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Seroepidemiology of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes (Bubalus bubalis) in the People's Republic of China
Veterinary Parasitology 143 (2007) 79–85 www.elsevier.com/locate/vetpar
Short communication
Seroepidemiology of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes (Bubalus bubalis) in the People’s Republic of China Jinhai Yu, Zhaofei Xia, Qun Liu *, Jing Liu, Jun Ding, Wei Zhang Parasitology Laboratory, Department of Veterinary Preventive Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, PR China Received 21 April 2006; received in revised form 24 June 2006; accepted 24 July 2006
Abstract A seroepidemiological survey of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes was carried out in the People’s Republic of China. Serum samples were obtained from dairy (n = 262, 9 herds in 9 provinces) and beef cattle (n = 10, 1 herd) and water buffaloes (n = 40) in China. All sera were tested for antibodies to N. caninum and T. gondii by an enzyme-linked immunosorbent assay (ELISA) and an indirect agglutination test (IAT), respectively. The overall seroprevalence of N. caninum in dairy cattle was 17.2% (45/262), and the herds seroprevalence of N. caninum was 88.9% (8/9), and antibodies to T. gondii were present in 6 cows (2.3%). None of the cows had antibodies against both T. gondii and N. caninum. Antibodies to T. gondii or N. caninum were not found in beef cattle or water buffaloes. The seroprevalence of N. caninum in aborting cows (20.2%) was higher than that in non-aborting cows (16.6%) with an odds ratio of 1.26 (95% CI, 0.54–2.95), but the difference was not statistically significant (P > 0.05). There was no apparent association of N. caninum seropositivity with age or number of pregnancies. This is the first report on the seroprevalence of N. caninum in cattle and water buffaloes in China. # 2006 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Enzyme-linked immunosorbent assay (ELISA); Toxoplasma gondii; Indirect agglutination test (IAT); Seroepidemiology; Cattle; Water buffaloes
1. Introduction Neospora caninum is an intracellular protozoan, which has similar morphologic and biologic characteristics to Toxoplasma gondii. Both parasites are distributed worldwide and can cause abortion in cattle. (Dubey, 1999; Dubey et al., 1998; Lapointe et al., 2003; Trees and Williams, 2005). Bovine abortions due to either parasite could result in a reduction of milk
* Corresponding author. Tel.: +86 10 6273 2807; fax: +86 10 6273 2804. E-mail address: [email protected] (Q. Liu). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.07.031
production and culling of animals, and thus substantial economic loss (Hobson et al., 2002; Romero et al., 2005; Thurmond and Hietala, 1996). Several researchers have reported the seroprevalence of N. caninum infection in water buffaloes in developing countries, such as Egypt (Dubey et al., 1998), southern Vietnam (Huong et al., 1998) and Brazil (Gennari et al., 2005), where water buffaloes are economically important domestic animals. N. caninum could be transmitted vertically from pregnant cows to the fetus (transplacental transmission) or horizontally between cows and cows or from cows to dogs (postnatal transmission) (Akca et al., 2005; Barber and Trees, 1998; Bartels et al., 2005; Bergeron et al.,
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province. These water buffaloes were kept by different farmers. Blood samples were centrifuged at 1000 g for 10 min and serum was obtained, frozen and stored at 20 8C until analysis. The breed, age, and pregnancy and abortion history were obtained through a questionnaire, which was sent to the local veterinarians at the time of blood collection. They were also the keepers of the animals’ health records. The owners were agreed with publication the data.
2000; Davison et al., 2001). Transplacental transmission is considered the major route of N. caninum transmission in cattle (Antony and Williamson, 2001), but it was suggested that horizontal transmission is required for the disease to persist in a herd. Investigations of the prevalence of N. caninum infection in heifers and adult cattle of the same herd may provide more information on the route of transmission. In the last two decades, China has kept a large number of dairy and beef cattle. However, there is no report about the epidemiology of neosporosis in cattle in China. The aim of this study was to investigate the seroprevalence of N. caninum and T. gondii infections in cattle and water buffaloes in China.
