Veterinary Parasitology 205 (2014) 361–364
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Detection of Neospora caninum-DNA in feces collected from dogs in Shenyang (China) and ITS1 phylogenetic analysis Jianhua Li a,1 , Pengfei He a,b,1 , Yanhui Yu a , Ling Du a , Pengtao Gong a , Guocai Zhang a , Xichen Zhang a,∗ a
Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, China Key Laboratory of Animal Clinical Diagnosis Technology, Ministry of Agriculture, Inter Mongolia Agricultural University, Huhhot 010018, China b
a r t i c l e
i n f o
Article history: Received 22 April 2014 Received in revised form 22 June 2014 Accepted 28 June 2014 Keywords: Neospora caninum Nested-PCR Dog Nc5 ITS1
a b s t r a c t Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Dog is known as a definitive host of N. caninum and involved in transmitting infections to intermediate hosts by shedding oocysts. To investigate the epidemiology of dog neosporosis in China, 212 dog feces specimens in Shenyang were screened by nested-PCR using Nc5 primers and confirmed by N. caninum ITS1 PCR. The positive rate of N. caninum DNA was 34.90% (74/212). There were no significant correlations in prevalence of Neospora infections between different ages and genders. N. caninum DNA positive samples were further examined by PCR using Hammondia heydorni-specific primers. 37 out of 74 N. caninum DNA positive samples were also H. heydorni DNA positive. Only Nc5 primers positive and H. heydorni primers negative samples were used for ITS1 gene sequence analysis. Sequencing results from the 37 N. caninum positive samples revealed that ITS1 gene has 96–100% similarity with N. caninum sequences deposited in Genbank. Also, the presence of a new genotype indicated genetic polymorphism of N. caninum in infected dog feces in Shenyang of China. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Neospora caninum is an obligate intracellular protozoan of the phylum apicomplexa with a wide range of intermediate hosts (Dubey, 2003). N. caninum causes neuromuscular disease in dogs and abortions in cattle (Dubey and Lindsay, 1996). Dogs, coyotes, foxes, and wolves have been found as the natural or experimental definitive hosts and N. caninum sexual cycle occurs in the gastrointestinal tract, resulting in the passage of unsporulated oocysts in feces (Basso et al.,
∗ Corresponding author. Tel.: +86 431 87981351; fax: +86 431 87981351. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.vetpar.2014.06.036 0304-4017/© 2014 Elsevier B.V. All rights reserved.
2001; Gondim et al., 2004; King et al., 2010; Wapenaar et al., 2006). Dogs can be co-infected with clinically important N. caninum and the non-pathogenic Hammondia heydorni that are closely related morphologically and biologically, which are not distinguishable microscopically (Dubey, 1993; Jan et al., 2002; McAllister et al., 1998). As definitive hosts of N. caninum, farm dogs can transmit N. caninum postnatally by ingestion of tachyzoites infected tissues or tissue cysts or by ingestion of sporulated oocysts contaminated food or drinking water (Dubey, 2003), which is a risk factor for seropositivity in cattle (Corbellini et al., 2006; Dijkstra et al., 2002; Schares et al., 2004). N. caninum infection in dairy cows, beef cattle, yaks and water Buffaloes has been investigated in different regions of China (Yu et al., 2007, 2008). Although seroprevalence of N. caninum has been reported at 31.25% in Shepherd Dogs of
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Table 1 Prevalence of N. caninum infection and Co-infection with Hammondia heydorni in naturally infected dog in Shenyang. Gender and age
Feces (n)
Nc-positive (n)
Nc-prevalence (%)
Nc + Hh-positive (n)
Nc + Hh-prevalence (%)
FA FJ MA MJ Total
59 64 52 37 212
23 22 17 12 74
38.98 34.38 32.69 32.43 34.90
2 21 7 7 37
0.03 0.33* 0.13 0.19 0.17
Notes: FA, female over 1 year old; FJ, female less than 1 year old; MA, male over 1 year old; MJ, male less than 1 year old. Nc: N. caninum; Hh: Hammondia heydorni. * P < 0.05.
