Neospora caninum: In vitro culture of tachyzoites in MCF-7 human breast carcinoma cells

doi:10.1016/j.exppara.2010.06.006

Experimental Parasitology

Volume 126, Issue 4, December 2010, Pages 536-539

https://doi.org/10.1016/j.exppara.2010.06.006 Get rights and content

Abstract

The development of Neospora caninum tachyzoites, an apicomplexan protozoan parasite, was studied in vitro using the human breast carcinoma cell 7 (MCF-7) as the host cell line. The extracellular NC-1 tachyzoites in MCF-7 cells were observed and counted daily for 6 consecutive days post-infection to establish the growth curve. The intracellular parasites were observed by acridine orange staining using Laser scanning confocal microscope. The results indicated that NC-1 tachyzoites invaded MCF-7 cells and multiplied intracellularly. The number of extracellular NC-1 tachyzoites started to increase rapidly around day 3 and reached the maximum number around day 4. Results from the present study suggested that MCF-7 cells were susceptible to NC-1 tachyzoites and could be used as an alternative cell line for in vitro studies.

Introduction

Neospora caninum, an apicomplexan protozoan parasite that closely related to Toxoplasma gondii, is of significant economic importance as this parasite can cause neurological diseases and abortions in numerous animals. It is also an important cause of reproductive diseases in cattle worldwide (Dubey, 2003). N. caninum was first described and named by Dubey et al. (1988) and now has been reported in various species of livestock, including cattle, sheep, goat, horse, and deer as reviewed by Dubey and Lindsay (1996).

The production of antigenic materials and the study of the relationship between host cells and the parasites require in vitro cultivation of the protozoan. In vitro cultures are also used to screen potential chemotherapeutic agents (Hemphill et al., 1996, Lindsay et al., 1996, Strohbusch et al., 2008, Leepin et al., 2008) or to perform genetic manipulation on the parasite (Howe et al., 1997, Radke et al., 2000).

Up to now, N. caninum has been successfully cultured in different host cells, such as Vero cell line (Angela, 2002, François et al., 2002, Kang et al., 2008, Kim et al., 2000, Naguleswaran et al., 2002, Okeoma et al., 2004, Vonlaufen et al., 2004), human colon cancer caco-2 cells (Omata et al., 2005).

In the present study, human breast carcinoma cell line (MCF-7) was tested as the host cell line for NC-1 tachyzoites culture in vitro. The results showed that this human derived tumor cell line was susceptible to NC-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

Section snippets

Cells culture

MCF-7 and Vero cells were plated in 6-well cell culture plates (Corning Incorporated, USA) with glass cover slips at a 1 × 10⁵/ml in RPMI-1640 (Invitrogen Co., GIBCO™, Grand Island, NY, USA) medium with 10% fetal bovine serum (FBS) (Haoyang biological manufacture Co., Ltd., Tianjin, China), 4 mM glutamine, 2.30 mg/ml NaHCO₃, 2.38 mg/ml HEPES (pH 7.2), 50 U/ml penicillin, and 50 mg/ml streptomycin at 37 °C with 5% CO₂.

Purification of N. caninum tachyzoites

The tachyzoites of NC-1 isolate (Dubey et al., 1988) were maintained in Vero cells as

Results and discussion

In the present study, the dynamic growth and proliferation process of NC-1 tachyzoites in MCF-7 cells in vitro was studied from days 1 to 6. NC-1 tachyzoites invaded MCF-7 cells and grew well. It took about 4 days for the parasites to reach the growth plateau under the current culture conditions.

The life cycle of N. caninum was typified by three infectious stages: tachyzoites, tissue cysts, and oocysts. Tachyzoites and tissue cysts were the two stages found in the intermediate hosts and both

Acknowledgments

The authors especially thank Dr. Wenbin Tuo (Animal and Natural Resources Institute, United States Department of Agriculture) for providing the N. caninum (NC-1) tachyzoite. This work was supported by Major Program of Preventive and Control for National Sever Infective Diseases (No. 2008ZX10004-001).

