Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing Pasteurella multocida OmpH Protein for Fowl Cholera Prevention in Ducks.

Fowl cholera is caused by the bacterium Pasteurella multocida, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from P. multocida integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of P. multocida OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent P. multocida X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases.

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.

Simultaneous Protective Immune Responses of Ducks against Duck Plague and Fowl Cholera by Recombinant Duck Enteritis Virus Vector Expressing Pasteurella multocida OmpH Gene.

Duck enteritis virus and Pasteurella multocida are major duck pathogens that induce duck plague and fowl cholera, respectively, in ducks and other waterfowl populations, leading to high levels of morbidity and mortality. Immunization with live attenuated DEV vaccine containing P. multocida outer membrane protein H (OmpH) can provide the most effective protection against these two infectious diseases in ducks. We have recently reported the construction of recombinant DEV expressing P. multocida ompH gene using the CRISPR/Cas9 gene editing strategy with the goal of using it as a bivalent vaccine that can simultaneously protect against both infections. Here we describe the findings of our investigation into the systemic immune responses, potency and clinical protection induced by the two recombinant DEV-ompH vaccine constructs, where one copy each of the ompH gene was inserted into the DEV genome at the UL55-LORF11 and UL44-44.5 intergenic regions, respectively. Our study demonstrated that the insertion of the ompH gene exerted no adverse effect on the DEV parental virus. Moreover, ducklings immunized with the rDEV-ompH-UL55 and rDEV-ompH-UL44 vaccines induced promising levels of P. multocida OmpH-specific as well as DEV-specific antibodies and were completely protected from both diseases. Analysis of the humoral and cellular immunity confirmed the immunogenicity of both recombinant vaccines, which provided strong immune responses against DEV and P. multocida. This study not only provides insights into understanding the immune responses of ducks to recombinant DEV-ompH vaccines but also demonstrates the potential for simultaneous prevention of viral and bacterial infections using viral vectors expressing bacterial immunogens.

Recombinant Ehrlichia canis GP19 Protein as a Promising Vaccine Prototype Providing a Protective Immune Response in a Mouse Model.

The intracellular bacterium Ehrlichia canis is the causative pathogen of canine monocytic ehrlichiosis (CME) in dogs. Despite its veterinary and medical importance, there is currently no available vaccine against this pathogen. In this study, the recombinant GP19 (rGP19) was produced and used as a recombinant vaccine prototype in a mouse model against experimental E. canis infection. The efficacy of the rGP19 vaccine prototype in the part of stimulating B and T cell responses and conferring protection in mice later challenged with E. canis pathogen were evaluated. The rGP19-specific antibody response was evaluated by ELISA after E. canis challenge exposure (on days 0, 7, and 14 post-challenge), and demonstrated significantly higher mean antibody levels in rGP19-immunized mice compared with adjuvant-immunized and naive mice. Significantly lower ehrlichial loads in blood, liver, and spleen DNA samples were detected in the immunized mice with rGP19 by qPCR. The up-regulation of IFNG and IL1 mRNA expression were observed in mice immunized with rGP19. In addition, this study detected IFN-γ-producing memory CD4+ T cells in the rGP19-immunized mice and later infected with E. canis on day 14 post-infection period using flow cytometry. The present study provided a piece of evidence that rGP19 may eliminate E. canis by manipulating Th1 and B cell roles and demonstrated a promising strategy in vaccine development against E. canis infection in the definitive host for further study.

Incidence of hemoparasitic infections in cattle from central and northern Thailand.

