CVI Accepts, published online ahead of print on 18 January...

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Title: ELISAs based on Neospora caninum dense granule protein 7 and profilin for 1

estimating the stage of neosporosis 2

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Running title: Serodiagnosis for estimating the stage of neosporosis 4

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Jun HIASA1, Maki NISHIMURA1, Kazuhito ITAMOTO2, Xuenan XUAN1, Hisashi 6

INOKUMA 3 and Yoshifumi NISHIKAWA 1, * 7

8 1 National Research Center for Protozoan Diseases, Obihiro University of Agriculture 9

and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan 10 2 Laboratory of Veterinary Clinical Diagnosis, Department of Veterinary Surgery, 11

Animal Medical Center of Yamaguchi University, 1677–1 Yoshida, Yamaguchi 12

753–8515, Japan 13 3 Department of Clinical Veterinary Medicine, Obihiro University of Agriculture and 14

Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan 15

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*Corresponding author: Yoshifumi NISHIKAWA, Ph.D., National Research Center for 17

Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 18

2-13, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Tel: +81-155-49-5886; Fax: 19

+81-155-49-5643; E-mail: [email protected] 20

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05669-11 CVI Accepts, published online ahead of print on 18 January 2012

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SUMMARY 22

Neospora caninum is an intracellular protozoan parasite that causes bovine and canine 23

neosporosis, characterized by fetal abortion and neonatal mortality, and neuromuscular 24

paralysis, respectively. Although many diagnostic methods to detect parasite-specific 25

antibodies or parasite DNA have been reported, to date, no effective serodiagnostic 26

techniques for estimating pathological status have been described. Our study aimed to 27

elucidate the relationship between parasite-specific antibody response, parasite 28

activation, and neurological symptoms caused by N. caninum infection using 29

recombinant antigen-based enzyme-linked immunosorbent assay. Among 30

experimentally infected mice, anti-N. caninum profilin (NcPF) antibody was only 31

detected in neurological symptomatic animals. Parasite numbers within the brain of the 32

symptomatic mice were significantly higher than that of asymptomatic animals. In 33

addition, anti-NcPF and anti-NcGRA7 antibodies were mainly detected at the acute 34

stage in experimentally infected dogs, while anti-NcSAG1 antibody was produced 35

during both acute and chronic stages. Furthermore, among anti-NcSAG1 antibody 36

positive clinical dogs, the positive rates of anti-NcGRA7 and anti-NcPF antibodies in 37

the neurological symptomatic dogs were significantly higher than those in the 38

non-neurological symptomatic animals. Our results suggested that the levels of 39

anti-NcGRA7 and anti-NcPF antibodies reflected parasite activation and neurological 40

symptoms in dogs. In conclusion, antibodies against NcGRA7 and NcPF may have 41

potential as suitable indicators for estimating the pathological status of neosporosis. 42

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INTRODUCTION 45

Neospora caninum is an intracellular apicomplexan protozoan parasite that infects a 46

range of host species (10). To date, domestic dogs (22) and coyotes (15) are known 47

definitive hosts, and cattle, sheep, water buffalo, horses, bison, and white-tailed deer are 48

known intermediate hosts (6). Bovine neosporosis is typically characterized by fetal 49

abortion and neonatal mortality (7), and several reports suggest that dogs infected with 50

N. caninum exhibit neuromuscular paralysis (3, 25). Drugs such as sulfonamides, 51

clindamycin, pyrimethamine or ponazuril are available for treatment of canine 52

neosporosis (25), and treatment needs to commence promptly before the development 53

of severe clinical symptoms. 54

To detect N. caninum infection, many serological diagnostic methods such as the 55

indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay 56

(ELISA) have been developed (9). There is accumulating evidence that ELISAs using 57

recombinant antigens derived from N. caninum exhibit high specificity and sensitivity 58

for serodiagnosis (9). This is especially the case for N. caninum surface antigen 59