2.2. Determination of antibodies to N. caninum Antibodies to N. caninum were determined using the CIVTEST bovis Neospora ELISA kit (Hipra, Spain) according to the manufacturer’s instructions. Positive and negative controls were provided in the kit. The tests were performed in the Veterinary College of China Agricultural University. Briefly, the N. caninum specific antigen was coated on a 96-well ELISA plate. After incubation of the diluted serum sample (1:100) in the test well and subsequent washing, a conjugate was added. The plate was washed again, and then a chromogenic enzyme substrate was added. The optical density (OD) at 405 nm was read using a photometer (BIO-RAD). A relative rate percent (IRPC) value was obtained using the following formula:
2. Materials and methods 2.1. Serum samples Blood samples were collected from the jugular or caudal vein by local veterinary practitioners from 262 dairy cows in 9 herds of 9 provinces in China from May to December in 2005. The number of serum samples from each province is shown in Table 1. The animals of each herd were randomly selected, and one blood sample was collected from each animal. We collected one blood sample according to the rate (1:10) in the herd. The nine provinces selected for the study covered the south (Guangdong, Guangxi), northeast (Harbin), northwest (Xinjiang), north (Beijing, Tianjin), inner north (Shanxi), east (Shandong), and the central plain (Sichuan) of China. The location of the herds is shown in Fig. 1. Serum samples of 10 beef cattle were obtained from a herd in Shandong province. Sera of 40 water buffaloes came from a pasturing area of Yangzhou in Jiangsu
ðOD405 sample mean OD405 negative controlsÞ 100 IRPC ¼ mean OD405 positive control mean OD405 negative control The sera were considered negative to N. caninum if the IRPC value was less than 6.0, and positive if IRPC > 10.0.
Table 1 Seroprevalence of N. caninum and T. gondii infection in dairy cattle Regions
Beijing Tianjin Shanxi Helongjiang Xinjiang Sichuan Guangxi Guangdong Shandong Total *
2
Number of samples
N. caninum
T. gondii *
Number of positive serum
Seroprevalence (%)
Number of positive serum
Seroprevalence (%)
32 29 26 30 37 42 28 18 20
11 2 6 8 7 5 2 4 0
34.4 6.9 23.1 26.7 18.9 11.9 7.1 22.2 0.0
0 0 1 1 2 2 0 0 0
0.0 0.0 3.8 3.3 5.4 4.8 0.0 0.0 0.0
262
45
17.2
6
2.3
x = 18.695, P = 0.0166.
J. Yu et al. / Veterinary Parasitology 143 (2007) 79–85
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Fig. 1. Map of PR China showing the location of the nine herds in nine provinces and a pasturing area of Yangzhou in Jiangsu province from which samples were collected.
2.3. Determination of antibodies to T. gondii All serum samples were also tested for antibodies to T. gondii using a commercial indirect agglutination test (IAT, Lanzhou Veterinary Institute of China Academy of Agriculture and Science) according to the manufacturer’s instructions. 2.4. Statistical analysis The data were analysed using the software of SAS (Statistical Analysis System, version 8.0). Chi-square analysis in the FREQ procedure was used to compare the seroprevalence of aborting cows with that of nonaborting cows, using the odd ratios to express it, and to analyze the difference in seroprevalence between regions, between age groups and between pregnancy groups. 3. Results
gondii and N. caninum. Detailed results are shown in Table 1. The herd prevalence of N. caninum infection in dairy cows ranged from 0.0% to 34.4%, and differences among the regions were statistically significant (x2 = 18.695, P = 0.0166). Serum samples taken from a herd in Beijing that exhibited persistent abortions had the highest seroprevalence (11/32, 34.4%). Antibodies to T. gondii or N. caninum were not detected in beef cattle or water buffaloes. 3.2. Seroprevalence of N. caninum in aborting and non-aborting cows Of the 262 cows, 187 had a pregnancy history record for the analysis of the association of abortion and N. caninum infection (Table 2). The seroprevalence of N. caninum in aborting cows (20.2%) was higher than that Table 2 Seroprevalence of aborting and non-aborting cows Cows
3.1. Seroprevalence of T. gondii and N. caninum N. caninum antibodies were detected in 45 (17.2%) of 262 cows, and T. gondii antibodies in 2.3% tested animals and the herds seroprevalence of N. caninum was 88.9% (8/9). None of the cows had antibodies against both T.
Number Number of Seroprevalence* of animals positive serum (%)
Aborting cows 94 Non-aborting cows 93
19 15
20.2 16.1
Cows with no history records
11
14.7
*
75
x2 = 0.524, P = 0.469.
