中国科技论文在线 http://www.paper.edu.cn

SEROPREVALENCE OF ANTIBODIES AGAINST NEOSPORA CANINUM IN PÈRE DAVID’S DEER (ELAPHURUS DAVIDIANUS) IN BEIJING# 5 HAN Fangjie1, FU Yong1, ZHANG Changsheng2, LIU Qun1, LIU Jing1** (1. Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China 100193; 2. Beijing Museum of Natural History, Beijing, China 100050) 10 Abstract: Neospora caninum is an apicomplexan protozoan that causes neuromuscular disease in canids and abortion in cattle worldwide. In China, sera from 49 Père David’s deer (Elaphurus davidianus) were investigated for the presence of antibodies against N. caninum using indirect fluorescence antibody tests (IFAT) and western blots. N. caninum antibodies were found in 26.53% (13 positive/49 tested) of the Père David’s deer according IFAT and the western blots. Western blot 15 analysis revealed there was seroreactivity against immunodominant N. caninum antigens, and the bands were 16, 25, and 37 kDa in size together with other visible bands. This is the first study of antibodies against N. caninum in Père David’s deer from China. Key words: Neospora caninum; Père David’s deer; Seroprevalence

0 Introduction 20 Neospora caninum is an obligate intracellular protozoan parasite with a worldwide distribution and a wide range of hosts. It is primarily a disease of cattle and dogs, and may cause diseases in domestic and wild animals [1]. Serological evidence in domestic, wild, and zoo animals indicates that many species have been exposed to this parasite. Wild canidae, including the Australian dingo (Canis familiaris), the coyote (Canis latrans), and the gray wolf (Canis lupus), are the definitive 25 hosts of N. caninum and shed oocysts to the environment through their feces [2-6]. Additionally, a wide range of mammals, especially ruminants, are intermediate hosts, and they become infected with N. caninum by ingesting oocysts from the environment or placental transfer from a mother to a fetus. Then, canidae are infected when they prey on ruminants. This sylvatic cycle is thought to be important in the epidemiology of N. caninum [3]. Cervidae, including white-tailed deer 30 (Odocoileus virginianus), sika deer (Cervus nippon), black-tailed deer (Odocoileus hemionus columbianus), mule deer (Odocoileus hemionus hemionus), red deer (Cervus elaphus), roe deer (Capreolus capreolus), moose (Alces alces), fallow deer (Dama dama) and axis deer (Axis axis) may be reservoir hosts of N. caninum in the wild [7-15]. Père David's deer is one of the world’s most rare and endangered deer species. This species has 35 been placed on the IUCN (International Union for Conservation of Nature) red list of threatened species. They are also considered to be first-grade state protected animals in China. This deer is native to the middle-lower reaches of the Yangtze River basin in China and became extirpated in the wild at least 1,200 years ago. Since then, the deer have been kept in captivity [16]. Père David's deer became extinct in China toward the end of the 19th century during the Qing Dynasty. 40 Twenty-two deer were reintroduced to Beijing from Britain in the 1980s and were raised in Beijing Milu Yuan, which is the place where the final local extinction of the deer occurred as well

Foundations: the Ph.D. Program Foundation of the Ministry of Education of China (No. 20130008120010); the Natural Science Foundation of China (No.31302075) Brief author introduction:Han Fangjie (1989-), Female, Graduate student, Parasitology research Correspondance author: LIU Jing (1978-), Female, Associate professor, Parasitology research. E-mail: [email protected]

- 1 - 中国科技论文在线 http://www.paper.edu.cn

as the location of the historical distribution of wild Père David's deer. Beijing Milu Yuan is 14 km from Beijing and covers more than 60 hectares. It is the first nature reserve for Père David's deer and includes pond, swamps, pasture and forest land. This reservation has gradually become an 45 ecological museum for studying Chinese cervidae animals, and the reservation offers Père David's deer to zoos or other protection zones in China. The deer are breeding and rejuvenating their population in Beijing Milu Yuan. They are free-range animals in the reservation. They are free to gather food and mate to ensure their constitution will not degenerate or their fertility will not diminish. The population recently increased to more than 200 deer. Cervidae animals can be 50 infected with N. caninum in the wild, and may subsequently experience abortion. Therefore, neosporosis may be a threat to the increasing population of Père David's deer. Reports on the prevalence of N. caninum in Père David's deer are limited to one study that detected 28 sera in animals from a zoo in Chomutov, Czech Republic [17]. The prevalence of neosporosis in the population of Père David's deer is unknown, and the prevalence of N. caninum 55 infection in Père David's deer in China has never been investigated. The aim of this study was to determine the prevalence of antibodies against N. caninum in Père David's Deer from Beijing. This is the first study to provide serological evidence of infection and exposure to N. caninum in Père David's deer in China. The results could reveal if neosporosis should be taken into account as a potential abortion risk factor for the breeding program of Père 60 David's deer. 1 Materials and Methods