NcSAG1 and the dense granule protein NcGRA7, which are effective antigens for 60

serodiagnosis of this parasite in cattle (1, 4, 18). To date, a serodiagnostic method for 61

the suitable indicator of clinical symptoms caused by N. caninum infection has not been 62

developed, and requires clinical evaluation in the canine host. The reason for the 63

difficulty in development of clinical diagnosis methods is that N. caninum is often 64

asymptomatic in immunocompetent hosts. 65

Detection of parasite activation may be required to estimate the clinical symptoms 66

caused by N. caninum infection. Importantly, the antibody response against N. caninum 67

varies between the acute (tachyzoite stage) and chronic (bradyzoite stage) stages in 68


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animals (1). NcGRA7 protein is an immunodominant antigen shared by both tachyzoite 69

and bradyzoite (2, 27), whereas NcSAG1 is expressed in the tachyzoite and is 70

down-regulated during tachyzoite-to-bradyzoite stage conversion (28). In addition, N. 71

caninum profilin (NcPF) is a cytosolic and actin-binding protein that has potential as a 72

serodiagnostic marker. Toxoplasma gondii profilin (TgPF), a homologous protein to 73

NcPF, stimulates innate immune response in mice by binding Toll-like receptor 11 74

(TLR11) on dendritic cells leading to release of inflammatory cytokine IL-12 (26, 29, 75

30). 76

Our study aimed to develop a serodiagnostic method for estimating the infection 77

status of dogs potentially infected with N. caninum because the serum specific 78

antibodies levels will likely correlate with clinical symptoms or with a given disease 79

stage. We focused on the difference in N. caninum-specific antibody production 80

between neurological symptomatic and asymptomatic animals to assess the use of 81

recombinant NcGRA7 and NcPF-based ELISAs for evaluating the pathological status 82

of canine neosporosis. 83

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MATERIALS AND METHODS 87

Parasite preparation. Tachyzoites of N. caninum Nc-1 strain (12) were propagated 88

in monkey kidney adherent fibroblasts (Vero cells) cultured in Eagle's minimum 89

essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 8% 90

heat-inactivated fetal bovine serum. Purification of tachyzoites involved washing the 91

parasites and host-cell debris in cold phosphate-buffered saline (PBS), and the final 92

pellet was resuspended in cold PBS before being passed through a 27-gauge needle and 93

a 5.0-μm-pre filter (Millipore, Bedford, MA, USA). 94

Construction and expression of recombinant NcPF. Complementary 95

deoxyribonucleic acid (cDNA) was synthesized from ribonucleic acid isolated with TRI 96

reagent (Sigma-Aldrich) using a SuperScript™ First-strand Synthesis System for 97

reverse transcription-polymerase chain reaction (RT-PCR) (Invitrogen, Carlsbad, CA, 98

USA). cDNA was used as a template to amplify the coding region of NcPF (accession 99

number BK006901). 100

Recombinant NcPF (rNcPF), which consisted of 163 amino acids (aa), was cloned 101

using a designed set of oligonucleotide primers that included a EcoR I restriction 102

enzyme site in the forward primer (5′- ATG AAT TCA TGT CGG ACT GGG ATC CCG 103

TT -3′) and an Xho I site in the reverse primer (5′- TAC TCG AGT TAA TAG CCA 104

GAC TGG TGA AG -3′). PCR products were digested with EcoR I and Xho I before 105

being ligated into the glutathione S-transferase (GST)-fusion protein in the Escherichia 106

coli expression vector pGEX-4T1 (GE Healthcare, Buckinghamshire, UK), which had 107

been digested with the same set of restriction enzymes (pGEX-NcPF). Plasmid 108

nucleotide sequences were determined using an ABI 3100 DNA sequencer (Applied 109

Biosystems, Foster City, CA, USA). rNcPF was expressed as glutathione S-transferase 110