Wulan county of Qinghai province (Niu and Ma, 2008), N. caninum prevalence in dogs as the definitive host and phylogenetic analysis is rarely reported in China. As for N. caninum DNA detection, PCR, nested-PCR and real time PCR using primers based on Nc5 or ITS1 sequences have been used for examining samples of brain, feces and other tissues (Ellis et al., 1998; Ghalmi et al., 2008; Moskwa et al., 2008; Sánchez et al., 2009; Truppel et al., 2010; Yamage et al., 1996). The aim of this study was to survey N. caninum infection in farm guard dogs and police dogs as definitive host in the rural area of Shenyang. N. caninum DNAs extracted from fecal specimens from 212 dogs were screened using a nested-PCR assay based on Nc5 region and the results were further verified by ITS1 sequences analysis.
2. Materials and methods 2.1. Fecal specimens Fecal specimens (212) were obtained from farmers guard dogs and police dog from October to December 2012 in rural area of Shenyang, China. All specimens were assigned to two age groups as follows: over 1 year old (59 females and 52 males), and less than 1 year old (64 females and 37 males).
2.2. N. caninum DNA detection from fecal specimens The fecal specimens DNAs were extracted using the FastDNA spin kit for feces (AXYGEN, USA) following the manufacturer’s instructions. For the detection of N. caninum DNA, the N. caninum specific gene Nc5 was amplified by nested PCR using primers: (OF: 5 -CAGTCAACCTACGTCTTC-3 ) and (OR: 5 GTGCGTCCAATCCTGTAA-3 ) in the primary PCR (328 bp) and primers: (IF: 5 -GTT GCT CTG CTG ACG TGT CGT TG-3 ) and (IR: 5 -CTCA ACA CAG AAC ACT GAA CTC TCG-3 ) in the secondary PCR (224 bp) (Yamage et al., 1996). The primary PCR condition was as followings: an initial denaturation step at 94 ◦ C for 3 min followed by 35 cycles of 94 ◦ C for 1 min, 53 ◦ C for 1 min, 72 ◦ C for 2 min and a final extension step at 72 ◦ C for 10 min. The condition for the secondary PCR was identical to the primary PCR, except that the annealing temperature was increased to 63 ◦ C. N. caninum NC-1 served as the positive control and PCR reactions without DNA served as the negative control. N. caninum DNA positive samples were further screened for
H. heydorni DNA by PCR using H. heydorni-specific primers JS4-JS5 according to Slapeta et al. (2002). 2.3. ITS1 sequences amplification of N. caninum and phylogenetic analysis ITS-1 sequences were amplified by PCR using tim3/tim11 primers from N. caninum DNA positive and H. heydorni DNA negative samples as described previously (Ellis et al., 1998). PCR products were sequenced by Sangon Biotech Co., Ltd (Shanghai) and analyzed with ClustalW (Version 2.1). Maximum parsimony approaches combined with the branch and bound algorithm were used to find the most parsimonious tree(s) by MEGA (Version 5). 2.4. Statistical analysis Data were analyzed using SPSS 15.0. Chi-square analysis was used to analyze the differences in prevalence between ages and genders. 3. Results 3.1. Prevalence of N. caninum infection in naturally infected dogs in Shenyang To assess N. caninum canine infection, 212 dog feces specimens in Shenyang of China were screened by nestedPCR using Nc5 primers. A specific 224 bp fragment was amplified in 74 out of 212 feces and the overall prevalence was 34.90% (Table 1). The positive rates were 36.58% (45/123) in female, 32.58% (29/89) in male, 36.04% (40/111) in dogs over 1 year old, 33.66% (34/101) in dogs less than 1 year old. There was no significant difference between ages and genders. 3.2. Co-infection of N. caninum with H. heydorni H. heydorni DNA was found in 37 out of 74 N. caninumpositive samples by PCR using H. heydorni-specific primers. Prevalence of N. caninum co-infection with H. heydorni was 50.00% (37/74) or 17.45% (37/212) (Table 1). 3.3. Phylogenetic analysis based on N. caninum ITS1 Sequences Only Nc5 primers positive and H. heydorni primers negative samples were used for ITS1 gene sequence analysis.