1.1 Serum samples Blood samples were collected from the jugular vein of 49 Père David's deer by veterinary practitioners at the Beijing Milu Ecological Research Center. The deer were selected to be sent to 65 other zoos or protection zones. The blood samples were centrifuged at 3500 rpm for 10 min, and the serum was obtained and stored at -20°C until analysis. 1.2 Determination of antibodies against N. caninum by western blot Tachyzoites of NC-1 were collected from Vero cell cultures, filtered through a 5 μm membrane, centrifuged for 10 min at 1800 rpm and lysed with RIPA Lysis Buffer (Beyotime, China) in ice for 70 30 min. Then, the samples were centrifuged for 10 min at 12000 rpm and the precipitation was discarded. The crude lysate was diluted with a 5× loading buffer (250 mM Tris–HCl pH6.8, 10% SDS, 5% β-mercaptoethanol, 25% glycerin and 0.1% bromophenol blue) and boiled for 5 min. Next, 20 μg protein fractions were placed in lanes and separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [18]. Following electrophoresis, the 75 proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The PVDF membranes were blocked in a buffer containing 5% nonfat dry milk in PBS at 37°C for 1 h following incubation with Pere David’s deer serum (dilution 1:300) at 37°C for 1 h. The membranes were washed in PBST (0.5‰ Tween 20) five times. Then, the PVDF membrane was incubated with a rabbit anti-deer IgG (H+L) horseradish peroxidase 80 (HRP)-labelled secondary antibody (PKL, USA) 1:1,000 at 37°C for 1 h and washed in PBST. A western blot assay was performed using the Mini-PROTEAN II Multiscreen Apparatus (BIORAD, USA). The immunoreactive proteins were detected with ECL chemiluminescence reagents (CoWin Biotech, China) [19].

- 2 - 中国科技论文在线 http://www.paper.edu.cn

85 1.3 Determination of antibodies against N. caninum and T. gondii with the indirect fluorescent antibody test (IFAT) All of the deer sera were analyzed with IFAT as previously described by Cui [20]. Cell culture-derived NC-1 tachyzoites or RH tachyzoites were mounted on coverslips, dried and fixed with 3% (vol/vol) paraformaldehyde in PBS for 20 min at room temperature, and then the cells 90 were blocked in PBS with 3% (wt/vol) BSA for 1 h at 37°C. The coverslips were incubated with Père David's deer serum diluted at 1:100 in 1% BSA-PBS with the primary antibodies for 1 h at 37°C, washed and incubated with fluorescein isothiocyanate (FITC)-labelled rabbit anti-deer IgG (H+L) (KPL, USA) diluted at 1:100 in 1% BSA-PBS as the secondary antibody for 1 h at 37°C. Then, the coverslips were mounted on a glass slide. Finally, the parasites were observed using an 95 inverted fluorescence microscope (Olympus IX71). 1.4 Data analysis The degree of agreement between the serological methods was estimated by calculating the kappa value using SPSS software (Statistical Analysis System, Version 17.0). 2 Results 100 A total of 13 (26.53%) out of the 49 serum samples contained antibodies against N. caninum, and the samples were confirmed as positive for N. caninum infection by western blot. Those sera generated a wide range of bands between 15 to 130 kDa. The same bands were detected at 16 kDa, 25 kDa and 37 kDa in almost all of the positive sera (Fig. 1), which demonstrated that the corresponding antigens were immunodominant. IFAT indicated that 13 (26.53%) out of the 49 105 serum contained antibodies against N. caninum.

Figure 1. Western blot analysis of the antibody responses in Père David's deer against N. caninum (NC-1).

Tachyzoite lysates of the NC-1 strain were incubated with sera from Père David's deer. Lanes 1-6, 8, 11-15 110 include positive sera samples; Lanes 7, 9-10 have negative sera.

- 3 - 中国科技论文在线 http://www.paper.edu.cn

N. caninum generated bright green fluorescence around tachyzoites when the samples were incubated with positive serum (Fig. 2A) compared to negative serum (Fig. 2B).

