An Improved Isolation Technique for Bovine<i>Neospora</i>Species

Yamane, Ichirō; Shibahara, Tomoyuki; Kokuho, Takehiro; Shimura, Kameo; Hamaoka, Toshiyuki; Haritani, Makoto; Conrad, Patricia A.; Park, Chon Ho; Sawada, Masumi; Umemura, Takashi

doi:10.1177/104063879801000411

Cited by 21 publications

(19 citation statements)

References 13 publications

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“…Many tachyzoites were found and principal pathogenic changes were identified in the uterus and pancreas at an early stage of infection. This finding was similar to those in previous studies in which tachyzoites were identified in the placenta when 2 ϫ 10 6 tachyzoites were inoculated subcutaneously at late midgestation 13 and in the pancreas when tachyzoites were inoculated into immunosuppressed 8 and immunodeficient 24 mice. In other studies, many tachyzoites and/or lesions were observed in the central nervous system, skeletal muscles, liver, lung, and peripheral nerves.…”

Section: Ifn-␥ Il-4 and Il-6 Production In Jpa1-infected Micesupporting

confidence: 91%

“…Athymic nude mice are also expected to be useful for isolating and maintaining Neospora, given their high susceptibility to Neospora infections. 24 Congenic wild type mice inoculated with Neospora did not exhibit any clinical signs. This result agrees with those of some previous reports 6,8,9 and disagrees with those of one study, in which seven of 12 congenic wild type mice inoculated with 2 ϫ 10 5 tachyzoites of Neospora (NC-1 strain) died 26-70 days after infection.…”

Section: Ifn-␥ Il-4 and Il-6 Production In Jpa1-infected Micementioning

confidence: 95%

“…Athymic nude mice were also useful for isolating the parasite from bovine tissues infected with Neospora. 19,24 Furthermore, interferon (IFN)-␥ knockout mice 14 and mice treated with antibody against IFN-␥ 8 were highly susceptible to Neospora infection and developed severe clinical and pathologic changes. Therefore, cytokines may suppress the propagation of Neospora tachyzoites and may be related to susceptibility to infection.…”

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Pathological and Immunological Findings of Athymic Nude and Congenic Wild Type BALB/c Mice Experimentally Infected withNeospora caninum

et al. 1999

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Abstract. Neospora is a cyst-forming coccidian parasite that causes abortions and neuromuscular disorders in a wide variety of mammals. Japanese bovine isolate JPA1 was inoculated intraperitoneally into BALB/c nu/ nu (athymic nude) and BALB/c (congenic wild type) female mice to examine the distribution of parasites and resistance mechanisms to Neospora infection. All the athymic nude mice died within 28 days after intraperitoneal injection of 2 ϫ 10 5 JPA1 tachyzoites, whereas all the congenic wild type mice survived without exhibiting any clinical signs. Tachyzoites were identified in the uterus and pancreas and later spread to many other organs. Most tachyzoites identified in the necrotic foci were localized in the epithelium of the venules and capillaries. Nude mice developed high level of serum interferon-␥ and interleukin-6 as infection proceeded. Inflammatory response to Neospora infection might be mediated by Th1-type dependent cellular immunity.

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Section: Ifn-␥ Il-4 and Il-6 Production In Jpa1-infected Micesupporting

confidence: 91%

Section: Ifn-␥ Il-4 and Il-6 Production In Jpa1-infected Micementioning

confidence: 95%

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confidence: 99%

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Pathological and Immunological Findings of Athymic Nude and Congenic Wild Type BALB/c Mice Experimentally Infected withNeospora caninum

et al. 1999

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“…Purification of tachyzoites: The tachyzoites cultured were purified according to an improved method of Yamane et al [25]. Briefly, Vero cells infected with the tachyzoites were disrupted and mixed with Dulbecco's phosphate buffered saline (PBS -; NISSUI PHARMACEUTICAL Co.) containing 30% isotonic percoll (Amersham Pharmacia Biotech, England), and then PBS -containing 50% and 80% isotonic percoll respectively, were injected under the layer and centrifuged at 1,250 × g for 30 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Monoclonal Antibodies Preventing Invasion of Neospora caninum Tachyzoites into Host Cells