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The PCR products of N. caninum ITS1 gene were sequenced to further confirm N. caninum infection. All 37 N. caninum ITS1 sequences revealed 96% -100% of similarity with N. caninum sequences deposited in the Genbank and were clustered into N. caninum group using ClustalW analysis. The mutation regions were found in MJ85 and MA 138 ITS1 sequences by MEGA analysis, indicating two genotypes: the predominate genotype (35 ITS1 sequences) and a new genotype (MJ85 and MA 138 ITS1 sequences).
sequences deposited in Genbank, suggesting that it was the epidemic genotype in Shenyang. Genetic mutations found in MJ85 and MA 138 ITS1 sequences suggested that genetic polymorphism of N. caninum isolates existed in that region. In conclusion, this is the first reported detection of N. caninum DNA in dog feces and epidemic genotype in China. Further epidemiological studies are required to determine the distribution of N. caninum infection in canine definitive hosts in China.
4. Discussion
Acknowledgement
Dogs are known as a definitive host of N. caninum. Animals can be infected through ingestion of sporulated oocysts shed in the feces of acutely infected dogs in contaminated food or drinking, which has led to proposed dog control mechanism on farms for reducing infection transmission to livestock (Dubey et al., 2007). However, N. caninum cannot be distinguished from Hammondia Neospora-like oocysts (HNLO) microscopically according to morphology in fecal samples from dogs (Schares et al., 2001). Molecular detection assays such as PCR are superior because of higher sensitivity and specificity (Jenkins et al., 2002). It has been suggested that N. caninum-specific PCRs based on Nc5 or ITS1 are suitable methods for distinguishing between N. caninum and HNLO DNA in fecal samples from dogs (Jenkins et al., 2002; Palavicini et al., 2007; Sager et al., 2006). In this study, N. caninum-specific nested-PCR using Nc5 primers were applied for N. caninum DNA detection in infected dog feces in Shenyang of China. Positive specimens were further confirmed by N. caninum ITS1 PCR. Seroprevalence of N. caninum has been reported at 31.25% (25/80) using ELISA in Shepherd Dogs of Wulan county of Qinghai province (Niu and Ma, 2008). PCR has been used in N. caninum DNA detection in dog feces in many countries. In the present study, a high prevalence of N. caninum infection (34.90%) was found in dogs in Shenyang of China. No significant correlations were found in prevalence of Neospora infection between different genders and ages in infected dogs in Shenyang when they acted as definitive host in N. caninum life cycle. Based on current data, changes in regulation have been proposed to separate the living and waste treatment areas of farm animals from dogs. The ribosomal ITS1 gene, located between 18S and 5.8S, is a fast evolving gene and more suitable for genotyping than 18S RNA. ITS1 sequences for NC-PR (DQ997618.1), NC-Illinois (AY259041.1), NC-SweB1 (EU564167.1), NCGNCBA2 (FJ176222.1), NC-BR/MG-3(HM229412.1), NCSP-(HQ542299.1) NC-Tanami (HQ873010.1) and NC-AL (DQ997618.1) had been deposited in Genbank. Neospora and H. heydorni are not only morphologically indistinguishable; their ITS1 sequences also share very high homology. To identify only Neospora positive among often co-infected samples, as shown in the present study with 50% coinfection rate (38/74), positive samples identified with Nc5 specific primers were further screened with Hammondia specific primers; only Nc5 primer positive and Hammondia primer negative samples were used for ITS1 gene sequence analysis. All 37 N. caninum ITS1 sequences were classified to N. caninum group and the predominate subgroup with 35 isolates share very high homology with N. caninum ITS1