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(GST) fusion proteins in the E. coli DH5α strain (Takara Bio Inc., Shiga, Japan). GST 111

tags of the recombinant proteins were removed with thrombin protease (GE Healthcare) 112

according to the manufacturer's instructions. 113

Expression of recombinant proteins of NcSAG1 and NcGRA7. Recombinant 114

NcSAG1 (rNcSAG1) and NcGRA7 (rNcGRA7) proteins were expressed in E. coli as 115

GST fusion proteins and then purified using Glutatione Sepharose 4B as described 116

previously (4, 16). 117

Mice and infection. Twenty-one (first trial) and 31 (second trial) seven-week-old 118

female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). All mice used 119

in the present study were treated under the guiding principles for the care and use of 120

research animals promulgated by Obihiro University of Agriculture and Veterinary 121

Medicine, Japan. They were intraperitoneally inoculated with 1 × 106 tachyzoites of the 122

N. caninum Nc-1 strain. Survival rates and clinical findings of the infected mice were 123

monitored until 49 days and 44 days after the infection in first and second trials, 124

respectively. Five and six mice from the first and second trials, respectively, exhibited 125

clinical signs of neosporosis, including head tilting, limb paralysis, circling motion and 126

febrile response (starry stiff coat). Five and six asymptomatic mice were identified in 127

the first and second trials, respectively. Eleven and nineteen mice from first and second 128

trials, respectively, died before the end of monitoring. 129

Serum (20 μl) was obtained weekly from mice via the tail vein and used to measure 130

levels of N. caninum-specific antibodies by ELISA. Blood was centrifuged at 1,000 × g 131

for 10 min, and serum was collected and stored at -20°C until use. To confirm the lack 132

of an antibody response in uninfected mice, control sera were taken from all animals 133

three days before infection. Thereafter, all surviving mice were killed using a high level 134


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of anesthetic for postmortem examination at the end of monitoring. Tissue samples 135

(liver, kidney, brain, spleen, lung, and heart) were obtained for DNA extraction and 136

real-time PCR analysis. Tissues were stored at -20°C until use. 137

Dogs and infection. Four purebred female specific pathogen-free (SPF) beagle dogs 138

(14–15 months) were used in this study. All dogs were purchased from Chugai Medical 139

Animal Institute (Nagano, Japan) and were housed in separate rooms. Prior to 140

experiments, dogs were proven to be free of N. caninum-specific antibody by ELISA 141

based on the lysates of N. caninum (18) and rNcSAG1 as described below. They had 142

never consumed uncooked meat or meat by-products, and were fed dry dog food for the 143

duration of the experiment. Dogs were released into a room for several hours each day 144

to permit exercise. They were intravenously inoculated with 2 × 106 tachyzoites of N. 145

caninum Nc-1 strain. Survival rates and clinical findings of the infected dogs were 146

monitored until 24 weeks post- infection. Infected dogs showed no clinical symptom 147

and death until end of the monitoring. Blood was collected from the saphenous vein, 148

centrifuged at 1,000 × g for 10 min, then the serum was collected and stored at -20°C 149

until later use. Dog experiments were conducted under the guiding principles for the 150

care and use of research animals promulgated by the Obihiro University of Agriculture 151

and Veterinary Medicine, Japan. 152

Dog serum samples from animal hospitals. Clinical serum samples from dogs 153

(n=27) that exhibited neurological symptoms such as disturbance of motility were 154

obtained from the Animal Medical Center of Yamaguchi University, Japan. Clinical 155

serum samples from non-neurological symptomatic dogs (n=143) were collected from 156

animal hospitals located in 35 prefectures of Japan. All serum samples were screened to 157

detect N. caninum infection by rNcSAG1-based ELISA as described below. Eighteen 158


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neurological symptomatic and 45 non-neurological symptomatic dog samples were 159

considered N. caninum positive. 160

Measurement of N. caninum specific antibodies by ELISA. Fifty microliters of 161

purified rNcPF, rNcSAG1, rNcGRA7, and control GST, at a final concentration of 0.1 162