115 Figure 2. IFAT analysis of the antibody response in Père David's deer against N. caninum (NC-1)

A: Positive serum of N. caninum responds to NC-1; B: Negative serum of N. caninum responds to NC-1. Scale bar: 20 μm

Two serological tests showed nearly consistent results, which indicates good agreement (κ=0.895). 120 There are only two sera that have difference between them (Table 1). Because cross-reaction with closely related parasites, especially T. gondii, was considered to be a potential problem, all sera were also analyzed for antibodies against T. gondii using IFAT. There are two sera (4.08%) containing antibodies for T. gondii. No cross-reactions or co-infections were observed, i.e., N. caninum positive serum samples confirmed by western bolt or IFAT were negative for T. gondii 125 and vice versa.

Table 1. Results of the serological tests of Neospora caninum antibodies in Père David's deer sera. Comparison between western blot and IFAT results. Western blot Positive Negative Total Seroprevalence % IFAT Positive 12 1 13 Negative 1 35 36 26.53 Total 13 36 49 Agreement 0.895 (κ value) Seroprevalence % 26.53

130 3 Discussion It has been reported that N. caninum can infect a wide range of animals, including some cervidae [21]. The seroprevalence of N. caninum in 106 red deer was 11% [10]. Additionally, the prevalence for N. caninum was 8.4% in 368 white-tailed deer [22]. Recently, 2.9% of 335 farmed fallow deer (Dama dama) were confirmed as positive for N. caninum [12]. The only report of the prevalence of 135 N. caninum in Père David's deer was 25% of 28 deer from a zoo in the Czech Republic [17]. In the present study, we tested 49 sera samples from Père David’s deer and the positive percentage was

- 4 - 中国科技论文在线 http://www.paper.edu.cn

26.53% (13/49). To the best of our knowledge, this survey is the first report of N. caninum infection in Père David's deer from China, and the first evidence of N. caninum infection in Père David's deer in Asia. 140 In our study, the seroprevalence of N. caninum in Père David's deer was 26.53%, which was detected by western blot and IFAT. The IFAT was the first serological test used to demonstrate the presence of antibodies against N. caninum. This test has been regarded as a reference test for the development of other assays for the detection of N. caninum antibodies [23]. We compared the western blot and IFAT results, and kappa statistics showed a good agreement between them. 145 Slight differences between the assays may be due to differences in subjective judgment. Western blots provide direct visual evidence of antibodies bound to specific antigens, which provides greater confidence in the results. So far, only a few references were reported on Western blot analysis of N. caninum infection in wildlife. In 2005, it was revealed that 29, 43, and 67-94 kDa specific bands were found in the immunoblots of sera collected from experimentally infected 150 South American Camelids [24]. Anderson used western blots to investigate N. caninum in white-tailed deer and identified two of the immunodominant proteins as 35 and 25 kDa bands in addition to six other visible bands [25]. Detection of sera in infected grey wolves with immunoblot analysis revealed 17, 34, and 43 kDa bands [26]. Recently, western blots of serum samples revealed three heavily stained 16, 25, and 37 kDa bands in farmed fallow deer [12]. All the analyses were 155 consistent with the range of bands found in our study through western blots. Although Père David's deer were mainly kept in captivity in Beijing and limited to a reservation environment, the reservation is a free-range deer park and some related facilities provide opportunities for Père David's deer to be outdoors. Therefore, it is easy for the deer to come in contact with other animals, especially wild or stray dogs. Canids and livestock are present in areas 160 where neosporosis was detected in Beijing, China [27-29]. The Père David's deer most likely became infected by ingesting food or water contaminated with N. caninum oocysts excreted by canids. Therefore, Père David's deer may contribute to the life cycle of N. caninum by becoming an intermediate host and carrying the cysts of N. caninum. 4 Conclusion 165 In this paper, Père David’s deer in Beijing have 26.53% seroprevalence of antibodies against N. caninum. Père David's deer was reintroduced and raised in Beijing Milu Yuan. They will be released to the wild, thereby restoring the wild population. Until now, their offspring have been scattered all over China. It is possible that the threat of N. caninum will spread to other regions of China, particularly where the final hosts of N. caninum, dogs, are found. To determine whether 170 exposure to N. caninum will lead Père David's deer to experience abortion or other clinical symptoms will require additional research. Our results demonstrated that Père David’s deer are exposed to N. caninum in Beijing, China. This research provides some information on the epidemiology of N. caninum. Our analysis shows that different serological tests could be used reliably to determine the seroprevalence of N. caninum, thereby providing credible methods for 175 supervising N. caninum infection in other deer populations in China. Père David's deer may act as the wildlife hosts or reservoirs for N. caninum. To better understand the relationship between the deer and N. caninum, the pathogen needs to be isolated and more sera samples as well as detailed data on reproductive loss need to be collected.