This study is supported by Department of Jilin Provincial Science and Technology of China (nos. 20100222 and 20130521005JH). References Basso, W., Veturini, L., Venturini, M.C., Hill, D.E., Kwok, O.C.H., Shen, S.K., Dubey, J.P., 2001. First isolation of Neospora caninum from the feces of a naturally infected dog. J. Parasitol. 87, 612–618. Corbellini, L.G., Smith, D.R., Pescador, C.A., Schmitz, M., Correa, A., Steffen, D.J., Driemeier, D., 2006. Herd-level risk factors for Neospora caninum seroprevalence in dairy farms in southern Brazil. Prev. Vet. Med. 74, 130–141. Dijkstra, T., Barkema, H.W., Eysker, M., Hesselink, J.W., Wouda, W., 2002. Natural transmission routes of Neospora caninum between farm dogs and cattle. Vet. Parasitol. 105, 99–104. Dubey, J.P., 1993. Toxoplasma, Neospora, Sarcocystis and other tissue cystforming coccidia of humans and animals. In: Kreier, J.P. (Ed.), Parasitic Protozoa, vol. 6. , second ed. Academic Press, New York, NY, pp. 1–131. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., 2003. Review of Neospora caninum and neosporosis in animals. Korean J. Parasitol. 41, 1–16. Dubey, J.P., Schares, G., Ortega-Mora, L.M., 2007. Epidemiology and control of neosporosis and Neospora caninum. Clin. Microbiol Rev. 20, 323–367. Ellis, J.T., Amoyal, G., Ryce, C., Harper, P.A., Clough, K.A., Homan, W.L., Brindley, P.J., 1998. Comparison of the large subunit ribosomal DNA of Neospora and Toxoplasma and development of a new genetic marker for their differentiation based on the D2 domain. Mol. Cell. Probes 12, 1–13. Ghalmi, F., China, B., Kaidi, R., Daube, G., Losson, B., 2008. Detection of Neospora caninum in dog organs using real time PCR systems? Vet. Parasitol. 155 (1–2), 161–167. Gondim, L.F., McAllister, M.M., Pitt, W.C., Zemlicka, D.E., 2004. Coyotes (Canis latrans) are definitive hosts of Neospora caninum. Int. J. Parasitol. 34, 159–161. ˇSlapeta, Jan R., Modry, ´ David, Kyselová, Iveta, Hˇorejˇs, Radek, Lukeˇs, Julius, ˇ 2002. Dog shedding oocysts of Neospora caninum: Koudela, Bretislav, PCR diagnosis and molecular phylogenetic approach. Vet. Parasitol. 109, 157–167. Jenkins, M., Baszler, T., Björkman, C., Schares, G., Williams, D., 2002. Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion. Int. J. Parasitol. 32, 631–636. King, J.S., Slapeta, J., Jenkins, D.J., Al-Qassab, S.E., Ellis, J.T., Windsor, P.A., 2010. Australian dingoes are definitive hosts of Neospora caninum. Int. J. Parasitol. 40, 945–950. McAllister, M.M., Dubey, J.P., Lindsay, J.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473–1478. ´ ´ J., Cabaj, W., 2008. Studies on Neospora Moskwa, B., Gozdzik, K., Bien, caninum DNA detection in the oocytes and embryos collected from infected cows. Vet. Parasitol. 158, 370–375. Niu, X.Y., Ma, L.Q., 2008. Serodiagnosis of Neospora Caninum infection in shepherd dogs of Wulan county of Qinghai province. Chin. Anim. Husb. Vet. Med. 35, 124–125. Palavicini, P., Romero, J.J., Dolz, G., Jiménez, A.E., Hill, D.E., Dubey, J.P., 2007. Fecal and serological survey of Neospora caninum in farm dogs in Costa Rica. Vet. Parasitol. 149, 265–270. Sager, H., Moret, C.S., Müller, N., Staubli, D., Esposito, M., Schares, G., Hässig, M., Stärk, K., Gottstein, B., 2006. Incidence of Neospora caninum
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and other intestinal protozoan parasites in populations of Swiss dogs. Vet. Parasitol. 139, 84–92. Sánchez, G.F., Banda, R.V., Sahagun, R.A., Ledesma, M.N., Morales, S.E., 2009. Comparison between immunohistochemistry and two PCR methods for detection of Neospora caninum in formalin-fixed and paraffin-embedded brain tissue of bovine fetuses? Vet. Parasitol. 164 (2–4), 328–332. Schares, G., Heydorn, A.O., Cüppers, A., Conraths, F.J., Mehlhorn, H., 2001. Hammondia heydorni-like oocysts shed by a naturally infected dog and Neospora caninum NC-1 cannot be distinguished. Parasitol. Res. 87, 808–816. ¨ ¨ Schares, G., Barwald, A., Staubach, C., Ziller, M., Kloss, D., Schroder, R., ¨ Labohm, R., Drager, K., Fasen, W., Hess, R.G., Conraths, F.J., 2004. Potential risk factors for bovine Neospora caninum infection in Germany are not under the control of the farmers. Parasitology 129, 301–309. ´ ´ D., Horejs, R., Lukes, J., Slapeta, J.R., Koudela, B., Votypka, J., Modry, 2002. Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum. Vet. J. 163, 147–154.