μM, were coated onto ELISA plates (Nunc, Denmark) overnight at 4°C with a 163

carbonate-bicarbonate buffer (pH 9.6). Plates were washed once with PBS containing 164

0.05% Tween20 (PBS-T), and blocked with PBS containing 3% skim milk (PBS-SM) 165

for 1 h at 37°C. Plates were then washed once with PBS-T, and 50 μl of serum samples 166

diluted at 1:250 with PBS-SM were added to duplicate wells. To confirm the differences 167

in the serum antibody levels at 7 and 14 days after the infection between the assessed 168

mice groups, serum samples were diluted at 1:500 and 1:2,000 with PBS-SM for 169

detecting IgG1 and IgG2a antibodies, respectively. Plates were incubated at 37°C for 1 170

h. After washing six times with PBS-T, plates were incubated with horseradish 171

peroxidase (HRP)-conjugated antibodies diluted at 1:10, 000 with PBS-SM at 37°C for 172

1 h. HRP-conjugated goat anti-mouse IgG1, or IgG2a (Bethyl Laboratories, USA) was 173

used for mouse serum samples. HRP-conjugated goat anti-canine IgG (Bethyl 174

Laboratories) was used for dog serum samples. Plates were further washed six times, 175

before the substrate solution (0.1 M citric acid, 0.2 M sodium phosphate, 0.003% H2O2, 176

and 0.3 mg/ml 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); Sigma-Aldrich) 177

was added to each well in 100 μl aliquots. Absorbances at 415 nm were read after 1 h of 178

incubation at room temperature using an ELISA reader (Corona Microplate Reader 179

MTP-120; Corona, Tokyo, Japan). Absorbance values were determined as the difference 180

in the mean optical density at a value of 415 nm (OD415nm) between the recombinant 181

antigen (rNcSAG1, rNcGRA7) and the GST protein. For ELISA using rNcPF, the result 182


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was determined as the OD415nm value. The cutoff point of dog serum samples was 183

determined as the mean OD415nm value for standard Neospora-negative sera (eighteen 184

SPF dogs) plus two standard deviations. 185

DNA extraction. Collected mice tissues were thawed in a 10x volume of lysis 186

buffer (0.1 mM Tris-HCl, pH 9.0, 0.1 M NaCl, 1 mM EDTA, 1% 187

Sodium-Dodecyl-Sulfate) containing proteinase K (100 μg/ml; Sigma-Aldrich) and 188

incubated for four days at 50°C. Ribonuclease A (100 μg/ml; Sigma-Aldrich) was then 189

added and incubated for 1 h at 37°C. Tissue DNA was extracted by 190

phenol-chloroform-isoamyl alcohol followed by ethanol precipitation, and resuspended 191

in TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA). The DNA concentration was 192

adjusted to 50 ng/μl for each tissue sample and used as a template for real-time PCR 193

analysis. 194

Real-time PCR. Oligonucleotide primers for N. caninum Nc5 sequence (20) 195

(GenBank accession no. X84238) were designed to amplify a 76-bp DNA fragment (5). 196

The N. caninum Nc5 forward primer spans nucleotides 248-257 197

(5’-ACTGGAGGCACGCTGAACAC-3’) and the N. caninum Nc5 reverse primer spans 198

nucleotides 303-323 (5’-AACAATGCTTCGCAAGAGGAA-3’). The PCR mixture (25 199

μL total volume) contained 1× SYBR Green PCR Buffer, 2 mM MgCl2, 200 μM 200

concentrations of each deoxynucleoside triphosphate (dATP, dCTP, and dGTP), 400 μM 201

dUTP, 0.625 U AmpliTaq Gold DNA polymerase, 0.25 U AmpErase UNG 202

(uracil-N-glycosilase), all of which were included in the SYBR Green PCR Core Kit 203

