Identification of latent neosporosis in sheep in Tehran, Iran by polymerase chain reaction using primers specific for the Nc-5 gene

Little is known about latent infection and molecular characterisation of Neospora caninum in sheep (Ovis aries). In this study, 330 sheep samples (180 hearts and 150 brains) were analysed for N. caninum DNA by nested polymerase chain reaction (PCR) targeting the Nc-5 gene. Neospora caninum DNA was detected in 3.9% (13/330) of sheep samples. The parasite’s DNA was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. No clinical signs were recorded from infected or uninfected animals. Sequencing of the genomic DNA revealed 96% – 99% similarity with each other and 95.15% – 100% similarity with N. caninum sequences deposited in GenBank. To our knowledge, this is the first report on the use of PCR to identify latent neosporosis in sheep in Iran. The results of this study have the potential to contribute to our understanding of the role of N. caninum-infected sheep in the epidemiology of neosporosis.

Until now, different genes such as internal transcribed spacer sequences, 18S-like ribosomal DNA (small-subunit rDNA), and Nc-5 genes have been used for molecular diagnosis of neosporosis (reviewed by Al-Qassab, ; Goodswen, Kennedy & Ellis [2013]). However, studies have indicated that the Nc-5 gene is one of the most highly sensitive and specific for the detection of neosporosis (Almerıa et al. 2002;Dubey et al. 2014;Hughes et al. 2006;Kaufmann et al. 1996;Paula et al. 2004;Yamage, Flechtner & Gottstein 1996) because it is repeated in the N. caninum sequence (Al-Qassab et al. 2010). Hence, the main objective of this study was to investigate detection and molecular characterisation of latent neosporosis in sheep (Ovis aries) in Tehran, Iran, by polymerase chain reaction (PCR) using primers specific for the Nc-5 gene.

Animals and study area
A total of 330 samples from healthy slaughtered sheep (180 hearts and 150 brains) were purchased from an abattoir in Vavan (located in the vicinity of Tehran) from April to September 2014. The animals tested originated from different counties (Eslamshahr, Shahriar, Robatkarim), all of which are located between 50 km and 200 km from Tehran. These locations have hot summers and moderate winters. No clinical signs such as fever, lymphadenitis, nasal and ocular discharges, or jaundice were recorded in any of the animals before slaughter.
Little is known about latent infection and molecular characterisation of Neospora caninum in sheep (Ovis aries). In this study, 330 sheep samples (180 hearts and 150 brains) were analysed for N. caninum DNA by nested polymerase chain reaction (PCR) targeting the Nc-5 gene. Neospora caninum DNA was detected in 3.9% (13/330) of sheep samples. The parasite's DNA was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. No clinical signs were recorded from infected or uninfected animals. Sequencing of the genomic DNA revealed 96% -99% similarity with each other and 95.15% -100% similarity with N. caninum sequences deposited in GenBank. To our knowledge, this is the first report on the use of PCR to identify latent neosporosis in sheep in Iran. The results of this study have the potential to contribute to our understanding of the role of N. caninum-infected sheep in the epidemiology of neosporosis.

Identification of latent neosporosis in sheep in Tehran,
Iran by polymerase chain reaction using primers specific for the Nc-5 gene Read online: Scan this QR code with your smart phone or mobile device to read online.

DNA extraction
The whole brain and heart of each sheep were individually rinsed with distilled water, packaged, and refrigerated. Approximately 200 g -250 g of different segments of brain and heart were homogenised with a pestle and mortar in liquid nitrogen, and DNA was extracted using a phenolchloroform extraction method as described in our recent report (Abdoli et al. 2015). To prevent DNA crosscontamination, all materials that were used between different tissue samples were decontaminated with sodium hypochlorite solution (2.5%) and rinsed with distilled water. The concentration of DNA was determined by NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) for each sample. Overall, the DNA concentration ranged between 150 ng/μL and 200 ng/μL.