Truppel, J.H., Montiani-Ferreira, F., Lange, R.R., Vilani, R.G., Reifur, L., Boerger, W., da Costa-Ribeiro, M.C., Thomaz-Soccol, V., 2010. Detection of Neospora caninum DNA in capybaras and phylogenetic analysis. Parasitol. Int. 59, 376–379. Wapenaar, W., Jenkins, M.C., O’Handley, R.M., Barkema, H.W., 2006. Neospora caninum-like oocysts observed in feces of free-ranging red foxes (Vulpes vulpes) and coyotes (Canis latrans). J. Parasitol. 92, 1270–1274. Yamage, M., Flechtner, O., Gottstein, B., 1996. Neospora caninum: specific oligonucleotide primers for the detection of brain “cyst” DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). J. Parasitol. 82, 272–279. Yu, J.H., Xia, Z.F., Liu, Q., Liu, J., Ding, J., Zhang, W., 2007. Seroepidemiology of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes (Bubalus bubalis) in the People’s Republic of China. Vet. Parasitol. 143, 79–85. Yu, J.H., Xia, Z.F., Liu, Q., Liu, J., Ding, J., Zhang, W., 2008. Seroepidemiology of Neospora caninum and Toxoplasma gondii infection in yaks (Bos grunniens) in Qinghai, China. Vet. Parasitol. 152, 330–332.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Detection of Neospora caninum-DNA in feces collected from dogs in Shenyang (China) and ITS1 phylogenetic analysis Jianhua Li a,1 , Pengfei He a,b,1 , Yanhui Yu a , Ling Du a , Pengtao Gong a , Guocai Zhang a , Xichen Zhang a,∗ a
Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, China Key Laboratory of Animal Clinical Diagnosis Technology, Ministry of Agriculture, Inter Mongolia Agricultural University, Huhhot 010018, China b
a r t i c l e
i n f o
Article history: Received 22 April 2014 Received in revised form 22 June 2014 Accepted 28 June 2014 Keywords: Neospora caninum Nested-PCR Dog Nc5 ITS1
a b s t r a c t Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Dog is known as a definitive host of N. caninum and involved in transmitting infections to intermediate hosts by shedding oocysts. To investigate the epidemiology of dog neosporosis in China, 212 dog feces specimens in Shenyang were screened by nested-PCR using Nc5 primers and confirmed by N. caninum ITS1 PCR. The positive rate of N. caninum DNA was 34.90% (74/212). There were no significant correlations in prevalence of Neospora infections between different ages and genders. N. caninum DNA positive samples were further examined by PCR using Hammondia heydorni-specific primers. 37 out of 74 N. caninum DNA positive samples were also H. heydorni DNA positive. Only Nc5 primers positive and H. heydorni primers negative samples were used for ITS1 gene sequence analysis. Sequencing results from the 37 N. caninum positive samples revealed that ITS1 gene has 96–100% similarity with N. caninum sequences deposited in Genbank. Also, the presence of a new genotype indicated genetic polymorphism of N. caninum in infected dog feces in Shenyang of China. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Neospora caninum is an obligate intracellular protozoan of the phylum apicomplexa with a wide range of intermediate hosts (Dubey, 2003). N. caninum causes neuromuscular disease in dogs and abortions in cattle (Dubey and Lindsay, 1996). Dogs, coyotes, foxes, and wolves have been found as the natural or experimental definitive hosts and N. caninum sexual cycle occurs in the gastrointestinal tract, resulting in the passage of unsporulated oocysts in feces (Basso et al.,
∗ Corresponding author. Tel.: +86 431 87981351; fax: +86 431 87981351. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.vetpar.2014.06.036 0304-4017/© 2014 Elsevier B.V. All rights reserved.