(Applied Biosystems), 20 pmol of each primer, and 1 μl of DNA template (50 ng). 204

Amplification was performed by a standard protocol recommended by the manufacturer 205

(2 min at 50°C, 10 min at 95°C, 40 cycles at 95°C for 15 s, and 60°C for 1 min). 206


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Amplification, data acquisition, and data analysis were carried out using an ABI 7700 207

Prism Sequence Detector machine, and the calculated cycle threshold values (Ct) were 208

exported to Microsoft Excel for analysis. Quantification was determined from the Ct 209

values, which was defined as the cycle at which the fluorescence exceeds the standard 210

deviation of the mean baseline emission for the early cycles by 10 times. Parasite 211

number in the samples was calculated by interpolation of the standard curve, in which 212

Ct values were plotted against the log of known concentration of parasites. After 213

Neospora Nc5 sequence amplification, the melting curves of PCR products were 214

acquired by stepwise increase of the temperature from 55–95°C for 20 min. Data 215

analyses were performed using the Dissociation Curves software (version 1.0 f; Applied 216

Biosystems). 217

Statistical analysis. Significant difference (P<0.05) was calculated by chi-square 218

test and Student's t-test. 219

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RESULTS 221

N. caninum specific antibodies in experimentally infected mice. To examine the 222

relationship between neurological symptoms and N. caninum specific antibody 223

production, the IgG1 and IgG2a antibody responses against rNcSAG1, rNcGRA7 and 224

rNcPF in experimentally infected mice were measured by ELISA. Initially, N. 225

caninum-infected mice were grouped into dead, neurologically symptomatic and 226

asymptomatic animals. Furthermore, parasite numbers in the brain of neurological 227

symptomatic mice were significantly higher than those of asymptomatic mice (Fig. 1A). 228

This result revealed that parasites existed within brains associated with neurological 229

symptoms. In addition, there were no significant differences in the number of parasites 230

in the spleen, heart and lungs of neurologically symptomatic and asymptomatic mice 231

(Fig. 1B-D). 232

Levels of IgG1 and IgG2a antibody against rNcSAG1 increased from seven days 233

post-infection, thereafter, these antibodies remained at high levels until the end of the 234

experiment (Fig. 2A and B). The detection of anti-rNcGRA7 antibody production was 235

delayed by one week compared with that of the anti-rNcSAG1 antibody (Fig. 2C and D). 236

Furthermore, we tested the sera at higher dilutions to confirm the differences in the serum 237

antibody levels between the assessed mice groups (Fig. 3). Antibody levels of 238

anti-rNcSAG1 were higher than those of anti-rNcGRA7 at 7 and 14 days after infection. 239

However, there were no marked differences in antibody responses against rNcGRA7 240

and rNcSAG1 among dead, neurologically symptomatic and asymptomatic mice (Fig. 241

2A-D and Fig. 3). Interestingly, antibodies against rNcPF showed unique production 242

dynamics. The anti-NcPF IgG1 antibody was detected in 3/5 neurologically 243

symptomatic mice and in 2/11 dead mice, while the levels of this antibody were low in 244


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the asymptomatic mice (Fig. 2E). Moreover, high anti-NcPF IgG2a antibody production 245

was observed in 4/5 neurological symptomatic mice and 7/11 dead mice, while only one 246

mouse from the asymptomatic mice produced this antibody (Fig. 2F). 247

248

Anti-N. caninum specific antibodies in experimentally infected dogs. ELISA was 249

used to measure IgG antibody responses against rNcGRA7, rNcSAG1 and rNcPF in 250

experimentally infected dogs (Fig. 4) to investigate changes in antibody responses. All 251

dogs infected with N. caninum produced IgG antibodies against the lysates of this 252

protozoan (data not shown). Serum antibody levels against rNcSAG1 peaked at 21 days 253

after infection. Thereafter, the antibody level gradually decreased (Fig. 3A). The 254