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Table 3 Seroprevalence of N. caninum in different age groups of dairy cows Age (year)
Number of animals
Number of positive serum
Seroprevalence* (%)
1 2 3 4 5 6 7 8 >8
3 29 17 30 56 37 11 14 15
1 5 6 6 9 5 4 4 1
33.3 17.2 35.3 20 16.1 13.5 36.4 28.6 6.7
50
4
8
Unknown *
2
x = 8.782, P = 0.361.
in non-aborting cows (16.1%), but the difference was not statistically significant. The odds ratio for aborting cows with N. caninum infection was 1.26 (95% CI, 0.54–2.95). Of the 19 seropositive and aborting cows, 1, 10 and 7 cows aborted during the first, second and third trimesters of gestation, respectively, and the abortion time was unknown for one cow. 3.3. Distribution of seropositive cows in different age groups As shown in Table 3, the seroprevalence varied in different age groups, ranging from 6.7% to 36.4%. There were no statistically significant differences among the age groups (x2 = 8.782, P = 0.3610) or association of N. caninum infection with age. 3.4. Association between seroprevalence and number of pregnancies Of the 262 cows tested, 158 cows had calved, and 24 were heifers, while the pregnancy history of 80 cows Table 4 Seroprevalence of N. caninum in different pregnancy groups Number of pregnancies
Number of cows
Number of positive serum
Seroprevalence* (%)
0 1 2 3 4 5 6 >7
24 21 64 25 25 14 6 3
3 8 12 4 6 5 1 0
12.5 38.1 18.8 16 24 35.7 16.7 0
80
3
3.8
Unknown *
2
x = 8.006, P = 0.332
was unknown. The seroprevalence of N. caninum infection in different pregnancy groups is shown in Table 4. The seroprevalence of cows that had given one birth was the highest (38.1%), but differences between pregnancy groups were not statistically significant (x2 = 8.006, P = 0.332). The prevalence of seropositivity in heifers was 12.5%. 4. Discussion Since the discovery of N. caninum in the late 1980s, a number of serological assays have been developed for the detection of antibodies to N. caninum. The ELISA by Hipra, Spain used in this study is a commercially available method, which has been validated and previously used by von Blumroder et al. (2004). The seroprevalence of T. gondii and N. caninum in cattle and water buffaloes has been studied in many countries. It varied between countries, regions, herds and even at different times of the same herds. This is the first study of seroepidemiology of N. caninum in cattle and water buffaloes in China. The overall seroprevalence in dairy cattle in 9 regions/provinces was 17.2%, similar to the reported prevalence in Brazil (17.8%) (Corbellini et al., 2006) and in Argentina (16.6%) (Moore et al., 2002). Herd seroprevalence in our survey varied from 0.0% to 34.4%, and the differences were statistically significant between herds or regions. N. caninum positive sera were found in eight out of nine regions, indicating that neosporosis in dairy cattle is widespread in China. There have been many reports on the association between seroprevalence of neosporosis and abortion. Koiwai et al. (2005) found that seropositive cattle of N. caninum infection were 9.2 times more likely to abort compared to seronegative cattle, and a similar odds ratio (8.0) was observed by Vaclavek et al. (2003). However, relatively low odds ratios, 1.1 and 2.0, were reported by Garcia-Vazquez et al. (2005) and Hobson et al. (2005), respectively. Although the association of abortion with neosprora seropositivity in our study was not very strong (odds ratio, 1.3), there was some evidence of neosporosis-related abortion. A relatively high prevalence of neosporosis (34.4%) was observed in a Beijing dairy farm, which had persistent abortions. Higher seroprevalence of N. caninum in abortion positive herds than abortion control herds was also reported by Hobson et al. (2005). Our study found that abortions in N. caninum positive cows occurred mostly in the second and third trimesters, consistent with the finding by Weston et al. (2005), who reported that abortions occurred
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predominantly between Days 120 and 152 gestation among N. caninum seropositive dairy cows. Predominantly mid-term abortions due to N. caninum were probably related to the low immune response of cows during this period of gestation. In this study, only one blood sample was taken from each cow, and blood samples were not collected at a certain time after abortion. Jenkins et al. (1997) demonstrated long persistence of antibodies to N. caninum after abortion, suggesting the time of blood sampling after abortion is not critical for the detection of anti-neospora antibodies. It was unlikely that aborting cows due to N. caninum infection were not detected in this study. A Netherland’s study