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Transcriptome and histopathological changes in mouse brain infected with Neospora caninum.

Nishimura M, Tanaka S, Ihara F, Muroi Y, Yamagishi J, Furuoka H, Suzuki Y, Nishikawa Y - Sci Rep (2015)

Bottom Line: A GOstat analysis predicted that the upregulated genes were involved in the host immune response.Genes whose expression correlated positively and negatively with parasite numbers were involved in the host immune response, and neuronal morphogenesis and lipid metabolic processes, respectively.These results suggest that changes in the gene expression profile associated with neuronal functions as well as immune responses can contribute to the pathogenesis in N. caninum-infected animals.

View Article: PubMed Central - PubMed

Affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan.

ABSTRACT
Neospora caninum is a protozoan parasite that causes neurological disorders in dogs and cattle. It can cause nonsuppurative meningoencephalitis and a variety of neuronal symptoms are observed, particularly in dogs. However, the pathogenic mechanism, including the relationship between the parasite distribution and the clinical signs, is unclear. In this study, to understand the pathogenic mechanism of neosporosis, parasite distribution and lesions were assessed in the brain of mice infected with N. caninum (strain Nc-1). Host gene expression was also analyzed with RNA sequencing (RNA-Seq). The histopathological lesions in the frontal lobe and the medulla oblongata were significantly more severe in symptomatic mice than in asymptomatic mice, although no association between the severity of the lesions and parasite numbers was found. In infected mice, the expression of 772 mouse brain genes was upregulated. A GOstat analysis predicted that the upregulated genes were involved in the host immune response. Genes whose expression correlated positively and negatively with parasite numbers were involved in the host immune response, and neuronal morphogenesis and lipid metabolic processes, respectively. These results suggest that changes in the gene expression profile associated with neuronal functions as well as immune responses can contribute to the pathogenesis in N. caninum-infected animals.

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Related in: MedlinePlus

Flow diagram illustrating the number of mice used in each analysis.(A) For the histopathological analysis, 25 mice were infected with N. caninum. Because eight of the 25 mice died before sampling or may not have been infected, the severity of the brain lesions in nine different areas (olfactory system, frontal lobe, caudate putamen, hippocampus, hypothalamus, amygdala, periaqueductal gray, medulla oblongata and cerebellum) was estimated in 17 mice (ten symptomatic and seven asymptomatic mice). (B) For the detection of N. caninum and the RNA-Seq analysis, 14 mice were used (four uninfected and 10 infected mice). Thirty-nine days after inoculation, six of the 10 infected mice showed clinical signs of neosporosis, but four animals did not. To detect N. caninum in different areas of the brain, six infected mice were selected (four symptomatic and two asymptomatic mice). The brains of these six infected mice were divided into eight different areas (olfactory system, frontal lobe, caudate putamen, hippocampus, hypothalamus, amygdala, periaqueductal gray, and cerebellum), and used for DNA extraction and quantitative PCR analysis of the parasite. Additionally, to detect N. caninum in the medulla oblongata, DNA was extracted from paraffin-embedded brain tissues containing the medulla oblongata and cerebellum (n = 17, from mice shown in Figure 1A) and subjected to quantitative PCR. For the detection of N. caninum in whole-brain samples and the RNA-Seq analysis, four uninfected and four infected (two symptomatic and two asymptomatic mice) brains were used. DNA and RNA were extracted from each brain sample and used for the quantitative PCR analysis of the parasite numbers and the RNA-Seq analysis.
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f1: Flow diagram illustrating the number of mice used in each analysis.(A) For the histopathological analysis, 25 mice were infected with N. caninum. Because eight of the 25 mice died before sampling or may not have been infected, the severity of the brain lesions in nine different areas (olfactory system, frontal lobe, caudate putamen, hippocampus, hypothalamus, amygdala, periaqueductal gray, medulla oblongata and cerebellum) was estimated in 17 mice (ten symptomatic and seven asymptomatic mice). (B) For the detection of N. caninum and the RNA-Seq analysis, 14 mice were used (four uninfected and 10 infected mice). Thirty-nine days after inoculation, six of the 10 infected mice showed clinical signs of neosporosis, but four animals did not. To detect N. caninum in different areas of the brain, six infected mice were selected (four symptomatic and two asymptomatic mice). The brains of these six infected mice were divided into eight different areas (olfactory system, frontal lobe, caudate putamen, hippocampus, hypothalamus, amygdala, periaqueductal gray, and cerebellum), and used for DNA extraction and quantitative PCR analysis of the parasite. Additionally, to detect N. caninum in the medulla oblongata, DNA was extracted from paraffin-embedded brain tissues containing the medulla oblongata and cerebellum (n = 17, from mice shown in Figure 1A) and subjected to quantitative PCR. For the detection of N. caninum in whole-brain samples and the RNA-Seq analysis, four uninfected and four infected (two symptomatic and two asymptomatic mice) brains were used. DNA and RNA were extracted from each brain sample and used for the quantitative PCR analysis of the parasite numbers and the RNA-Seq analysis.

