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www.researchgate.net/profile/Yoshifumi-Nishikawa 

researchmap: 

https://researchmap.jp/read0136808

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Masters and doctoral course in Obihiro University

Laboratory Topics

 A protozoan is an infectious pathogen that, like a cancer cell, is composed of a single eukaryotic cell and multiplies indefinitely in the animal body. Our laboratory conducts basic research on the following pathogenic protozoa.

 Plasmodium spp., which is important in the medical field, affects 300 to 500 million people worldwide and kills as many as 2 million people annually. Toxoplasma gondii infects 30% of the world's population, causing miscarriages and congenital toxoplasmosis in newborns. In the livestock industry, Neospora caninum causes abortions in cattle and Cryptosporidium parvum causes diarrhea in calves, which reduces livestock productivity and causes losses to the agricultural economy. In addition, meat derived from slaughter animals infected with Toxoplasma and cattle feces containing Cryptosporidium can be a source of infection to humans.

Microscopic observation of Toxoplasma

https://www.facebook.com/yoshifumi.nishikawa/videos/1506841893126589

https://www.facebook.com/yoshifumi.nishikawa/videos/680832896795853

Infection dynamics of intracellular protozoan parasites 

 Intracellular protozoan parasites are able to infect and multiply by skillfully exploiting host cells. These parasites actively invade host cells and form parasitophorous vacuole (PV) to escape the host's immune system and to obtain nutrients from host cells.

 The presence of numerous secreted proteins such as MIC, ROP, and GRA has been studied to date to determine the factors on the parasite side involved in adhesion to and invasion of host cells. Parasite-infected host cells become resistant to host stress responses by skillfully altering host signaling pathways in order to divide and multiply in the host cell by parasite-derived molecules. In addition, we believe that a mechanism exists to force the death of immune-responsive cells that are harmful to the parasites.

 We are searching for novel parasite-derived factors to unravel the mystery of this survival strategy of parasites.

Elucidation of the pathogenesis of toxoplasmosis

 Toxoplasma is an intracellular parasitic protozoan that causes fatal pneumonia and encephalitis in immunocompromised patients and can cause miscarriage and birth defects in pregnant women. This protozoan parasite infects feline animals as its definitive host and nearly all mammals and birds, including humans, as intermediate hosts. Toxoplasma forms cysts in the nerve and muscle cells of the host, resulting in a persistent latent infection. Although usually considered harmless to humans with healthy immune systems, chronic infection has been shown to affect animal behavior and has been reported to increase the risk of psychiatric disorders, self-harm, and traffic accidents in humans.

In order to elucidate the pathogenesis of toxoplasmosis, our laboratory has accumulated basic data by creating pathological models of encephalitis, eye damage, and congenital infection. One of the interesting findings from this process is that Toxoplasma causes behavioral changes in mice.

　Changes in host behavior due to Toxoplasma infection have been known for some time, and it would be interesting to speculate that these behavioral changes activate the Toxoplasma life cycle. Experimental models of maze learning have shown that Toxoplasma-infected mice have poorer learning ability and memory. In addition, infected mice are less alert to the unknown and insensitive to the smell of cats. These behavioral changes in infected mice and rats are fatal to their escape from cats, which are their complement, but from the protozoan side, they allow them to reach their definitive hosts more efficiently. Studies of behavioral changes in humans caused by Toxoplasma infection also have a long history. There are reports that people infected with Toxoplasma have been implicated in schizophrenia, personality changes, and increased risk of car accidents. Although there are some differences between men and women, they are more prone to psychomotor disturbances and anxiety, and may exhibit hallucinations, cognitive impairment, and other symptoms. Although certain observations have been made regarding the mechanisms involved in Toxoplasma infection-induced behavioral changes in host animals, many of these mechanisms remain a mystery.

 Our research focuses on the mechanism of host manipulation by protozoan infection and its relationship to brain pathology. So far, we have detected parasites in a wide range of regions (cortex, striatum, hippocampus, amygdala, and thalamus) in the brains of infected mice, and necrotic lesions were observed especially in the cortex. In addition, large-scale gene expression changes occurred in the infected brain, suggesting abnormal activation of the neuroimmune system and neuronal dysfunction. These findings suggest that the pathogenesis of Toxoplasma encephalopathy is triggered by an interaction between pathogenic factors on the part of the protozoan parasite and neuroimmunity. Similarly, it is speculated that pathogenic factors on the part of the protozoan may be involved in eye damage and adverse effects of infection during pregnancy on mother and child, and we are working to elucidate the pathogenesis of toxoplasmosis.

