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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.

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.

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 10 8 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.

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.

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.