2001; Gondim et al., 2004; King et al., 2010; Wapenaar et al., 2006). Dogs can be co-infected with clinically important N. caninum and the non-pathogenic Hammondia heydorni that are closely related morphologically and biologically, which are not distinguishable microscopically (Dubey, 1993; Jan et al., 2002; McAllister et al., 1998). As definitive hosts of N. caninum, farm dogs can transmit N. caninum postnatally by ingestion of tachyzoites infected tissues or tissue cysts or by ingestion of sporulated oocysts contaminated food or drinking water (Dubey, 2003), which is a risk factor for seropositivity in cattle (Corbellini et al., 2006; Dijkstra et al., 2002; Schares et al., 2004). N. caninum infection in dairy cows, beef cattle, yaks and water Buffaloes has been investigated in different regions of China (Yu et al., 2007, 2008). Although seroprevalence of N. caninum has been reported at 31.25% in Shepherd Dogs of
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Table 1 Prevalence of N. caninum infection and Co-infection with Hammondia heydorni in naturally infected dog in Shenyang. Gender and age
Feces (n)
Nc-positive (n)
Nc-prevalence (%)
Nc + Hh-positive (n)
Nc + Hh-prevalence (%)
FA FJ MA MJ Total
59 64 52 37 212
23 22 17 12 74
38.98 34.38 32.69 32.43 34.90
2 21 7 7 37
0.03 0.33* 0.13 0.19 0.17
Notes: FA, female over 1 year old; FJ, female less than 1 year old; MA, male over 1 year old; MJ, male less than 1 year old. Nc: N. caninum; Hh: Hammondia heydorni. * P < 0.05.
Wulan county of Qinghai province (Niu and Ma, 2008), N. caninum prevalence in dogs as the definitive host and phylogenetic analysis is rarely reported in China. As for N. caninum DNA detection, PCR, nested-PCR and real time PCR using primers based on Nc5 or ITS1 sequences have been used for examining samples of brain, feces and other tissues (Ellis et al., 1998; Ghalmi et al., 2008; Moskwa et al., 2008; Sánchez et al., 2009; Truppel et al., 2010; Yamage et al., 1996). The aim of this study was to survey N. caninum infection in farm guard dogs and police dogs as definitive host in the rural area of Shenyang. N. caninum DNAs extracted from fecal specimens from 212 dogs were screened using a nested-PCR assay based on Nc5 region and the results were further verified by ITS1 sequences analysis.
2. Materials and methods 2.1. Fecal specimens Fecal specimens (212) were obtained from farmers guard dogs and police dog from October to December 2012 in rural area of Shenyang, China. All specimens were assigned to two age groups as follows: over 1 year old (59 females and 52 males), and less than 1 year old (64 females and 37 males).
2.2. N. caninum DNA detection from fecal specimens The fecal specimens DNAs were extracted using the FastDNA spin kit for feces (AXYGEN, USA) following the manufacturer’s instructions. For the detection of N. caninum DNA, the N. caninum specific gene Nc5 was amplified by nested PCR using primers: (OF: 5 -CAGTCAACCTACGTCTTC-3 ) and (OR: 5 GTGCGTCCAATCCTGTAA-3 ) in the primary PCR (328 bp) and primers: (IF: 5 -GTT GCT CTG CTG ACG TGT CGT TG-3 ) and (IR: 5 -CTCA ACA CAG AAC ACT GAA CTC TCG-3 ) in the secondary PCR (224 bp) (Yamage et al., 1996). The primary PCR condition was as followings: an initial denaturation step at 94 ◦ C for 3 min followed by 35 cycles of 94 ◦ C for 1 min, 53 ◦ C for 1 min, 72 ◦ C for 2 min and a final extension step at 72 ◦ C for 10 min. The condition for the secondary PCR was identical to the primary PCR, except that the annealing temperature was increased to 63 ◦ C. N. caninum NC-1 served as the positive control and PCR reactions without DNA served as the negative control. N. caninum DNA positive samples were further screened for
H. heydorni DNA by PCR using H. heydorni-specific primers JS4-JS5 according to Slapeta et al. (2002). 2.3. ITS1 sequences amplification of N. caninum and phylogenetic analysis ITS-1 sequences were amplified by PCR using tim3/tim11 primers from N. caninum DNA positive and H. heydorni DNA negative samples as described previously (Ellis et al., 1998). PCR products were sequenced by Sangon Biotech Co., Ltd (Shanghai) and analyzed with ClustalW (Version 2.1). Maximum parsimony approaches combined with the branch and bound algorithm were used to find the most parsimonious tree(s) by MEGA (Version 5). 2.4. Statistical analysis Data were analyzed using SPSS 15.0. Chi-square analysis was used to analyze the differences in prevalence between ages and genders. 3. Results 3.1. Prevalence of N. caninum infection in naturally infected dogs in Shenyang To assess N. caninum canine infection, 212 dog feces specimens in Shenyang of China were screened by nestedPCR using Nc5 primers. A specific 224 bp fragment was amplified in 74 out of 212 feces and the overall prevalence was 34.90% (Table 1). The positive rates were 36.58% (45/123) in female, 32.58% (29/89) in male, 36.04% (40/111) in dogs over 1 year old, 33.66% (34/101) in dogs less than 1 year old. There was no significant difference between ages and genders. 3.2. Co-infection of N. caninum with H. heydorni H. heydorni DNA was found in 37 out of 74 N. caninumpositive samples by PCR using H. heydorni-specific primers. Prevalence of N. caninum co-infection with H. heydorni was 50.00% (37/74) or 17.45% (37/212) (Table 1). 3.3. Phylogenetic analysis based on N. caninum ITS1 Sequences Only Nc5 primers positive and H. heydorni primers negative samples were used for ITS1 gene sequence analysis.