showed similar seroprevalence across all age groups, suggesting that the infection had most likely been perpetuated by vertical transmission (Wouda et al., 1999). However, a Danish study showed that seropositivity increased with age (Jensen et al., 1999). Neither of these findings was observed in our study, in which there was no significant association between the age of the animals and seroprevalence. The varied seroprevalence in different age groups suggested the possibility of horizontal (postnatal) transmission in the investigated herds. With regard to the relationship between the number of pregnancies and seroprevalence of N. caninim in cattle, Jensen et al. (1999) found that seropositivity rose with increasing numbers of pregnancy, but a positive relationship was not observed in our study. There were many reports on the seroepidemiology of N. caninum in water buffaloes (Fujii et al., 2001; Guarino et al., 2000; Hilali et al., 1998; Rodrigues et al., 2004, 2005) and beef cattle (Haddad et al., 2005; Kim et al., 2002; Tennent-Brown et al., 2000). The seroprevalence of N. caninum varied between studies and herds (0–70.9% in water buffaloes and 2.8–4.1% in beef cattle), but there have been no published reports on the seroprevalence in these animals in China. Our study detected no antibodies to N. caninum or T. gondii in water buffaloes or beef cattle. However, only a limited number of buffaloes and beef cattle were studied in this survey, and further studies with a larger sample size are needed to investigate the epidemiology of N. caninum and T. gondii infections in these livestock. The seroprevalence of T. gondii in dairy cows was 2.3%, which is in line with results obtained by Lin et al. (1990), but it was much lower than that (10.5–27.9%) observed in other countries (Huong et al., 1998; Samad et al., 1997; van Knapen et al., 1995). None of the cows had antibodies against both T. gondii and N. caninum. This indicated that the assays for T. gondii and N. caninum antibodies were specific for the corresponding
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protozoan (i.e. no cross-reactions), and the cattle were not infected with both parasites. Several researchers had reported similar results (Bjorkman et al., 1994; Lally et al., 1996), but others (Bae et al., 2000; Huong et al., 1998; Ooi et al., 2000) found antibodies to both parasites in the serum of dairy cows. In conclusion, this is the first seroprevalence study of N. caninum in cattle and water buffaloes in the People’s Republic of China. The results suggested that neosporosis in dairy cattle is widespread in China. It may play an important role in the abortion of dairy cattle, although the association between abortion and seropositivity was not statistically significant. Further studies with a large number of samples are necessary to confirm whether N. caninum infection is a significant factor of abortions in dairy cattle in China. The current study found no seropositivity for N. caninum and T. gondii in water buffaloes and beef cattle, but the number of samples investigated was small, and the epidemiology needs further investigation. Acknowledgements We thank local veterinary practitioners for collecting blood samples and Dr. Jin Zhu, Therapeutic Goods Administration, Canberra, Australia, for revising the manuscript. We wish to thank Hipra Corp., Spain for providing the CIVTEST bovis Neospora ELISA kit. This study was supported by grants from the National Natural Science Foundation of China (30371080), Natural Science Foundation of Beijing (6042016) and the National Key Technologies R & D Program (2004BA514A18B). References Akca, A., Gokce, H.I., Guy, C.S., McGarry, J.W., Williams, D.J., 2005. Prevalence of antibodies to Neospora caninum in local and imported cattle breeds in the Kars province of Turkey. Res. Vet. Sci. 78, 123–126. Antony, A., Williamson, N.B., 2001. Recent advances in understanding the epidemiology of Neospora caninum in cattle. N. Z. Vet. J. 49, 42–47. Bae, J.S., Kim, D.Y., Hwang, W.S., Kim, J.H., Lee, N.S., Nam, H.W., 2000. Detection of IgG antibody against Neospora caninum in cattle in Korea. Kor. J. Parasitol. 38, 245–249. Barber, J.S., Trees, A.J., 1998. Naturally occurring vertical transmission of Neospora caninum in dogs. Int. J. Parasitol. 28, 57–64. Bartels, C.J., van Maanen, C., van der Meulen, A.M., Dijkstra, T., Wouda, W., 2005. Evaluation of three enzyme-linked immunosorbent assays for detection of antibodies to Neospora caninum in bulk milk. Vet. Parasitol. 131, 235–246. Bergeron, N., Fecteau, G., Pare, J., Martineau, R., Villeneuve, A., 2000. Vertical and horizontal transmission of Neospora caninum in dairy herds in Quebec. Can. Vet. J. 41, 464–467.
