganisms. However, most rumen microorganisms are obligate anaerobes, which require special cultivation conditions. Presently, the metagenomic techniques, which enable isolation and characterization of microbial genes directly from environmental samples, have been applied to overcome these problems. In this study, the sequence-based screening approach was successfully applied to identify a novel β-glucosidase gene, Br2, from a bovine rumen metagenomic sample. A 1338-bp complete coding sequence of Br2 encodes a 51-kDa GH1 β-glucosidase of 445 amino acid residues with 59% sequence identity to a β-glucosidase from Cellulosilyticum ruminicola JCM 14822. The recombinantly expressed Br2 exhibited an optimal activity at pH 6.5 and 40°C, reflecting its rumen bacterial origin, and relatively higher catalytic efficiencies toward glucoside and fucoside substrates than other glycosides, similar to many previously reported bacterial β-glucosidases. Our sequence-based screening approach can be applied to identify other genes of interest from environmental samples.