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The PCR products of N. caninum ITS1 gene were sequenced to further confirm N. caninum infection. All 37 N. caninum ITS1 sequences revealed 96% -100% of similarity with N. caninum sequences deposited in the Genbank and were clustered into N. caninum group using ClustalW analysis. The mutation regions were found in MJ85 and MA 138 ITS1 sequences by MEGA analysis, indicating two genotypes: the predominate genotype (35 ITS1 sequences) and a new genotype (MJ85 and MA 138 ITS1 sequences).
sequences deposited in Genbank, suggesting that it was the epidemic genotype in Shenyang. Genetic mutations found in MJ85 and MA 138 ITS1 sequences suggested that genetic polymorphism of N. caninum isolates existed in that region. In conclusion, this is the first reported detection of N. caninum DNA in dog feces and epidemic genotype in China. Further epidemiological studies are required to determine the distribution of N. caninum infection in canine definitive hosts in China.
4. Discussion
Acknowledgement
Dogs are known as a definitive host of N. caninum. Animals can be infected through ingestion of sporulated oocysts shed in the feces of acutely infected dogs in contaminated food or drinking, which has led to proposed dog control mechanism on farms for reducing infection transmission to livestock (Dubey et al., 2007). However, N. caninum cannot be distinguished from Hammondia Neospora-like oocysts (HNLO) microscopically according to morphology in fecal samples from dogs (Schares et al., 2001). Molecular detection assays such as PCR are superior because of higher sensitivity and specificity (Jenkins et al., 2002). It has been suggested that N. caninum-specific PCRs based on Nc5 or ITS1 are suitable methods for distinguishing between N. caninum and HNLO DNA in fecal samples from dogs (Jenkins et al., 2002; Palavicini et al., 2007; Sager et al., 2006). In this study, N. caninum-specific nested-PCR using Nc5 primers were applied for N. caninum DNA detection in infected dog feces in Shenyang of China. Positive specimens were further confirmed by N. caninum ITS1 PCR. Seroprevalence of N. caninum has been reported at 31.25% (25/80) using ELISA in Shepherd Dogs of Wulan county of Qinghai province (Niu and Ma, 2008). PCR has been used in N. caninum DNA detection in dog feces in many countries. In the present study, a high prevalence of N. caninum infection (34.90%) was found in dogs in Shenyang of China. No significant correlations were found in prevalence of Neospora infection between different genders and ages in infected dogs in Shenyang when they acted as definitive host in N. caninum life cycle. Based on current data, changes in regulation have been proposed to separate the living and waste treatment areas of farm animals from dogs. The ribosomal ITS1 gene, located between 18S and 5.8S, is a fast evolving gene and more suitable for genotyping than 18S RNA. ITS1 sequences for NC-PR (DQ997618.1), NC-Illinois (AY259041.1), NC-SweB1 (EU564167.1), NCGNCBA2 (FJ176222.1), NC-BR/MG-3(HM229412.1), NCSP-(HQ542299.1) NC-Tanami (HQ873010.1) and NC-AL (DQ997618.1) had been deposited in Genbank. Neospora and H. heydorni are not only morphologically indistinguishable; their ITS1 sequences also share very high homology. To identify only Neospora positive among often co-infected samples, as shown in the present study with 50% coinfection rate (38/74), positive samples identified with Nc5 specific primers were further screened with Hammondia specific primers; only Nc5 primer positive and Hammondia primer negative samples were used for ITS1 gene sequence analysis. All 37 N. caninum ITS1 sequences were classified to N. caninum group and the predominate subgroup with 35 isolates share very high homology with N. caninum ITS1
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