Background: Hemoparasites, such as Babesia spp., Theileria spp. and Anaplasma spp., can negatively affect the health of farm animals resulting in significant losses in production. These losses inherently affect the economics of the livestock industry. Since increases in the severity of vector-borne diseases in the southeast Asian region have been reported, investigations of parasitic epidemiology in Thailand will be necessary to improve the existing parasite control strategies for blood parasitic infections. This study aims to investigate incidences of bovine hemoparasites throughout central and northern Thailand by focusing on areas of high-density cattle populations. Methods: Blood parasitic infections among cattle were screened and identified by microscopic examination. Anemia status was then determined by evaluation of the packed cell volume (PCV) of each animal. Furthermore, blood parasites were detected and identified by genus and species-specific primers through the polymerase chain reaction method. Amplicons were subjected to DNA sequencing; thereafter, phylogenetic trees were constructed to determine the genetic diversity and relationships of the parasite in each area. Results: A total of 1,066 blood samples were found to be positive for blood parasitic infections as follows: 13 (1.22%), 389 (36.50%), and 364 (34.15%) for Babesia bovis, Theileria orientalis, and Anaplasma marginale, respectively. Furthermore, multiple hemoparasitic infections in the cattle were detected. The hematocrit results revealed 161 hemoparasitic infected samples from 965 blood samples, all of which exhibiting indications of anemia with no significant differences. Sequence analysis of the identified isolates in this study revealed that B. bovis rap-1, four separate clades of T. orientalis msps, and A. marginale msp4 exhibited considerable sequence similarity to homologous sequences from isolates obtained from other countries. Sequence similarity ranged between 98.57-100%, 83.96-100%, and 97.60-100% for B. bovis rap-1, T. orientalis msps, and A. marginale msp4, respectively. Conclusion: In this study, the analyzed incidence data of cattle hemoparasitic infection in Thailand has provided valuable and basic information for the adaptation of blood-borne parasitic infections control strategies. Moreover, the data obtained from this study would be useful for future effective parasitic disease prevention and surveillance among cattle.

Immunization of Cattle With Recombinant Structural Ectodomains I and II of Babesia bovis Apical Membrane Antigen 1 [BbAMA-1(I/II)] Induces Strong Th1 Immune Response.

Both strong innate and adaptive immune responses are an important component of protection against intraerythrocytic protozoan parasites. Resistance to bovine babesiosis is associated with interferon (IFN)-γ mediated responses. CD4+ T cells and macrophages have been identified as major effector cells mediating the clearance of pathogens. Previously, the apical membrane antigen 1 (AMA-1) was found to significantly induce the immune response inhibiting B. bovis merozoite growth and invasion. However, a detailed characterization of both humoral and cellular immune responses against the structure of B. bovis AMA-1 (BbAMA-1) has not yet been established. Herein, the present study aimed to express the recombinant BbAMA-1 domain I+II protein [rBbAMA-1(I/II)], which is the most predominant immune response region, and to characterize its immune response. As a result, cattle vaccinated with BbAMA-1(I/II) significantly developed high titters of total immunoglobulin (Ig) G antibodies and a high ratio of IgG2/IgG1 when compared to control groups. Interestingly, the BbAMA-1(I/II)-based formulations produced in our study could elicit CD4+ T cells and CD8+ T cells producing IFN-γ and tumor necrosis factor (TNF)-α. Collectively, the results indicate that immunization of cattle with BbAMA-1(I/II) could induce strong Th1 cell responses. In support of this, we observed the up-regulation of Th1 cytokine mRNA transcripts, including IFN-γ, TNF-α, Interleukin (IL)-2 and IL-12, in contrast to down regulation of IL-4, IL-6 and IL-10, which would be indicative of a Th2 cytokine response. Moreover, the up-regulation of inducible nitric oxide synthase (iNOS) was observed. In conclusion, this is the first report on the in-depth immunological characterization of the response to BbAMA-1. According to our results, BbAMA-1 is recognized as a potential candidate vaccine against B. bovis infection. As evidenced by the Th1 cell response, it could potentially provide protective immunity. However, further challenge-exposure with virulent B. bovis strain in immunized cattle would be needed to determine its protective efficacy.

Effect of GP19 Peptide Hyperimmune Antiserum on Activated Macrophage during Ehrlichia canis Infection in Canine Macrophage-like Cells.

In terms of its veterinary importance, vaccine development against Ehrlichia canis is needed. However, the effect of developing vaccines on humoral immune response against E. canis infection is still unknown. Novel GP194-43 was synthesized according to E. canis GP19 epitope prediction. To restrict any loss and/or illness in the host animal, rabbits were used in this study to produce GP194-43 hyperimmune sera. The effect of GP194-43 hyperimmune sera on neutralization was examined in vitro by determining the inhibition of E. canis infection of the macrophage-like cell line (DH82) in the presence of the sera. Four groups of DH82 cells received differing treatments. These included E. canis experimentally infected DH82 cells, E. canis-infected DH82 cells with control rabbit serum (untreated group), E. canis-infected DH82 cells with GP194-43 rabbit antiserum (treated group) and uninfected cells (negative control group), respectively. The treated group developed a decrease (p < 0.01) in the percentage of E. canis infected cells after 3 days post-infection at 48.57 ± 1.28. In addition, real-time PCR analyses of cytokine mRNA expression involved with the macrophage, humoral, and cellular immune responses were conducted. The findings revealed an upregulated expression of IFNG in the treated group during the infection. This study demonstrated neutralization in the GP194-43 peptide hyperimmune sera of immunized rabbits. Notably, IFN-γ production could be effectively promoted in canine macrophages in relation to the activation of macrophages and adaptive immune responses. The results of this study indicate the potential for the use of this immunogen in further investigations involving immunized and infected dogs as E. canis host species.