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Short communication
Seroepidemiology of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes (Bubalus bubalis) in the People’s Republic of China Jinhai Yu, Zhaofei Xia, Qun Liu *, Jing Liu, Jun Ding, Wei Zhang Parasitology Laboratory, Department of Veterinary Preventive Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, PR China Received 21 April 2006; received in revised form 24 June 2006; accepted 24 July 2006
Abstract A seroepidemiological survey of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes was carried out in the People’s Republic of China. Serum samples were obtained from dairy (n = 262, 9 herds in 9 provinces) and beef cattle (n = 10, 1 herd) and water buffaloes (n = 40) in China. All sera were tested for antibodies to N. caninum and T. gondii by an enzyme-linked immunosorbent assay (ELISA) and an indirect agglutination test (IAT), respectively. The overall seroprevalence of N. caninum in dairy cattle was 17.2% (45/262), and the herds seroprevalence of N. caninum was 88.9% (8/9), and antibodies to T. gondii were present in 6 cows (2.3%). None of the cows had antibodies against both T. gondii and N. caninum. Antibodies to T. gondii or N. caninum were not found in beef cattle or water buffaloes. The seroprevalence of N. caninum in aborting cows (20.2%) was higher than that in non-aborting cows (16.6%) with an odds ratio of 1.26 (95% CI, 0.54–2.95), but the difference was not statistically significant (P > 0.05). There was no apparent association of N. caninum seropositivity with age or number of pregnancies. This is the first report on the seroprevalence of N. caninum in cattle and water buffaloes in China. # 2006 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Enzyme-linked immunosorbent assay (ELISA); Toxoplasma gondii; Indirect agglutination test (IAT); Seroepidemiology; Cattle; Water buffaloes
1. Introduction Neospora caninum is an intracellular protozoan, which has similar morphologic and biologic characteristics to Toxoplasma gondii. Both parasites are distributed worldwide and can cause abortion in cattle. (Dubey, 1999; Dubey et al., 1998; Lapointe et al., 2003; Trees and Williams, 2005). Bovine abortions due to either parasite could result in a reduction of milk
* Corresponding author. Tel.: +86 10 6273 2807; fax: +86 10 6273 2804. E-mail address: [email protected] (Q. Liu). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.07.031
production and culling of animals, and thus substantial economic loss (Hobson et al., 2002; Romero et al., 2005; Thurmond and Hietala, 1996). Several researchers have reported the seroprevalence of N. caninum infection in water buffaloes in developing countries, such as Egypt (Dubey et al., 1998), southern Vietnam (Huong et al., 1998) and Brazil (Gennari et al., 2005), where water buffaloes are economically important domestic animals. N. caninum could be transmitted vertically from pregnant cows to the fetus (transplacental transmission) or horizontally between cows and cows or from cows to dogs (postnatal transmission) (Akca et al., 2005; Barber and Trees, 1998; Bartels et al., 2005; Bergeron et al.,
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province. These water buffaloes were kept by different farmers. Blood samples were centrifuged at 1000 g for 10 min and serum was obtained, frozen and stored at 20 8C until analysis. The breed, age, and pregnancy and abortion history were obtained through a questionnaire, which was sent to the local veterinarians at the time of blood collection. They were also the keepers of the animals’ health records. The owners were agreed with publication the data.
2000; Davison et al., 2001). Transplacental transmission is considered the major route of N. caninum transmission in cattle (Antony and Williamson, 2001), but it was suggested that horizontal transmission is required for the disease to persist in a herd. Investigations of the prevalence of N. caninum infection in heifers and adult cattle of the same herd may provide more information on the route of transmission. In the last two decades, China has kept a large number of dairy and beef cattle. However, there is no report about the epidemiology of neosporosis in cattle in China. The aim of this study was to investigate the seroprevalence of N. caninum and T. gondii infections in cattle and water buffaloes in China.