Ref：https://www.extended-phenotype.org

Ref: h

ttp://www.ted.com/talks/ed_yong_suicidal_wasps_zombie_roaches_and_other_tales_of_parasites?language=ja#t-43450

Analysis of Toxoplasma isolates from around the world to find virulence factors 

 Toxoplasma differs in genetic characteristics from region to region, and its genetic polymorphisms have been postulated to play a role in pathogenicity. Unique genetic exchanges across continents between Toxoplasma strains from mainland Japan and strains from China (Eurasia) and North America have been identified, as well as genetic links between Toxoplasma endemic to Okinawa and Latin American-type strains. Toxoplasma found especially in Central and South America has been reported to cause visual impairment and pneumonia even in humans with normal immune function, suggesting the existence of a highly virulent strain, but its etiology has not yet been elucidated. Therefore, we believe that comparative analysis of the highly pathogenic South American, Japanese, Chinese, Southeast Asian, and North American strains in terms of genome and virulence will lead to the discovery of a parasite virulence factor associated with the Japanese strain.

Elucidation of the pathogenic mechanism of neosporosis 

 Neospora caninum is an intracellular protozoan parasite whose definitive host is a canine animal and intermediate hosts are cattle, sheep, goats, and deer. After infection of the host cell, the parasite releases various proteins from its secretory organelle to form parasitophorous vacuole (PV) and establish parasitism. In nature, infection is established orally, and the parasite spreads from the digestive system to the rest of the body, leading to latent infection in the central nervous system and muscle tissues. This infection is transmitted by horizontal infection through oocysts shed in the feces of the definitive host and vertical infection in the intermediate host. In cattle, Neospora causes a high rate of abortions, stillbirths, and abnormal births characterized by neurological symptoms in calves. Vertical transmission of Neospora can take place over many generations, and this is the main cause of the spread of the parasite and abortions, thus posing a barrier to the development of a vaccine. Our research aims to elucidate the mechanism of vertical transmission of Neospora. We hypothesize that vertical transmission is established by hijacking the environment of the gestational uterus and placenta by parasite transmission factors.

Vaccine development for protozoan diseases 

 Much remains to be done in the development of vaccines against protozoan infections, with the exception of a few, such as malaria. Many pathogenic protozoa skillfully evade host immunity, making it difficult to develop effective vaccines. Recent studies have shown that Plasmodium parasitizes within erythrocytes and that host defense mechanisms are triggered by immune complex formation and phagocytosis by macrophages in the spleen, while Toxoplasma and Neospora parasitize within various cells, including macrophages, and that Th1 immunity, including T cell-dependent activation of macrophages along with Th2 immunity It has been shown that Th1 immunity is an important elimination effector. Therefore, the next important research issue to be faced is “how to induce strong Th1 immunity specific to parasites”. In other words, the technological seeds that can artificially induce Th1 immunity are the key to successful development of a protozoan vaccine. It is also a challenging research subject because there is no effective vaccine that can prevent diarrhea in calves caused by Cryptosporidium. We are developing vaccines against these protozoan diseases by fabricating lipid nanoparticles and multifunctional liposomes encapsulating vaccine antigens. 

Drug discovery research against protozoan diseases 

 Since livestock protozoan diseases cause significant economic losses to the global livestock industry, there has always been a need to establish urgent countermeasures against these diseases. The emergence of insecticide-resistant species and contamination of the environment and food supply have become problems, making it difficult to combat protozoan diseases and vectors. Based on the results of our fundamental research “Understanding parasite infections and diseases,” we are academically developing an evaluation system consisting of an in vitro drug screening system, a vector model animal infection evaluation system, and a micro/macro disease analysis model evaluation system, and are also conducting disease state evaluation in natural hosts in collaboration with international partner institutions. The center has also established a “drug discovery research center for livestock protozoan diseases” with a view to social implementation by conducting pathogenetic evaluations on natural hosts in collaboration with international partner institutions. In other words, the center is promoting basic research to develop complete therapeutics against protozoan diseases by finding compounds that specifically suppress the infection pathology that will be the actual therapeutic target.

Development of a socially implementable diagnostic system for livestock protozoan diseases 

We are developing a diagnostic system that can be adapted to diagnostic and epidemiological studies in the field. We are working with farms, clinical veterinarians, and livestock health centers to provide a socially-implementable diagnostic system to the field to understand the status of Neospora, Toxoplasma, and Cryptosporidium contamination in Japan and around the world, and to propose countermeasures.