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development.

BACKGROUND: Bovine babesiosis caused by Babesia bovis (B. bovis) has had a significant effect on the mobility and mortality rates of the cattle industry worldwide. Live-attenuated vaccines are currently being used in many endemic countries, but their wide use has been limited for a number of reasons. Although recombinant vaccines have been proposed as an alternative to live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates in the development of a vaccine against diseases caused by apicomplexan parasite species. In Plasmodium falciparum (P. falciparum) AMA-1 (PfAMA-1), several antibodies against epitopes in the plasminogen, apple, and nematode (PAN) motif of PfAMA-1 domain I significantly inhibited parasite growth. Therefore, the purpose of this study was to predict an epitope from the PAN motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was then bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Subsequently, in vitro growth- and the invasion-inhibitory effects of the rabbit antiserum were immunologically characterized. RESULTS: Our results demonstrated that the predicted BbAMA-1 epitope was located on the surface-exposed α-helix of the PAN motif in domain I at the apex area between residues 181 and 230 with six polymorphic sites. Subsequently, sBbAMA-1 elicited antibodies capable of recognizing the native BbAMA-1 in immunoassays. Furthermore, anti-serum against sBbAMA-1 was immunologically evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the rabbit anti-sBbAMA-1 serum at a dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50-70% on days 3 and 4 of cultivation, along with the invasion of merozoites by approximately 60% within 4 h of incubation when compared to the control groups. CONCLUSION: Our results indicate that the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

Immune response in dairy cattle against combined foot and mouth disease and haemorrhagic septicemia vaccine under field conditions.

BACKGROUND: Foot-and-mouth disease (FMD) and Haemorrhagic septicemia (HS) are two important diseases that are known to have caused significant economic losses to the cattle industry. Accordingly, vaccinations have been recognized as an efficient method to control and prevent both of the above-mentioned diseases. This study aimed to determine the immune response to FMD virus antigens and the recombinant outer membrane protein of HS (rOmpH) of Pasteurella multocida in cattle administered as a combination vaccine and compare antibody titers with the two vaccines given independently, under field conditions. Dairy cattle were divided into three groups. Each group was immunized with different vaccine types according to the vaccination program employed in this study. Antibody responses were determined by indirect ELISA, liquid phase blocking ELISA (LPB-ELISA) and viral neutralization test (VNT). Furthermore, the cellular immune responses were measured by lymphocyte proliferation assay (LPA). RESULTS: The overall antibody titers to HS and FMDV were above cut-off values for the combined FMD-HS vaccine in this study.The mean antibody titer against HS after the first immunization in the combined FMD-HS vaccine groups was higher than in the HS vaccine groups. However, no statistically significant differences (p > 0.05) were observed between groups. Likewise, the antibody titer to the FMDV serotypes O/TAI/189/87 and Asia 1/TAI/85 determined by LPB-ELISA in the combined vaccine were not statistically significantly different when compared to the FMD vaccine groups. However, the mean VNT antibody titer of combined vaccine against serotype O was significantly higher than the VN titer of FMD vaccine groups (p < 0.05). Moreover, the LPA results showed that all vaccinated groups displayed significantly higher than the negative control (p < 0.05). Nevertheless, no differences in the lymphocyte responses were observed in comparisons between the groups (p > 0.05). CONCLUSIONS: The combined FMD-HS vaccine formulated in this study could result in high both antibody and cellular immune responses without antigenic competition. Therefore, the combined FMD-HS vaccine can serve as an alternative vaccine against both HS and FMD in dairy cattle under field conditions.