Uchida

Ike

Kurotaki

et al. 2004

The Journal of Veterinary Medical Science

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ABSTRACT. Twelve monoclonal antibodies (MAbs) against Neospora caninum tachyzoites were produced to specify the antigens related to the invasion of tachyzoites into host cells. In the assay to evaluate the inhibition activity, all these MAbs prevented the cultured Vero cells from the invading by the tachyzoites. These MAbs recognized approximately a 73 kDa antigen in Western blot analysis. Immunofluorescence assay and immune electron microscopy revealed that this 73 kDa antigen is a part of the surface antigens of N. caninum tachyzoite, and that the tachyzoite antigen identified plays an important role for invasion of host cells. KEY WORDS: invasion process, monoclonal antibody, Neospora caninum, surface antigen.J. Vet. Med. Sci. 66(11): 1355-1358, 2004 Neospora caninum is a coccidian protozoon first isolated from the brain of a puppy by Dubey in 1988, which developed paralysis in the hind limbs [5]. Dogs infected with this parasite mainly show neurological symptoms and infected cows are attached with abortion and stillborn, so serious economical damages are produced [4]. In 1998, McAllister et al. reported that definitive host of the protozoon is dogs [14]. However, the life cycle of N. caninum is not completely clarified yet, and the procedures for prevention and treatment of neosporosis also are not established. The pathogenesis of N. caninum is known to be caused by multiplication of parasites resulting destruction of many kinds of host cells by invasion [2,3,20]. To develop the prevention and treatment of neosporosis, analyses of antigens related with the invasion process into host cells are required. Antigens of some protozoan parasites belonging to phylum Apicomplexa, such as Toxoplasma gondii participate in invading the host cells and were thought to be surface antigens, secretory proteins and those in the structures of apical complex such as microneme, rhoptry and dense granule [6,11,15,18]. Also in N. caninum, some antigens, possibly affecting the invasion, were studied, and the antigens on the body surface [9,10] In this study, we prepared monoclonal antibodies (MAbs) against N. caninum tachyzoites which obstruct the invasion of tachyzoites into host cells, and further, we tried to specify the antigen of N. caninum concerning with attachment and invasion into the host cells. MATERIALS AND METHODSParasites: Neospora caninum JPA-1 strain used was isolated and passaged by Yamane et al. in the National Institute of Animal Health, Japan [23]. The isolate was inoculated into monolayer Vero cells maintained in Eagle's minimum essential medium (MEM; NISSUI PHARMACEUTICAL Co., Japan) supplemented with 10% fetal calf serum (FCS; HARLAN SERA-LAB, England), 2 mM L-glutamine (Wako Pure Chemical Industries, Japan), 1% MEM Vitamin solution (GIBCO BRL, U.S.A.), 13 mM sodium hydrogen carbonate (NaHCO 3 ) (Wako Pure Chemical Industries), and 4 µl/ml Fungizone (GIBCO BRL). The tachyzoites cultured were used for examination. Purification of tachyzoites:The tachyzoites cultured were purified according to an improved metho...

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“…The parasites grown in Vero cells were purified using density gradient methods as previously reported [27]. Brain cells in Since the first in vitro isolation of Neospora caninum from dogs in 1988 [8], tachyzoites of N. caninum have been cultured in vitro in bovine macrophages, bovine cardiopulmonary arterial endothelial cells [7], monkey kidney cells and many other cell lines as well as foetal mouse brain tissue [9].…”

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The inhibitory Effect of Interferon Gamma and Tumor Necrosis Factor Alpha on Intracellular Multiplication of Neospora caninum in Primary Bovine Brain Cells.

Yamane

Kitani

Kokuho

et al. 2000

The Journal of Veterinary Medical Science

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ABSTRACT. Primary culture of bovine brain cells was examined for its susceptibility to Neospora caninum infections, and this model was used to investigate the effects of bovine interferon gamma (IFN-γ) and tumor necrosis factors alpha (TNF-α) on tachyzoite growth. Tachyzoites of N. caninum grew well in this culture, and tachyzoite growth in astroglia and microglia were confirmed by immunocytochemical staining. IFN-γ inhibited the tachyzoite growth, and this inhibition was not reversed by the addition of nitric oxide antagonist. TNF-α, to a lesser extent, also inhibited the tachyzoite growth. Th-1 type cytokines may play an important role in host defense mechanisms in N. caninum infection.-KEY WORDS: brain culture, cytokine, Neospora caninum.

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An Improved Isolation Technique for BovineNeosporaSpecies

Cited by 21 publications

References 13 publications

Pathological and Immunological Findings of Athymic Nude and Congenic Wild Type BALB/c Mice Experimentally Infected withNeospora caninum

Pathological and Immunological Findings of Athymic Nude and Congenic Wild Type BALB/c Mice Experimentally Infected withNeospora caninum

Monoclonal Antibodies Preventing Invasion of Neospora caninum Tachyzoites into Host Cells

The inhibitory Effect of Interferon Gamma and Tumor Necrosis Factor Alpha on Intracellular Multiplication of Neospora caninum in Primary Bovine Brain Cells.

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