Mentions: Pathological severity was evaluated in 17 mice, and 10 of the 17 mice showed clinical signs of neosporosis including febrile responses and leg paralysis 39 days after infection (See Figure 1A). Seven of 10 symptomatic mice showed neurological signs, including circling motion, head tilting, and leg paralysis. We analyzed the nine area of brain histopathologically and for the quantification of parasite load. Histopathological lesions, including perivascular cuff, mononuclear cellular meningitis, glial cell activation, and focal necrosis, were observed in the brains of all 17 mice, which are similar to the lesions found in dogs891415. Each focal lesion was scored for severity using a scale from 1 to 4 (See Figure 2A–D). The total pathological scores for all areas in the brain are shown in Figure 2E. The total scores for the frontal lobe and medulla oblongata were significantly higher in the symptomatic mice than in the asymptomatic mice. Although there were no significant differences between the symptomatic and asymptomatic mice in the cerebellum, two symptomatic mice showed very high scores.


Transcriptome and histopathological changes in mouse brain infected with Neospora caninum.

Nishimura M, Tanaka S, Ihara F, Muroi Y, Yamagishi J, Furuoka H, Suzuki Y, Nishikawa Y - Sci Rep (2015)

Flow diagram illustrating the number of mice used in each analysis.(A) For the histopathological analysis, 25 mice were infected with N. caninum. Because eight of the 25 mice died before sampling or may not have been infected, the severity of the brain lesions in nine different areas (olfactory system, frontal lobe, caudate putamen, hippocampus, hypothalamus, amygdala, periaqueductal gray, medulla oblongata and cerebellum) was estimated in 17 mice (ten symptomatic and seven asymptomatic mice). (B) For the detection of N. caninum and the RNA-Seq analysis, 14 mice were used (four uninfected and 10 infected mice). Thirty-nine days after inoculation, six of the 10 infected mice showed clinical signs of neosporosis, but four animals did not. To detect N. caninum in different areas of the brain, six infected mice were selected (four symptomatic and two asymptomatic mice). The brains of these six infected mice were divided into eight different areas (olfactory system, frontal lobe, caudate putamen, hippocampus, hypothalamus, amygdala, periaqueductal gray, and cerebellum), and used for DNA extraction and quantitative PCR analysis of the parasite. Additionally, to detect N. caninum in the medulla oblongata, DNA was extracted from paraffin-embedded brain tissues containing the medulla oblongata and cerebellum (n = 17, from mice shown in Figure 1A) and subjected to quantitative PCR. For the detection of N. caninum in whole-brain samples and the RNA-Seq analysis, four uninfected and four infected (two symptomatic and two asymptomatic mice) brains were used. DNA and RNA were extracted from each brain sample and used for the quantitative PCR analysis of the parasite numbers and the RNA-Seq analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300462&req=5