A Preliminary Study of the Cross-Reactivity of Canine MAGE-A with Hominid Monoclonal Antibody 6C1 in Canine Mammary Gland Tumors: An Attractive Target for Cancer Diagnostic, Prognostic and Immunotherapeutic Development in Dogs.

Melanoma-associated antigen-A (MAGE-A), a family of cancer/testis antigens, has been recognized as a potential target molecule for cancer immunotherapy. However, there has been very little information available with regard to this antigen in dogs. This study aimed to investigate the expression of MAGE-A in canine mammary gland tumors (CMTs) using immunohistochemistry and immunoblotting with human monoclonal MAGE-A antibody 6C1. The present study has provided evidence of cross-reactivity of the canine MAGE-A expression with the human MAGE-A antibody in CMTs. The MAGE-A antigens were expressed in moderate- and high-grade malignant CMTs (22.22%, 2/9), but no expression was observed in benign CMTs. The immunohistochemical staining of canine MAGE antigen in CMT cells showed nuclear and nuclear-cytoplasmic expression patterns that may be involved with the mitotic cell division of tumor cells. Molecular weights of the canine MAGE-A antigen presented in this study were approximately 42-62 kDa, which were close to those of other previous studies involving humans and dogs. The findings on this protein in CMTs could supply valuable oncological knowledge for the development of novel diagnostic, prognostic and immunotherapeutic tumor markers in veterinary medicine.

Evaluation of an in-house indirect enzyme-linked immunosorbent assay of feline panleukopenia VP2 subunit antigen in comparison to hemagglutination inhibition assay to monitor tiger antibody levels by Bayesian approach.

BACKGROUND: Feline panleukopenia virus (FPV) is an etiologic pathogen of feline panleukopenia that infects all members of Felidae including tigers (Panthera tigris). Vaccinations against FPV among wild felid species have long been practiced in zoos worldwide. However, few studies have assessed the tiger immune response post-vaccination due to the absence of a serological diagnostic tool. To address these limitations, this study aimed to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the monitoring of tiger antibody levels against the feline panleukopenia vaccine by employing the synthesized subunit capsid protein VP2. An in-house horseradish peroxidase (HRP) conjugated rabbit anti-tiger immunoglobulin G (IgG) polyclonal antibody (HRP-anti-tiger IgG) was produced in this study and employed in the assay. It was then compared to a commercial HRP-conjugated goat anti-cat IgG (HRP-anti-cat IgG). Sensitivity and specificity were evaluated using the Bayesian model with preferential conditional dependence between HRP-conjugated antibody-based ELISAs and hemagglutination-inhibition (HI) tests. RESULTS: The posterior estimates for sensitivity and specificity of two indirect ELISA HRP-conjugated antibodies were higher than those of the HI test. The sensitivity and specificity of the indirect ELISA for HRP-anti-tiger IgG and HRP-anti-cat IgG were 86.5, 57.2 and 86.7%, 64.6%, respectively, while the results of the HI test were 79.1 and 54.1%. In applications, 89.6% (198/221) and 89.1% (197/221) of the tiger serum samples were determined to be seropositive by indirect ELISA testing against HRP-anti-tiger and HRP-anti-cat, respectively. CONCLUSION: To the best of our knowledge, the specific serology assays for the detection of the tiger IgG antibody have not yet been established. The HRP-anti-tiger IgG has been produced for the purpose of developing the specific immunoassays for tigers. Remarkably, an in-house indirect ELISA based on VP2 subunit antigen has been successfully developed in this study, providing a potentially valuable serological tool for the effective detection of tiger antibodies.

An Intranasal Vaccination with a Recombinant Outer Membrane Protein H against Haemorrhagic Septicemia in Swamp Buffaloes.