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Neospora caninum, an obligate intracellular parasitic protozoan discovered by Dubey in 1988, is the pathogen of neosporosis, which causes neurological symptoms in dogs and abortions in cows. Since there is no effective drug or vaccine against N. caninum, a deeper understanding of the molecules critical to parasite survival inside host cells is necessary. This study aimed to determine the role of N. caninum peroxiredoxin 1 (NcPrx1) in maintaining redox homeostasis and virulence of N. caninum. By determining the localization of NcPrx1 protein and establishing NcPrx1 gene knockout strain (ΔNcPrx1), the roles of NcPrx1 in N. caninum for invasion, replication, growth, oxidative stress, as well as pathogenicity were investigated. Our results showed that a predicted Alkyl Hydroperoxide1 (AHP1) domain was found in the amino acid sequence of NcPrx1, which displayed a high degree of similarity to homologs of several protozoa. Immunofluorescence assay (IFA) indicated that NcPrx1 was a cytoplasmic protein in N. caninum tachyzoites. Compared to wild type (WT) strain, ΔNcPrx1 strain showed reduced plaque area, invasion and egress rates. Reactive oxygen species (ROS) and malondialdehyde (MDA) were accumulated, and total antioxidant capacity (T-AOC) was attenuated in ΔNcPrx1 tachyzoites, which indicated that ΔNcPrx1 strain was more sensitive to oxidative stress. Furthermore, ΔNcPrx1 strain-infected C57BL/6 mice showed improved survival rate, reduced parasite burden, alleviated pathological changes in tissues, and decreased secretions of IL-6, IL-12, TNF-α, and IFN-γ in serum compared to the WT strain group. These findings suggested that NcPrx1 was a virulence factor of N. caninum which played an important role in maintaining the redox homeostasis of the parasite.
In vitro cultivation methods for coccidian parasite research
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Citation Excerpt :
Abstract No. 34.). More recent studies also showed a successful infection of human breast carcinoma cell 7 (MCF-7) (Lv et al., 2010) and CaCo2 cells, in which an anti-N. caninum antibody also inhibited parasite growth in vitro (Omata et al., 2005).
The subclass Coccidia comprises a large group of protozoan parasites, including important pathogens of humans and animals such as Toxoplasma gondii, Neospora caninum, Eimeria spp., and Cystoisospora spp. Their life cycle includes a switch from asexual to sexual stages and is often restricted to a single host species. Current research on coccidian parasites focuses on cell biology and the underlying mechanisms of protein expression and trafficking in different life stages, host cell invasion and host-parasite interactions. Furthermore, novel anticoccidial drug targets are evaluated. Given the variety of research questions and the requirement to reduce and replace animal experimentation, in vitro cultivation of Coccidia needs to be further developed and refined to meet these requirements. For these purposes, established culture systems are constantly improved. In addition, new in vitro culture systems lately gained considerable importance in research on Coccidia. Well established and optimized in vitro cultures of monolayer cells can support the viability and development of parasite stages and even allow completion of the life cycle in vitro, as shown for Cystoisospora suis and Eimeria tenella. Furthermore, new three-dimensional cell culture models are used for propagation of Cryptosporidium spp. (close relatives of the coccidians), and the infection of three-dimensional organoids with T. gondii also gained popularity as the interaction between the parasite and host tissue can be studied in more detail. The latest advances in three-dimensional culture systems are organ-on-a-chip models, that to date have only been tested for T. gondii but promise to accelerate research in other coccidians. Lastly, the completion of the life cycle of C. suis and Cryptosporidium parvum was reported to continue in a host cell-free environment following the first occurrence of asexual stages. Such axenic cultures are becoming increasingly available and open new avenues for research on parasite life cycle stages and novel intervention strategies.
Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum
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Viability of the partially purified tachyzoites was evaluated by trypan blue (Gibco) exclusion test (Chamberland and Current, 1990). NC-1 tachyzoites (obtained from in vitro described above) were adjusted to a concentration of 2 × 106/ml in PBS and 1 ml was added to the cells in each 25 cm2 tissue culture flask (Lv et al., 2010 with some modifications). Both cell lines were cultured at an equal concentration of 106 cells per each separate flask (parasite to cell ratio was 2 tachyzoites: 1 cell; three 25 cm2 tissue culture flasks for each cell line).
In this study the tachyzoite yields of Neospora caninum were compared in two cell lines: Vero (African Green Monkey Kidney) and suspension culture of murine macrophage (J774) cell lines. Then, N. caninum were continuously passaged in these cell lines for 3 months and the effect of host cells on virulence of tachyzoites was assessed by broiler chicken embryonated eggs. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs with different dilutions (0.5 × 10⁴, 1.0 × 10⁴, 1.5 × 10⁴) of tachtzoites isolated from these cell cultures. The mortality pattern and pathological changes of the dead embryos and hatched chickens were noted. Tissue samples of brain, liver and heart were examined by histopathological and detection of DNA of parasite by polymerase chain reaction (PCR). Also, consecutive sections of the tissues examined histologically were used for immunohistochemical (IHC) examination. Embryos inoculated with tachyzoites derived from Vero cell line (group V) showed a higher mortality rate (100%) than the embryos that received tachyzoites derived from J774 cell line (group J) (10% mortality rate). The results of this study indicated that the culture of N. caninum in J774 cell led to a marked increase in the number of tachyzoite yields and rapid attenuation in comparison to Vero, so the results were confirmed by IHC and PCR. This study is the first report of the significant effect of host cell on the attenuation of virulence of N. caninum tachyzoites. These findings could potentially provide a practical approach in the mass production of N. caninum tachyzoites, and also in producing live attenuated vaccine.
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¹: These authors contributed equally to this study.

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Neospora caninum: In vitro culture of tachyzoites in MCF-7 human breast carcinoma cells

Abstract

Introduction

Section snippets

Cells culture

Purification of N. caninum tachyzoites

Results and discussion

Acknowledgments

Veterinary Parasitology

Molecular and Biochemical Parasitology

Journal of Comparative Pathology

Veterinary Parasitology

International Journal of Parasitology

Veterinary Immunology and Immunopathology

Experimental Parasitology

International Journal of Parasitology

International Journal of Parasitology

International Journal of Parasitology

Vero cell surface proteoglycan interaction with the microneme protein NcMIC3 mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii

Veterinary Parasitology

Newly recognized fatal protozoan disease of dogs

Journal of the American Veterinary Medical Association

Review of Neospora caninum and neosporosis in animals

The Korean Journal of Parasitology

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells

Veterinary Research

Adhesion and invasion of bovine endothelial cells by Neospora caninum

Parasitology