antibody levels against rNcGRA7 peaked between 14–21 days after infection, which is 255

similar dynamics of the anti-rNcSAG1 antibody production (Fig. 3B). However, 256

antibody levels as a response against rNcGRA7 decreased quicker than those against 257

rNcSAG1 (Fig. 3A and B). Furthermore, anti-rNcPF antibodies were detected at 14–21 258

days after infection, and rapidly decreased (Fig. 3C). At 112 days after infection, serum 259

antibodies against rNcPF was not detectable, although production of antibody against 260

rNcSAG1 and rNcGRA7 was still observed (Fig. 3). We further observed that 261

anti-rNcPF antibody levels varied among animals. 262

263

Evaluation of anti-N. caninum specific antibody levels in dogs exhibiting 264

neurological symptoms. Our results suggested that anti-rNcPF antibody might be 265

associated with the progression of neurological symptom. To investigate the relationship 266

between the antibody response and neurological symptoms caused by N. caninum 267

infection, dog serum samples from animal hospitals were examined (Table. 1). To 268


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determine N. caninum infection status, serum samples were screened using a 269

rNcSAG1-based ELISA because anti-NcSAG1 antibody was detected at both acute and 270

chronic stages of the infection as shown in Fig. 4A. Serum samples were then grouped 271

into neurologically symptomatic and non-neurologically symptomatic dogs. Positive 272

rates of anti-rNcGRA7 and anti-rNcPF antibodies in the neurologically symptomatic 273

dogs were significantly higher than those in the non-neurologically symptomatic 274

animals (Table 1. P<0.05). Four anti-rNcGRA7 positive and four anti-rNcPF positive 275

dogs were found in nine anti-NcSAG1-negative and neurological symptomatic dogs (data 276

not shown). 277

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DISCUSSION 279

In order to effectively treat neosporosis that exhibits neurological symptoms, a rapid 280

and accurate diagnosis method is needed. Previous study suggested that neosporosis 281

symptoms were increased after immunosuppressive therapy for granulomatous 282

meningoencephalitis (11, 13). Although PCR analysis for detection of N. caninum from 283

cerebrospinal fluid (CSF) has been described (23, 24), CSF must typically be collected 284

under anesthesia, and is associated with considerable risk. Therefore, more effective and 285

safe serological diagnosis methods for neosporosis are required. While many serological 286

diagnosis methods to detect N. caninum infection have been reported, a technique that 287

also measures the pathological status has not been developed. 288

Development of diagnostic tools capable of discriminating between the active and 289

chronic stages of infection that relate to pathological status is one of major challenges 290

for the control of neosporosis. A potential candidate is a recombinant antigen-based 291

ELISA using rNcSAG1 and has previously been proven useful in both cattle (4) and 292

dogs (21). Our study has further shown that using experimentally infected dogs, the 293

levels of IgG antibody against rNcSAG1 are kept at high levels over an extended period 294

of time. In addition, antibody levels of anti-rNcSAG1 were higher than those of 295

anti-rNcGRA7 at the acute stage in experimentally infected mice, suggesting the higher 296

antigenicity of NcSAG1. These results suggested that the anti-rNcSAG1 antibody is 297

potentially a suitable marker for the broad detection of N. caninum infection at both 298

acute and chronic stages. Previous study indicated that anti-NcGRA7 IgG antibody was 299

also observed during acute infection in cattle (primo-infection, re-infection and 300

recrudescence) (1), while our study has further demonstrated the usefulness of 301

anti-rNcGRA7 antibody as a marker of N. caninum activation in dogs. Similar to the 302