- 5 - 中国科技论文在线 http://www.paper.edu.cn

180 Acknowledgements (Optional) We are grateful to Zhenyu Zhong (Beijing Milu Ecological Research Center) for offering serum samples and information.

References

[1] Dubey, J.P., Schares, G., Ortega-Mora, L.M. Epidemiology and control of neosporosis and Neospora caninum 185 [J]. Clinical microbiology reviews, 2007, 20(2): 323-367. [2] McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M. Dogs are definitive hosts of Neospora caninum [J]. International journal for parasitology, 1998, 28(9): 1473-1478. [3] Gondim, L.F., McAllister, M.M., Mateus-Pinilla, N.E., Pitt, W.C., Mech, L.D., Nelson, M.E. Transmission of Neospora caninum between wild and domestic animals [J]. The Journal of parasitology, 2004, 90(6): 1361-1365. 190 [4] Dubey, J.P , Jenkins, M.C., Rajendran, C., Miska, K., Ferreira, L.R., Martins, J., Kwok, O.C., Choudhary, S. Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum [J]. Veterinary Parasitology, 2011, 181(2-4): 382-387. [5] Dubey, J.P., Jenkins, M.C., Ferreira, L.R., Choudhary, S., Verma, S.K., Kwok, O.C., Fetterer, R., Butler, E., Carstensen, M. Isolation of viable Neospora caninum from brains of wild gray wolves (Canis lupus) [J]. Veterinary 195 Parasitology, 2014, 201(1-2): 150-153. [6] King, J.S., Slapeta, J., Jenkins, D.J., Al-Qassab, S.E., Ellis, J.T., Windsor, P.A. Australian dingoes are definitive hosts of Neospora caninum [J]. International journal for parasitology, 2010, 40(8): 945-950.. [7] Dubey, J.P., Hollis, K., Romand, S., Thulliez, P., Kwok, O.C., Hungerford, L., Anchor, C., Etter, D. High prevalence of antibodies to Neospora caninum in white-tailed deer (Odocoileus virginianus)[J]. International 200 journal for parasitology, 1999, 29(10):1709-1711. [8] Omata, Y., Ishiguro, N., Kano, R., Masukata, Y., Kudo, A., Kamiya, H., Fukui, H., Igarashi, M., Maeda, R., Nishimura, M., Saito, A. Prevalence of Toxoplasma gondii and Neospora caninum in sika deer from eastern Hokkaido, Japan [J]. Journal of wildlife diseases, 2005, 41(2): 454-458. [9] Gozdzik, K., Jakubek, E.B., Bjorkman, C., Bien, J., Moskwa, B., Cabaj, W. Seroprevalence of Neospora 205 caninum in free living and farmed red deer (Cervus elaphus) in Poland [J]. Polish journal of veterinary sciences, 2010, 13(1): 117-120. [10] Dubey, J.P., Mansfield, K., Hall, B., Kwok, O.C., Thulliez, P. Seroprevalence of Neospora caninum and Toxoplasma gondii in black-tailed deer (Odocoileus hemionus columbianus) and mule deer (Odocoileus hemionus hemionus)[J]. Veterinary Parasitology, 2008, 156(3-4): 310-313. 210 [11] Malmsten, J., Jakubek, E.B., Bjorkman, C. Prevalence of antibodies against Toxoplasma gondii and Neospora caninum in moose (Alces alces) and roe deer (Capreolus capreolus) in Sweden [J]. Vet Parasitol, 2011, 177(3-4):275-280. [12] Bien, J., Moskwa, B., Bogdaszewski, M., Cabaj, W. Detection of specific antibodies anti-Neospora caninum in the fallow deer (Dama dama)[J]. Research in veterinary science, 2012, 92(1): 96-98. 215 [13] Basso, W., More, G., Quiroga, M.A., Balducchi, D., Schares, G., Venturini, M.C. Neospora caninum is a cause of perinatal mortality in axis deer (Axis axis)[J]. Veterinary parasitology, 2014, 199(3-4): 255-258. [14] Almeria, S., Lopez-Gatius, F. Bovine neosporosis: clinical and practical aspects[J]. Research in veterinary science, 2013, 95(2): 303-309. [15] Pruvot, M., Hutchins, W., Orsel, K. Statistical evaluation of a commercial Neospora caninum competitive 220 ELISA in the absence of a gold standard: application to wild elk (Cervus elaphus) in Alberta [J]. Parasitology research, 2014, 113(8): 2899-2905. [16] Yang, D., Jiang, Z., Ma, J., Hu, H., Li, P. Causes of endangerment or extinction of some mammals and its relevance to the reintroduction of Pere David's deer in the Dongting Lake drainage area [J]. Shengwu Duoyangxing, 2005, 13(5): 451-461. 225 [17] Sedlak, K., Bartova, E. Seroprevalences of antibodies to Neospora caninum and Toxoplasma gondii in zoo animals [J]. Veterinary parasitology, 2006, 136(3-4): 223-231. [18] Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Nature, 1970, 227(5259): 680-685. [19] Lei, T., Wang, H., Liu, J., Nan, H., Liu, Q. ROP18 is a key factor responsible for virulence difference between 230 Toxoplasma gondii and Neospora caninum [J]. PloS one, 2014, 9, e99744. [20] Cui, X., Lei, T., Yang, D., Hao, P., Li, B., Liu, Q. Toxoplasma gondii immune mapped protein-1 (TgIMP1) is a novel vaccine candidate against toxoplasmosis [J]. Vaccine, 2012, 30(13): 2282-2287. [21] Dubey, J.P. Review of Neospora caninum and neosporosis in animals [J]. The Korean journal of parasitology, 2003, 41(1): 1-16. 235 [22] Olamendi-Portugal, M., Caballero-Ortega, H., Correa, D., Sanchez-Aleman, M.A., Cruz-Vazquez, C., Medina-Esparza, L., Ortega, S.J., Cantu, A., Garcia-Vazquez, Z. Serosurvey of antibodies against Toxoplasma gondii and Neospora caninum in white-tailed deer from Northern Mexico [J]. Veterinary parasitology, 2012, 189(2-4): 369-373. [23] Bjorkman, C., Uggla, A. Serological diagnosis of Neospora caninum infection [J]. International journal for 240 parasitology, 1999, 29(10): 1497-1507. [24] Wolf, D., Schares, G., Cardenas, O., Huanca, W., Cordero, A., Barwald, A., Conraths, F.J., Gauly, M., Zahner, H., Bauer, C. Detection of specific antibodies to Neospora caninum and Toxoplasma gondii in naturally infected

