Potential Development Ability of Residual Zoites, a Second-Generation Meront, Inducing Long-Term Infection by the Mouse Eimerian Parasite, Eimeria krijgsmanni

Acta Parasitologica https://doi.org/10.1007/s11686-024-00910-2 ORIGINAL PAPER Potential Development Ability of Residual Zoites, a Second‑Generation Meront, Inducing Long‑Term Infection by the Mouse Eimerian Parasite, Eimeria krijgsmanni Masanobu Mizuno1 · Satoru Kiyotake1 · Makoto Matsubayashi2 · Takane Kaneko3 · Hitoshi Hatai4 · Yoshikazu Fujimoto5 · Moe Ijiri5 · Hiroaki Kawaguchi6 · Toshihiro Matsui7 · Tomohide Matsuo1 Received: 11 December 2023 / Accepted: 13 August 2024 © The Author(s) 2024 Abstract Purpose Coccidiosis caused by eimerian parasites results in lethal watery or bloody diarrhea in hosts, and markedly impairs the growth of and feed utilization by host animals. We previously investigated detailed the life cycle of Eimeria krijgsmanni as a mouse eimerian parasite. Only second-generation meronts, as an asexual stage, were morphologically detected in the epithelium of the host cecum for at least 8 weeks after infection, even though oocyst shedding finished approximately 3 weeks after infection. The presence of zoites was of interest because infection by eimerian parasites is considered to be self-limited after their patent period. Methods To clarify the significance of residual second-generation meronts in E. krijgsmanni infection, we performed infection experiments using immunocompetent mice under artificial immunosuppression and congenital immunodeficient mice. Results The number of oocysts discharged and the duration of oocyst discharge both increased in immunosuppressed mice. In immunodeficient mice, numerous oocysts were shed over a markedly longer period, and oocyst discharge did not finish until 56 days after inoculation. Conclusions The present results suggest that the second-generation meronts survived in the epithelial cells of the cecum after the patent period, thereby contributing to extended infection as an asexual stage. The results obtained on E. krijgsmanni indicate that infections by Eimeria spp. are not self-limited and potentially continue for a long period of time. Keywords Eimeria · Mouse · Immunosuppression · Immunodeficiency · Latent infection * Tomohide Matsuo 1 Laboratory of Parasitology, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890‑0065, Japan 2 Laboratory of Veterinary Immunology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano 598‑5831, Osaka, Japan 3 Department of Life Sciences, Faculty of Life Sciences, Kyushu Sangyo University, Fukuoka 813‑8503, Japan 4 Farm Animal Clinical Skills and Diseases Control Center, Iwate University, Morioka 020‑8550, Iwate, Japan 5 Transboundary Animal Diseases Research Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890‑0065, Japan 6 Laboratory of Veterinary Pathology, School of Veterinary Medicine, Kitasato University, Towada 034‑8628, Aomori, Japan 7 Seisen University, Higashi Gotanda, Tokyo 141‑8642, Japan Introduction Eimeria spp., belonging to the phylum Apicomplexa, are the intracellular protozoan parasites, that are found in most classes of vertebrates worldwide [1] and exhibit high host specificity. Coccidiosis caused by eimerian parasites results in lethal watery or bloody diarrhea in hosts, and markedly impairs the growth of and feed utilization by host animals. Although drugs and vaccines are available for some species [2, 3], a number of limitations, such as safety issues, troublesome vaccine strategies, and the emergence of drug resistant strains have a negative impact on their effectiveness [4, 5]. Therefore, further research is needed to develop new methods for the prevention and/or treatment of infection by Eimeria spp. However, experiments on eimerian parasites may be technically challenging. Well-established methods to replicate life cycles using in vitro cultures, such as those for Toxoplasma gondii and Babesia spp., are not yet available Vol.:(0123456789) Acta Parasitologica for Eimeria spp. [6], and infection experiments using large animals, including cattle, are also challenging. Therefore, we hypothesized that infection experiments using mice may be useful to efficiently study on eimerian parasites. Fourteen species of murine Eimeria have been described to date [7, 8], and a number of studies have been published on Eimeria falciformis, E. papillata, and E. vermiformis. However, murine Eimeria infection remain unclear. We previously reported our findings on the host specificity [9], life cycle [10], and drug susceptibility [11, 12] of E. krijgsmanni as a mouse eimerian parasite. The life cycle of Eimeria spp. consists of asexual and sexual developmental stages and is self-limited, which means that parasites disappear and the host completely recovers from infection after the patent period finishes. In E. krijgsmanni, asexual stages followed by sexual stages involve four generations, and oocyst shedding finishes approximately 3 weeks after infection [10]. Furthermore, the host acquires resistance to subsequent infection at least 3 weeks after the primary infection [11]. We previously showed that only second-generation meronts (meront II), an asexual stage in the host, were morphologically detected in the epithelium of the cecum at least until 8 weeks after infection [10]. This was the first study to show the presence of special zoites detected for a long time after the patent period of infection by Eimeria spp. The present study investigated the significance of meront II in infection by E. krijgsmanni because our previous findings were based solely on morphological observations and, thus, it was unclear whether residual meront II were alive. We herein conducted infection experiments using immunocompetent mice under artificial immunosuppression. Additionally, congenital immunodepression model mice, namely, severe combined immunodeficiency (SCID) mice and nude mice, were used in infection experiments to investigate E. krijgsmanni infection in the absence of host immune responses. Materials and Methods Mice Five-week-old female immunocompetent (BALB/c) and immunodeficient (SCID and nude) mice (CREA Japan, Tokyo) were used in each experiment. All mice were confirmed by a fecal examination to be free of natural infection by Eimeria spp., and were raised under coccidian-free conditions until used in experiments. Parasites E. krijgsmanni was obtained from the Division of Tropical Diseases and Parasitology, Department of Infectious Diseases, Kyorin University School of Medicine, and maintained by routine passage through ICR mice (CLEA Japan, Tokyo) at the Laboratory of Parasitology, Joint Faculty of Veterinary Medicine, Kagoshima University. Oocysts were collected from the feces of infected mice and stored at 4 °C until used after sporulation in 2% potassium dichromate solution at 25 °C for several days. Infection and Assessment of Infection All mice were orally inoculated with 2.0 × 102 oocysts. The discharge of oocysts in the feces was examined using the sugar flotation (...truncated)