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previous study in cattle, the levels of IgG antibody against rNcGRA7 in experimentally 303

infected dogs decreased from 30 days after infection. These dynamics of anti-rNcGRA7 304

antibody production may relate to parasite activation. In clinical samples of 305

anti-rNcSAG1 antibody positive dogs, positive rates of IgG antibody against rNcGRA7 306

in neurologically symptomatic dogs were significantly higher than those in the 307

non-neurologically symptomatic animals. This result suggested that neurological signs 308

caused by N. caninum infection might coincident with parasite activation. 309

The dynamics of anti-rNcPF antibody production were different between mice and 310

dogs following N. caninum infection. In mice, anti-NcPF antibody was detected in 311

neurologically symptomatic animals, but not in asymptomatic ones. In contrast, 312

experimentally infected dogs produced anti-NcPF antibody only at the acute stage. The 313

kinetics of antibody production, as assessed by determining their serum levels in i.v. 314

infected dogs or i.p. infected mice may not be the same as in naturally infected animals 315

where the parasite disseminates from the gastrointestinal tract. Although such a 316

difference in anti-NcPF antibody production may be due to differences between the host 317

species or inoculation routes of the parasite, we currently have no exact explanation for 318

this. 319

Given that NcPF lacks a signal peptide and is a cytosolic protein (19), we 320

therefore speculated that large amounts of NcPF would be required to stimulate the 321

specific antibody production. During the acute stage of N. caninum infection, host 322

immune cells would control the parasite burden. For instance, T cells such as CD8+ T 323

cells may kill the N. caninum-infected host cells and macrophage killing of the parasites 324

supposedly occurs intracellularly, resulting in the release of NcPF from the dead 325

parasites. Otherwise, NcPF may be released from the large number of free parasites 326


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during the activation stage or during host cell invasion. Such a released NcPF might be 327

the antigen which can stimulate antibody production. In mice, high levels of anti-NcPF 328

antibody production may be due to the reactivation stage of N. caninum in 329

neurologically symptomatic animals. While in the experimentally N. caninum-infected 330

dogs, high levels of anti-NcPF antibody production at the acute stage may reflect host 331

immunity associated with infection control. In addition, NcPF itself may stimulate 332

immune responses because interferon-gamma production was induced in mice by NcPF 333

inoculation (19). 334

The number of parasite in brain of mice that exhibited neurological symptoms was 335

higher than that of asymptomatic animals. This result indicated that neurological 336

symptoms might be caused by N. caninum infection of the central neuron system (CNS). 337

Furthermore, anti-rNcPF antibody levels may correlate with the parasite number in CNS. 338

Brain lesions examined by magnetic resonance imaging in neurologically symptomatic 339

dogs were mainly found in both the cerebrum and cerebellum (Data not shown). 340

However, there were no statistically significant differences on the region of the brain 341

lesions between neurologically symptomatic anti-rNcSAG1 negative and positive dogs. 342

Furthermore, we analysed the brain lesion correlation with anti-NcGRA7 and anti-NcPF 343

serum positivity. However, there was no significant correlation (data not shown). This 344

result was supported by previous study that suggested neosporosis is an important cause 345

of progressive cerebellar ataxia and cerebellar atrophy in adult dogs (14). Thus, N. 346

caninum infection of the CNS, especially the cerebrum, is likely to cause neurological 347

symptoms in infected animals. 348

In summary, we have described the possibility of NcGRA7 and NcPF recombinant 349

proteins as useful diagnostic tools for dogs exhibited neurological signs. To date, 350


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definitive diagnosis of neosporosis has been conducted using immunohistochemical 351

staining of N. caninum in neural tissue. Reliable antemortem diagnosis of neosporosis is 352

necessary before an effective therapeutic strategy can be initiated. Our recombinant 353

antigen-based ELISA may replace soluble extract based ELISA or tachyzoite-based 354

IFAT in routine serodiagnosis and PCR assay to detect parasite DNA from CSF, and 355

may have the additional advantage of being capable of estimating pathological status. 356

Increased levels of antibodies against NcSAG1, NcGRA7 and NcPF in dogs exhibiting 357

neurological symptoms are most likely indicative of current N. caninum infection. In 358

addition, by measuring parasite activation using rNcGRA7 and rNcPF-based ELISAs, 359

we might detect neosporosis earlier than via the direct observation of symptoms. 360