2.2. Determination of antibodies to N. caninum Antibodies to N. caninum were determined using the CIVTEST bovis Neospora ELISA kit (Hipra, Spain) according to the manufacturer’s instructions. Positive and negative controls were provided in the kit. The tests were performed in the Veterinary College of China Agricultural University. Briefly, the N. caninum specific antigen was coated on a 96-well ELISA plate. After incubation of the diluted serum sample (1:100) in the test well and subsequent washing, a conjugate was added. The plate was washed again, and then a chromogenic enzyme substrate was added. The optical density (OD) at 405 nm was read using a photometer (BIO-RAD). A relative rate percent (IRPC) value was obtained using the following formula:
2. Materials and methods 2.1. Serum samples Blood samples were collected from the jugular or caudal vein by local veterinary practitioners from 262 dairy cows in 9 herds of 9 provinces in China from May to December in 2005. The number of serum samples from each province is shown in Table 1. The animals of each herd were randomly selected, and one blood sample was collected from each animal. We collected one blood sample according to the rate (1:10) in the herd. The nine provinces selected for the study covered the south (Guangdong, Guangxi), northeast (Harbin), northwest (Xinjiang), north (Beijing, Tianjin), inner north (Shanxi), east (Shandong), and the central plain (Sichuan) of China. The location of the herds is shown in Fig. 1. Serum samples of 10 beef cattle were obtained from a herd in Shandong province. Sera of 40 water buffaloes came from a pasturing area of Yangzhou in Jiangsu
ðOD405 sample mean OD405 negative controlsÞ 100 IRPC ¼ mean OD405 positive control mean OD405 negative control The sera were considered negative to N. caninum if the IRPC value was less than 6.0, and positive if IRPC > 10.0.
Table 1 Seroprevalence of N. caninum and T. gondii infection in dairy cattle Regions
Beijing Tianjin Shanxi Helongjiang Xinjiang Sichuan Guangxi Guangdong Shandong Total *
2
Number of samples
N. caninum
T. gondii *
Number of positive serum
Seroprevalence (%)
Number of positive serum
Seroprevalence (%)
32 29 26 30 37 42 28 18 20
11 2 6 8 7 5 2 4 0
34.4 6.9 23.1 26.7 18.9 11.9 7.1 22.2 0.0
0 0 1 1 2 2 0 0 0
0.0 0.0 3.8 3.3 5.4 4.8 0.0 0.0 0.0
262
45
17.2
6
2.3
x = 18.695, P = 0.0166.
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Fig. 1. Map of PR China showing the location of the nine herds in nine provinces and a pasturing area of Yangzhou in Jiangsu province from which samples were collected.
2.3. Determination of antibodies to T. gondii All serum samples were also tested for antibodies to T. gondii using a commercial indirect agglutination test (IAT, Lanzhou Veterinary Institute of China Academy of Agriculture and Science) according to the manufacturer’s instructions. 2.4. Statistical analysis The data were analysed using the software of SAS (Statistical Analysis System, version 8.0). Chi-square analysis in the FREQ procedure was used to compare the seroprevalence of aborting cows with that of nonaborting cows, using the odd ratios to express it, and to analyze the difference in seroprevalence between regions, between age groups and between pregnancy groups. 3. Results
gondii and N. caninum. Detailed results are shown in Table 1. The herd prevalence of N. caninum infection in dairy cows ranged from 0.0% to 34.4%, and differences among the regions were statistically significant (x2 = 18.695, P = 0.0166). Serum samples taken from a herd in Beijing that exhibited persistent abortions had the highest seroprevalence (11/32, 34.4%). Antibodies to T. gondii or N. caninum were not detected in beef cattle or water buffaloes. 3.2. Seroprevalence of N. caninum in aborting and non-aborting cows Of the 262 cows, 187 had a pregnancy history record for the analysis of the association of abortion and N. caninum infection (Table 2). The seroprevalence of N. caninum in aborting cows (20.2%) was higher than that Table 2 Seroprevalence of aborting and non-aborting cows Cows
3.1. Seroprevalence of T. gondii and N. caninum N. caninum antibodies were detected in 45 (17.2%) of 262 cows, and T. gondii antibodies in 2.3% tested animals and the herds seroprevalence of N. caninum was 88.9% (8/9). None of the cows had antibodies against both T.
Number Number of Seroprevalence* of animals positive serum (%)
Aborting cows 94 Non-aborting cows 93
19 15
20.2 16.1
Cows with no history records
11
14.7
*
75
x2 = 0.524, P = 0.469.