- 6 - 中国科技论文在线 http://www.paper.edu.cn

alpacas (Lama pacos), llamas (Lama glama) and vicunas (Lama vicugna) from Peru and Germany [J]. Veterinary parasitology, 2005, 130(1-2): 81-87. 245 [25] Anderson, T., DeJardin, A., Howe, D.K., Dubey, J.P., Michalski, M.L. Neospora caninum antibodies detected in Midwestern white-tailed deer (Odocoileus virginianus) by Western blot and ELISA [J]. Veterinary parasitology, 2007, 145(1-2): 152-155. [26] Bjorkman, C., Jakubek, E.B., Arnemo, J.M., Malmsten, J. Seroprevalence of Neospora caninum in gray wolves in Scandinavia [J]. Veterinary parasitology, 2010, 173(1-2): 139-142. 250 [27] Yu, X., Chen, N., Hu, D., Zhang, W., Li, X., Wang, B., Kang, L., Li, X., Liu, Q., Tian, K. Detection of Neospora caninum from farm-bred young blue foxes (Alopex lagopus) in China [J]. The Journal of veterinary medical science / the Japanese Society of Veterinary Science, 2009, 71(1): 113-115. [28] Liu, J., Yu, J., Wang, M., Liu, Q., Zhang, W., Deng, C., Ding, J. Serodiagnosis of Neospora caninum infection in cattle using a recombinant tNcSRS2 protein-based ELISA [J]. Veterinary parasitology, 2007, 143(3-4): 255 358-363.

北京麋鹿新孢子虫血清流行病学调查 韩方洁1，傅勇1，张昌盛2，刘群1，刘晶1 260 （1. 农业部动物流行病学与人畜共患病重点实验室，农业部国家动物寄生原虫实验室，中 国农业大学动物医学院，北京； 2. 北京自然科学博物馆，北京 100050） 摘要：新孢子虫病是能够引起犬神经肌肉系统疾病和牛流产的一种机会性原虫病，广泛分布 于世界各地，能感染多种野生动物。本研究利用免疫印迹法和间接荧光检测法，对北京地区 265 49 头麋鹿血清进行了新孢子虫抗体检测。结果显示，新孢子虫血清抗体阳性率为 26.53%， 且两种检测方法符合率良好。免疫印迹法可观察到阳性血清能够与新孢子虫全虫抗原在 16、 25 和 37kDa 有明显的反应条带。这是我国首次关于麋鹿的新孢子虫感染情况的报道。结果 提示新孢子虫病应该作为麋鹿流产的潜在因素加以考虑。 关键词：新孢子虫；麋鹿；血清流行病学 270 中图分类号：S855.9

- 7 -