Treatment of canine neosporosis is difficult and shows only partial effects (17). 361

Therefore, to obtain effective therapeutic effects, treatment should be started before 362

muscular contracture has occurred (8). The relevance of our findings for the clinical field 363

must take into account that dogs likely will be checked for neosporosis upon symptoms 364

are noticed. In addition, the usefulness of this method for the economic relevant bovine 365

host should be evaluated in the future studies. 366


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Acknowledgments 367

We thank Dr. J. P. Dubey (United States Department of Agriculture, Agriculture 368

Research Service, Livestock and Poultry Sciences Institute, and Parasite Biology and 369

Epidemiology Laboratory) for the gift of N. caninum Nc-1 isolate. This research was 370

supported by the Japan Society for the Promotion of Science through the Funding 371

Program for Next Generation World-Leading Researchers (NEXT Program), initiated 372

by the Council for Science and Technology Policy (2011/LS003). 373

374

Conflict of interest statement 375

The authors declare no conflict of interest. 376

377


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308:1626-1629.472


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Figure Legends 473

Fig. 1. Real-time PCR for determining N. caninum numbers in various organs of the 474

mice at 49 days after the infection. (A) Brain. (B) Spleen. (C) Heart. (D) Lungs. Group 475

1: mice exhibiting neurological symptoms (n=5). Group 2: mice exhibiting asymptom 476

(n=5). * Significant difference (P<0.05) was calculated by student’s t-test. The parasite 477

DNA was not detected in kidney and liver. Reproducibility of the data was confirmed by 478

two independent experiments, which both gave similar results. 479

480

Fig. 2. Production of IgG1 (A, C, E) and IgG2a (B, D, F) antibodies against rNcSAG1 481

(A, B), rNcGRA7 (C, D) and rNcPF (E, F) in mice infected with N. caninum 482

tachyzoites. Serum samples diluted at 1:250 were tested by ELISA. Blue lines indicate 483

asymptomatic mice. Red lines indicate neurological symptomatic mice. Black lines 484

correspond to mice that died along the experiment time course. Reproducibility of the 485

data was confirmed by two independent experiments, which both gave similar results. 486

487

Fig. 3. Comparison of the differences in the serum antibody levels against rNcSAG1 (A, 488

B) and rNcGRA7 (C, D) at 7 and 14 days post inoculation (dpi) between the assessed 489

mice groups. Serum samples diluted at 1:500 and 1:2,000 were tested by ELISA for 490

detection IgG1 (A, C) and IgG2a (B, D). A indicates asymptomatic mice. S indicates 491

neurological symptomatic mice. D corresponds to mice that died along the experiment 492

time course. 493

494

Fig. 4. Production of IgG antibodies against rNcSAG1 (A), rNcGRA7 (B) and rNcPF 495

(C) in dogs infected with N. caninum tachyzoites. Serum samples diluted at 1:250 were 496


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tested by ELISA. 497

498

499


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Table 1. rNcGRA7 and rNcPF-based ELISAs for clinical samples of dogs infected with N. caninum

Groups anti-rNcGRA7 antibody positive anti-rNcPF antibody positive

Neurological symptomatic 66.7% (12/18) a 66.7% (12/18) a

Non-neurologically symptomatic 35.6% (16/45) 26.7% (12/45)

Serum samples screened by rNcSAG1-based ELISA were used. Serum samples were grouped intoneurologically symptomatic (n=18) and non-neurologically symptomatic dogs (n=45). Percentage of positivesamples (number of positive/number of total rNcSAG1 positive) is shown and the significant difference wascalculated by chi-square test (P<0.05).

a, significant difference in the percentage positive between neurological symptomatic and non-neurologicalsymptomatic dogs for the same antigen.


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CVI Accepts, published online ahead of print on 18 January...

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