Hemorrhagic septicemia (HS) is an important infectious disease in cattle and buffaloes, caused by Pasteurella multocida B:2 and E:2. The intranasal recombinant OmpH-based vaccine was successfully used to protect dairy cattle from HS in a previous study. Thus, this study aimed to examine the protective ability of that vaccine among buffaloes. Four groups of Thai swamp buffaloes received different vaccines and were labeled as 100 or 200 µg of the rOmpH with CpG-ODN2007, commercial HS bacterin vaccine, and nonvaccinated control groups. Sera and whole blood were collected to examine the antibody levels and cellular immune response using indirect ELISA and MTT assay, respectively. Challenge exposure was performed with virulent P. multocida strain M-1404 serotype B:2 on day 72 of the experiment. The antibody titers to P. multocida among immunized buffaloes were significantly higher than in the control group (p < 0.01), especially the 200 µg of the rOmpH group. The stimulation index (SI) of the intranasally vaccinated groups revealed significantly higher levels than the nonvaccinated group (p < 0.01), but not different from the intramuscularly commercial HS vaccine. The clinical signs and high fever were observed after challenge exposure in the nonvaccinated group, while it was not observed among the 200 µg of rOmpH immunized buffaloes. The other immunized groups showed partial protection with transient fever. In conclusion, the rOmpH-based intranasal vaccine could elicit protective ability and induce antibody- and cell-mediated immune response against virulent P. multocida strain among swamp buffaloes.

Identification of potential canine mammary tumour cell biomarkers using proteomic approach: Differences in protein profiles among tumour and normal mammary epithelial cells by two-dimensional electrophoresis-based mass spectrometry.

Canine mammary tumours (CMTs) are regarded as invasive with a high rate of recurrent and metastasis in intact female dogs. Tumour diagnosis, therefore, is an important step in predicting and monitoring tumour progression. This study was designed to identify protein expression on CMTs by employing a proteomic approach. The primary cell culture from benign mixed tumour, simple carcinoma, complex carcinoma and normal mammary gland were established, and two-dimensional electrophoresis (2DE) was subsequently performed. The different spots on each sample type were collected for identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that cytokeratin 5 (CK5) and transketolase (TKT) were identified in benign mixed tumour cells and complex carcinoma cells. In contrast, cytokeratin 18 (CK18) and pyruvate kinase PKM were identified in simple carcinoma cells. Moreover, alpha-2-HS-glycoprotein tumour antigen was identified specifically in complex carcinoma cells. In addition, ATP-dependent 6-phosphofructokinase platelet type and elongation factor 2 proteins were observed in benign cells. In conclusion, all expressed proteins in this study have been recognized for acting as their expression that differs from healthy mammary epithelial cells. Expectantly, this study identified the expressed proteins that might be useful in further diagnostic biomarker studies on CMTs.

Protection against fowl cholera in ducks immunized with a combination vaccine containing live attenuated duck enteritis virus and recombinant outer membrane protein H of Pasteurella multocida.

Fowl cholera is a highly contagious disease within the global duck farming industry. This study aimed at formulating and evaluating the protective efficacy of a combination vaccine containing a recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain X-73 with a live attenuated duck plague vaccine into a single dose. Four groups of ducks received different treatments and the groups were labelled as non-vaccinated, combined vaccination, duck plague vaccination and rOmpH vaccination, respectively. The combined vaccination group was comprised of live attenuated duck plague commercial vaccine with 100 µg rOmpH to a total volume of 0.5 ml/duck/intramuscular administration. All groups were challenged with avian P. multocida strain X-73 via intranasal administration. In addition, blood samples were collected monthly over a period of 6 months to determine the appropriate antibody level by indirect ELISA. The indirect ELISA results in the combination vaccine group revealed that the average levels of the serum antibody against the duck enteritis virus (0.477 ± 0.155) and fowl cholera (0.383 ± 0.100) were significantly higher than those values in the non-vaccinated control group (0.080 ± 0.027 and 0.052 ± 0.017), respectively (P < 0.05). Moreover, all vaccinated ducks were effectively protected from fowl cholera. This preliminary study indicated that a combination vaccine did not affect the antibody response in the subjects while protecting the ducks against experimental P. multocida infection. This combination vaccine should be considered part of an alternative pre-treatment strategy that could replace the monovalent vaccine.

Two different genogroups of Ehrlichia canis from dogs in Thailand using immunodominant protein genes.