f1: Flow diagram illustrating the number of mice used in each analysis.(A) For the histopathological analysis, 25 mice were infected with N. caninum. Because eight of the 25 mice died before sampling or may not have been infected, the severity of the brain lesions in nine different areas (olfactory system, frontal lobe, caudate putamen, hippocampus, hypothalamus, amygdala, periaqueductal gray, medulla oblongata and cerebellum) was estimated in 17 mice (ten symptomatic and seven asymptomatic mice). (B) For the detection of N. caninum and the RNA-Seq analysis, 14 mice were used (four uninfected and 10 infected mice). Thirty-nine days after inoculation, six of the 10 infected mice showed clinical signs of neosporosis, but four animals did not. To detect N. caninum in different areas of the brain, six infected mice were selected (four symptomatic and two asymptomatic mice). The brains of these six infected mice were divided into eight different areas (olfactory system, frontal lobe, caudate putamen, hippocampus, hypothalamus, amygdala, periaqueductal gray, and cerebellum), and used for DNA extraction and quantitative PCR analysis of the parasite. Additionally, to detect N. caninum in the medulla oblongata, DNA was extracted from paraffin-embedded brain tissues containing the medulla oblongata and cerebellum (n = 17, from mice shown in Figure 1A) and subjected to quantitative PCR. For the detection of N. caninum in whole-brain samples and the RNA-Seq analysis, four uninfected and four infected (two symptomatic and two asymptomatic mice) brains were used. DNA and RNA were extracted from each brain sample and used for the quantitative PCR analysis of the parasite numbers and the RNA-Seq analysis.
Mentions: Pathological severity was evaluated in 17 mice, and 10 of the 17 mice showed clinical signs of neosporosis including febrile responses and leg paralysis 39 days after infection (See Figure 1A). Seven of 10 symptomatic mice showed neurological signs, including circling motion, head tilting, and leg paralysis. We analyzed the nine area of brain histopathologically and for the quantification of parasite load. Histopathological lesions, including perivascular cuff, mononuclear cellular meningitis, glial cell activation, and focal necrosis, were observed in the brains of all 17 mice, which are similar to the lesions found in dogs891415. Each focal lesion was scored for severity using a scale from 1 to 4 (See Figure 2A–D). The total pathological scores for all areas in the brain are shown in Figure 2E. The total scores for the frontal lobe and medulla oblongata were significantly higher in the symptomatic mice than in the asymptomatic mice. Although there were no significant differences between the symptomatic and asymptomatic mice in the cerebellum, two symptomatic mice showed very high scores.

Bottom Line: A GOstat analysis predicted that the upregulated genes were involved in the host immune response.Genes whose expression correlated positively and negatively with parasite numbers were involved in the host immune response, and neuronal morphogenesis and lipid metabolic processes, respectively.These results suggest that changes in the gene expression profile associated with neuronal functions as well as immune responses can contribute to the pathogenesis in N. caninum-infected animals.

View Article: PubMed Central - PubMed

Affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan.

ABSTRACT
Neospora caninum is a protozoan parasite that causes neurological disorders in dogs and cattle. It can cause nonsuppurative meningoencephalitis and a variety of neuronal symptoms are observed, particularly in dogs. However, the pathogenic mechanism, including the relationship between the parasite distribution and the clinical signs, is unclear. In this study, to understand the pathogenic mechanism of neosporosis, parasite distribution and lesions were assessed in the brain of mice infected with N. caninum (strain Nc-1). Host gene expression was also analyzed with RNA sequencing (RNA-Seq). The histopathological lesions in the frontal lobe and the medulla oblongata were significantly more severe in symptomatic mice than in asymptomatic mice, although no association between the severity of the lesions and parasite numbers was found. In infected mice, the expression of 772 mouse brain genes was upregulated. A GOstat analysis predicted that the upregulated genes were involved in the host immune response. Genes whose expression correlated positively and negatively with parasite numbers were involved in the host immune response, and neuronal morphogenesis and lipid metabolic processes, respectively. These results suggest that changes in the gene expression profile associated with neuronal functions as well as immune responses can contribute to the pathogenesis in N. caninum-infected animals.

Show MeSH
Related in: MedlinePlus