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Table 3 Seroprevalence of N. caninum in different age groups of dairy cows Age (year)
Number of animals
Number of positive serum
Seroprevalence* (%)
1 2 3 4 5 6 7 8 >8
3 29 17 30 56 37 11 14 15
1 5 6 6 9 5 4 4 1
33.3 17.2 35.3 20 16.1 13.5 36.4 28.6 6.7
50
4
8
Unknown *
2
x = 8.782, P = 0.361.
in non-aborting cows (16.1%), but the difference was not statistically significant. The odds ratio for aborting cows with N. caninum infection was 1.26 (95% CI, 0.54–2.95). Of the 19 seropositive and aborting cows, 1, 10 and 7 cows aborted during the first, second and third trimesters of gestation, respectively, and the abortion time was unknown for one cow. 3.3. Distribution of seropositive cows in different age groups As shown in Table 3, the seroprevalence varied in different age groups, ranging from 6.7% to 36.4%. There were no statistically significant differences among the age groups (x2 = 8.782, P = 0.3610) or association of N. caninum infection with age. 3.4. Association between seroprevalence and number of pregnancies Of the 262 cows tested, 158 cows had calved, and 24 were heifers, while the pregnancy history of 80 cows Table 4 Seroprevalence of N. caninum in different pregnancy groups Number of pregnancies
Number of cows
Number of positive serum
Seroprevalence* (%)
0 1 2 3 4 5 6 >7
24 21 64 25 25 14 6 3
3 8 12 4 6 5 1 0
12.5 38.1 18.8 16 24 35.7 16.7 0
80
3
3.8
Unknown *
2
x = 8.006, P = 0.332
was unknown. The seroprevalence of N. caninum infection in different pregnancy groups is shown in Table 4. The seroprevalence of cows that had given one birth was the highest (38.1%), but differences between pregnancy groups were not statistically significant (x2 = 8.006, P = 0.332). The prevalence of seropositivity in heifers was 12.5%. 4. Discussion Since the discovery of N. caninum in the late 1980s, a number of serological assays have been developed for the detection of antibodies to N. caninum. The ELISA by Hipra, Spain used in this study is a commercially available method, which has been validated and previously used by von Blumroder et al. (2004). The seroprevalence of T. gondii and N. caninum in cattle and water buffaloes has been studied in many countries. It varied between countries, regions, herds and even at different times of the same herds. This is the first study of seroepidemiology of N. caninum in cattle and water buffaloes in China. The overall seroprevalence in dairy cattle in 9 regions/provinces was 17.2%, similar to the reported prevalence in Brazil (17.8%) (Corbellini et al., 2006) and in Argentina (16.6%) (Moore et al., 2002). Herd seroprevalence in our survey varied from 0.0% to 34.4%, and the differences were statistically significant between herds or regions. N. caninum positive sera were found in eight out of nine regions, indicating that neosporosis in dairy cattle is widespread in China. There have been many reports on the association between seroprevalence of neosporosis and abortion. Koiwai et al. (2005) found that seropositive cattle of N. caninum infection were 9.2 times more likely to abort compared to seronegative cattle, and a similar odds ratio (8.0) was observed by Vaclavek et al. (2003). However, relatively low odds ratios, 1.1 and 2.0, were reported by Garcia-Vazquez et al. (2005) and Hobson et al. (2005), respectively. Although the association of abortion with neosprora seropositivity in our study was not very strong (odds ratio, 1.3), there was some evidence of neosporosis-related abortion. A relatively high prevalence of neosporosis (34.4%) was observed in a Beijing dairy farm, which had persistent abortions. Higher seroprevalence of N. caninum in abortion positive herds than abortion control herds was also reported by Hobson et al. (2005). Our study found that abortions in N. caninum positive cows occurred mostly in the second and third trimesters, consistent with the finding by Weston et al. (2005), who reported that abortions occurred
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predominantly between Days 120 and 152 gestation among N. caninum seropositive dairy cows. Predominantly mid-term abortions due to N. caninum were probably related to the low immune response of cows during this period of gestation. In this study, only one blood sample was taken from each cow, and blood samples were not collected at a certain time after abortion. Jenkins et al. (1997) demonstrated long persistence of antibodies to N. caninum after abortion, suggesting the time of blood sampling after abortion is not critical for the detection of anti-neospora antibodies. It was unlikely that aborting cows due to N. caninum infection were not detected in this study. A Netherland’s study showed similar seroprevalence