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis (CME). While there is a high prevalence of CME in Thailand, genetic diversity of E. canis is still poorly defined. This study examined the molecular characteristics of E. canis using PCR and phylogenetic analysis of the dsb, gp19 and gp36 genes. DNA was extracted from 220 whole blood samples of naturally infected dogs, and all had clinical signs compatible with tick-borne diseases. Of these, 16.4% (36/220) provided positive E. canis DNA via the dsb and gp19 genes. However, only 13 out of the 36 samples (36.1%) were positive for the gp36 gene. Sequences of the dsb gene had very high identity (99-100%) with previously deposited E. canis sequences. Sequences of the gp19 gene were similar to those from US and Taiwanese genogroups (98.8-99.5% identity). Elucidation of genetic characteristics of E. canis based on the gp36 gene displayed 91.4-99.1% shared identity. There were 426-429 bp of a 5' end pre-repeat tandem region, a 27 bp repetition with variable numbers of a tandem repeat (TR) region of 9 amino acid sequences (TEDSVSAPA), and a variable 3' end region with sequence length depending on the isolate (72-93 bp). Phylogenetic trees of E. canis, particularly using the gp36 amino acid sequences, showed that the Thai strains fell into two phylogenetic clades contained within other worldwide E. canis strains. Alignment and phylogenetic analysis suggested that E. canis strains from Thailand could be divided into two genogroups, the US and Taiwanese genogroups. This study provides the first characterization of the dsb and gp19 genes of E. canis in Thailand, the results support the conclusion that the gp36 is a potential target for genotyping and elucidation of phylogenetic relationships among E. canis strains.

Quercetin manipulates the expression of genes involved in the reactive oxygen species (ROS) process in chicken heterophils.

Chicken heterophils generate reactive oxygen species (ROS) molecules to defend against invading pathogens. The present study examined effects of quercetin on chicken heterophils. Heterophils were stimulated with PBS, 50 µM quercetin (QH), PMA or Escherichia coli (EC) and the resulting intracellular ROS molecules were determined. Flow cytometry results showed that cells stimulated with QH, PMA and EC had a higher ROS production. Increases in intracellular ROS molecules were identified in all treatment groups by fluorescence microscopy. Determination of the ability of quercetin to manipulate mRNA expression of ROS subunits was assessed using real-time RT-PCR. Quercetin and other stimulants up-regulated the majority of genes involved in ROS production: CYBB (NOX2), NCF1 (p47phox), NCF2 (p67phox), NOX1 and RAC2. The antioxidant property of QH was explored by measuring mRNA expression of CAT and SOD1. The data indicate increased levels of CAT with all treatments; however, only QH attenuated the expression of the SOD1 gene. To further investigate the effects of ROS-driven inflammation or cell death, IL6, CASP8 and MCL1 genes were preferentially tested. The inflammatory gene (IL6) was profoundly down-regulated in the QH- and PMA-treated groups while EC induced a strikingly high IL6 expression level. Investigation of the known apoptotic (CASP8) and anti-apoptotic (MCL1) genes found down-regulation of CASP8 in the QH- and PMA-treated groups which were contradicted to the MCL1 gene. In conclusion, quercetin can enhance ROS production by regulating the expression of genes involved in ROS production as well as in subsequent processes.

Low levels of genetic diversity associated with evidence of negative selection on the Babesia bovis apical membrane antigen 1 from parasite populations in Thailand.

Babesia bovis, a parasite infecting cattle and buffalo, continues to spread throughout the developing world. The babesial vaccine was developed to be a sustainable alternative treatment to control the parasite. However, genetic diversity is a major obstacle for designing and developing a safe and effective vaccine. The apical membrane antigen 1 (AMA-1) is considered to be a potential vaccine candidate antigen among immunogenic genes of B. bovis. To gain a more comprehensive understanding of B. bovis AMA-1 (BbAMA-1), three B. bovis DNA samples were randomly selected to characterize in order to explore genetic diversity and natural selection and to predict the antigen epitopes. The sequence analysis revealed that BbAMA-1 has a low level of polymorphism and is highly conserved (95.46-99.94%) among Thai and global isolates. The majority of the polymorphic sites were observed in domains I and III. Conversely, domain II contained no polymorphic sites. We report the first evidence of strong negative or purifying selection across the full length of the gene, especially in domain I, by demonstrating a significant excess of the average number of synonymous (dS) over the non-synonymous (dN) substitutions. Finally, we also predict the linear and conformational B-cell epitope. The predicted B-cell epitopes appeared to be involved with the amino acid changes. Collectively, the results suggest that the conserved BbAMA-1 may be used to detect regional differences in the B. bovis parasite. Importantly, the limitation of BbAMA-1 diversity under strong negative selection indicates strong functional constraints on this gene. Thus, the gene could be a valuable target vaccine candidate antigen.