University of Miyazaki

Calf diarrhea is a multifactorial disease that poses substantial challenges to the cattle industry, necessitating timely identification and characterization of causative agents to inform effective control strategies. This study investigated the prevalence, co-occurrence, and characteristics of Cryptosporidium and Eimeria infections in diarrheic calves. A total of 348 diarrheal fecal samples were collected from calves aged 1-30 days across 15 prefectures in Japan between February 2020 and February 2022. Preliminary diagnosis of calf diarrhea was performed using an immunochromatographic test (ICT) kit. Cryptosporidium spp. were detected in 36.5% (127/348) of samples. Using nested PCR and conventional PCR with species-specific primers, targeting these two major parasitic pathogens, we assessed seasonal variations, prevalence, and associations with calf age, breed, and sex. Overall, Cryptosporidium was detected in 58.6% of samples, while Eimeria spp. were found in 7.5%. Mixed infections involving both pathogens occurred in 4.9% of samples. The highest detection rates for both pathogens were observed in calves aged 8-15 days, with a significant age-related decline in mixed infections (p < 0.05). Seasonal variations were observed, with distinct prevalence patterns for each pathogen. Breed- and sex-related differences were minimal but are described in detail. Phylogenetic analysis confirmed C. parvum as the predominant Cryptosporidium species and E. bovis as the most common Eimeria species. These findings highlight the widespread presence of Cryptosporidium and Eimeria spp. in diarrheic calves in Japan and provide insights into age- and season-related infection dynamics, informing targeted control and management strategies.

Interleukin-1β is a pro-inflammatory cytokine with pluripotent roles in mediating immune response. In fish, IL-1β plays a major role in inflammatory responses to pathogenic infections, though its immunological functions through direct stimulation have not been studied in detail. In this study, two IL-1β genes (il1b1 and il1b2) from the Japanese medaka (Oryzias latipes) were identified and characterized. The broad immunostimulatory effects of recombinant IL-1β (OlrIL-1β) from Japanese medaka were explored using embryonic fibroblast-like cell line (OLHdrR-e3) and primary cultured kidney cells. Recombinant proteins were produced via prokaryotic expression based on predicted mature isoforms IL-1β1_Asp⁹⁵ (type II) and IL-1β2_Asp¹²¹ (type I) to investigate their distinct functional characteristics. Then, their immune-related gene expression, phagocytosis, and reactive oxygen species (ROS) production ability were evaluated in vitro and ex vivo. rIL-1β triggers the expression of not only pro-inflammatory cytokines such as il1b, tnfa, il6, il8, mcp1b, and csf1b, but also the antimicrobial peptide, def1b. Additionally, they upregulate the expression of cell specific markers, including macrophage- and granulocyte-specific genes mpeg1.1, mpx2, and epx, and nitric oxide synthase (NO) gene nos1. Moreover, medaka OlrIL-1β proteins exhibited robust phagocytosis and superoxide anion production ability than those in unstimulated control. In all evaluations, the OlrIL-1β proteins corresponding to the predicted cleavage sites exhibited strong immune activation, with type I OlIL-1β2_Asp¹²¹ demonstrating more potent response than type II. To our knowledge, this is the study to reveal the immunomodulatory functions of Japanese medaka IL-1β, highlighting its potential in immunotherapy for disease prevention and control in aquaculture.

The development of antibody-based fluorescent sensors relying on tryptophan-mediated quenching, such as Quenchbody (Q-body), often exhibits limited fluorescence responses because dye quenching depends on the location of tryptophan residues within the antibody. Here, we developed a competitive fluorescence immunoassay, termed an intermolecular Quenchbody (iQ-body), that utilizes intermolecular Förster resonance energy transfer (FRET) between an N-terminally fluorescent-labeled IgG antibody and an antigen-quencher conjugate. Anti-thyroxine IgG antibody (clone 6901 SPTN-5) showed minimal fluorescence changes upon antigen binding, despite N-terminal labeling with TAMRA. In contrast, the addition of an antigen-quencher conjugate (QSY9-X-T3) effectively quenched the TAMRA fluorescence via intermolecular FRET. Subsequent competitive displacement by thyroxine (T4) resulted in concentration-dependent fluorescence recovery. The iQ-body strategy provides a simple approach for constructing competitive fluorescence immunoassays for small-molecule targets using publicly available IgG antibodies.