across all age groups, suggesting that the infection had most likely been perpetuated by vertical transmission (Wouda et al., 1999). However, a Danish study showed that seropositivity increased with age (Jensen et al., 1999). Neither of these findings was observed in our study, in which there was no significant association between the age of the animals and seroprevalence. The varied seroprevalence in different age groups suggested the possibility of horizontal (postnatal) transmission in the investigated herds. With regard to the relationship between the number of pregnancies and seroprevalence of N. caninim in cattle, Jensen et al. (1999) found that seropositivity rose with increasing numbers of pregnancy, but a positive relationship was not observed in our study. There were many reports on the seroepidemiology of N. caninum in water buffaloes (Fujii et al., 2001; Guarino et al., 2000; Hilali et al., 1998; Rodrigues et al., 2004, 2005) and beef cattle (Haddad et al., 2005; Kim et al., 2002; Tennent-Brown et al., 2000). The seroprevalence of N. caninum varied between studies and herds (0–70.9% in water buffaloes and 2.8–4.1% in beef cattle), but there have been no published reports on the seroprevalence in these animals in China. Our study detected no antibodies to N. caninum or T. gondii in water buffaloes or beef cattle. However, only a limited number of buffaloes and beef cattle were studied in this survey, and further studies with a larger sample size are needed to investigate the epidemiology of N. caninum and T. gondii infections in these livestock. The seroprevalence of T. gondii in dairy cows was 2.3%, which is in line with results obtained by Lin et al. (1990), but it was much lower than that (10.5–27.9%) observed in other countries (Huong et al., 1998; Samad et al., 1997; van Knapen et al., 1995). None of the cows had antibodies against both T. gondii and N. caninum. This indicated that the assays for T. gondii and N. caninum antibodies were specific for the corresponding
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protozoan (i.e. no cross-reactions), and the cattle were not infected with both parasites. Several researchers had reported similar results (Bjorkman et al., 1994; Lally et al., 1996), but others (Bae et al., 2000; Huong et al., 1998; Ooi et al., 2000) found antibodies to both parasites in the serum of dairy cows. In conclusion, this is the first seroprevalence study of N. caninum in cattle and water buffaloes in the People’s Republic of China. The results suggested that neosporosis in dairy cattle is widespread in China. It may play an important role in the abortion of dairy cattle, although the association between abortion and seropositivity was not statistically significant. Further studies with a large number of samples are necessary to confirm whether N. caninum infection is a significant factor of abortions in dairy cattle in China. The current study found no seropositivity for N. caninum and T. gondii in water buffaloes and beef cattle, but the number of samples investigated was small, and the epidemiology needs further investigation. Acknowledgements We thank local veterinary practitioners for collecting blood samples and Dr. Jin Zhu, Therapeutic Goods Administration, Canberra, Australia, for revising the manuscript. We wish to thank Hipra Corp., Spain for providing the CIVTEST bovis Neospora ELISA kit. This study was supported by grants from the National Natural Science Foundation of China (30371080), Natural Science Foundation of Beijing (6042016) and the National Key Technologies R & D Program (2004BA514A18B). References Akca, A., Gokce, H.I., Guy, C.S., McGarry, J.W., Williams, D.J., 2005. Prevalence of antibodies to Neospora caninum in local and imported cattle breeds in the Kars province of Turkey. Res. Vet. Sci. 78, 123–126. Antony, A., Williamson, N.B., 2001. Recent advances in understanding the epidemiology of Neospora caninum in cattle. N. Z. Vet. J. 49, 42–47. Bae, J.S., Kim, D.Y., Hwang, W.S., Kim, J.H., Lee, N.S., Nam, H.W., 2000. Detection of IgG antibody against Neospora caninum in cattle in Korea. Kor. J. Parasitol. 38, 245–249. Barber, J.S., Trees, A.J., 1998. Naturally occurring vertical transmission of Neospora caninum in dogs. Int. J. Parasitol. 28, 57–64. Bartels, C.J., van Maanen, C., van der Meulen, A.M., Dijkstra, T., Wouda, W., 2005. Evaluation of three enzyme-linked immunosorbent assays for detection of antibodies to Neospora caninum in bulk milk. Vet. Parasitol. 131, 235–246. Bergeron, N., Fecteau, G., Pare, J., Martineau, R., Villeneuve, A., 2000. Vertical and horizontal transmission of Neospora caninum in dairy herds in Quebec. Can. Vet. J. 41, 464–467.
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