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.

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.

CRISPR/Cas9 Editing of Duck Enteritis Virus Genome for the Construction of a Recombinant Vaccine Vector Expressing ompH Gene of Pasteurella multocida in Two Novel Insertion Sites.

Duck enteritis virus (DEV) and Pasteurella multocida, the causative agent of duck plague and fowl cholera, are acute contagious diseases and leading causes of morbidity and mortality in duck. The NHEJ-CRISPR/Cas9-mediated gene editing strategy, accompanied with the Cre-Lox system, have been employed in the present study to show that two new sites at UL55-LORF11 and UL44-44.5 loci in the genome of the attenuated Jansen strain of DEV can be used for the stable expression of the outer membrane protein H (ompH) gene of P. multocida that could be used as a bivalent vaccine candidate with the potential of protecting ducks simultaneously against major viral and bacterial pathogens. The two recombinant viruses, DEV-OmpH-V5-UL55-LORF11 and DEV-OmpH-V5-UL44-44.5, with the insertion of ompH-V5 gene at the UL55-LORF11 and UL44-44.5 loci respectively, showed similar growth kinetics and plaque size, compared to the wildtype virus, confirming that the insertion of the foreign gene into these did not have any detrimental effects on DEV. This is the first time the CRISPR/Cas9 system has been applied to insert a highly immunogenic gene from bacteria into the DEV genome rapidly and efficiently. This approach offers an efficient way to introduce other antigens into the DEV genome for multivalent vector.

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.

Construction and characterization of an OmpH-deficient mutant of Pasteurella multocida strain X-73.

A capsule-defective mutant strain PBA129 of Pasteurella multocida was constructed by electroporation of phagemid containing the coding region of the antisense RNA of the ompH gene into the wild type strain X-73 (serovar A:1) of P. multocida. The pathogenicity and protective potency of the mutant against homologous and heterologous challenge in mice and chickens were characterized. Greyish colonies of the mutant, indicating lower capsule thickness, on selective dextrose starch agar were observed under an obliquely transmitted light stereomicroscope and compared to iridescent colonies of the wild type strain X-73. Strain PBA129 had lower capsule thickness than the wild type strain as observed with an electron microscope. Strain PBA129 was apparently attenuated, as mice and chickens inoculated with the bacteria at 108 CFU survived. Protection was observed in both mice and chickens inoculated with strain PBA129 upon challenge exposure to avian P. multocida strains X-73 and P-1059 (serovar A:3), respectively. In conclusion, the mutant strain PBA129 of P. multocida strain X-73 was completely attenuated, and it was possible to induce sufficient protection against avian P. multocida strains.