Nested polymerase chain reaction
Nested PCR was conducted using specific primers for the Nc-5 gene. The first round of PCR was conducted using a pair of N. caninum-specific primers, Np21plus (5'-CCCAGTGCGTCCAATCCTGTAAC-3') and Np6plus (5'-CTCGCCAGTCCAACCTACGTCTTCT-3') (Muller et al. 1996). Nested PCR was performed with the primers Np6 (5'-CAGTCAACCTACGTCTTCT-3') and Np7 (5'-GGGTGAACCGAGGGAGTTG-3') (Hughes et al. 2006). Each amplification was performed in 20-μL reaction mixtures containing 10 μL of 2x master mixes (DFS Master Mix, BIORON GmbH, Ludwigshafen, Germany), each of the respective primers (10 pmol for the first round reaction and 25 pmol for nested PCR), 7 μL of distilled water, and 1 μL of template DNA. One microlitre of the first round product was used as the template for nested PCR. For each reaction, a negative control (double distilled water) and a positive control (DNA extracted from the Nc-5 strain of N. caninum) were included. Amplification was performed with initial denaturation for 5 minutes at 94 °C, followed by 40 cycles at 94 °C for 40 seconds (denaturation), annealing at 62 °C in the first round, and 56 °C in nested PCR for 40 seconds, extension at 72 °C for 40 seconds, and final extension at 72 °C for 10 minutes. PCR products were electrophoresed on a 1.5% agarose gel stained with safe stain (Sinaclon, Tehran, Iran) and visualised under ultraviolet trans-illumination.

Nucleotide sequence analysis
Four positive PCR products (from the second reaction) were amplified with a master mix containing Pfu DNA polymerase (Thermo Fisher Scientific, Waltham, USA, cat. no. EP0501), extracted from the gel (Vivantis gel purification kit, Selangor Darul Ehsan, Malaysia) according to the manufacturer's protocols. Then the products were sequenced in the forward and reverse directions by Sequetech (Mountain View, CA, USA) (Abdoli et al. 2015). The sequences were edited with BioEdit sequence alignment editor (Hall 1999), aligned with Nc-5 partial sequences from other hosts by ClustalX2.12 (Larkin et al. 2007) and compared with sequences of N. caninum available in GenBank. Phylogenetic trees were inferred and evolutionary analyses were conducted using the Tamura three-parameter option of the neighbour-joining model with MEGA6 software (http://www.megasoftware. net/) (Tamura et al. 2013). The bootstrap scores were calculated for 1000 replicates (Tamura et al. 2013).

Results
Neospora caninum DNA was detected in 13 out of 330 sheep samples (3.9%). The infection rates in the heart and brain samples were 6.7% (12/180) and 0.7% (1/150), respectively. Four nucleotide sequences of the Nc-5 gene with a length of 227 bp (Figure 1) were submitted to the GenBank database (GenBank accession numbers KR106181, KR106182, KR106183, KR106184). The results demonstrated our sequences shared 96% -99% similarity with each other (Figures 2 and 3) and 95% -100% similarity with N. caninum deposited in GenBank (Appendix Figure 1). Phylogenetic trees showed intraspecific variations between our isolates and other N. caninum specimens deposited in GenBank ( Figure 2

Discussion
Although an association between ovine abortion and neosporosis has been reported in different studies (Dubey & Lindsay 1990;Howe et al. 2008Howe et al. , 2012Jolley et al. 1999;Moreno et al. 2012;Pena et al. 2007), there is little information describing molecular detection of latent neosporosis in sheep. Here, we found a total infection rate of 3.9% (13/330) in our sheep samples. Interestingly, 12 out of 13 positive samples were detected in the hearts and one positive sample was diagnosed in the brain. In previous studies, the seroprevalence of N. caninum has been reported in a range of 1.1% -8.3% of sheep from the west of Iran (Ezatpour et al. 2015;Gharekhani & Heidari 2014). Moreover, N. caninum DNA was detected in 8.5% (Asadpour et al. 2013) and 0.9% of aborted ovine fetuses  in Iran (Sasani et al. 2013 (Ş uteu et al. 2013). In the majority of studies, N. caninum was detected in brain samples from aborted or naturally infected sheep (Asadpour et al. 2013;Bishop et al. 2010;Dubey & Lindsay 1990;Sasani et al. 2013;Silva et al. 2009). In this regard, Silva and colleagues detected N. caninum DNA in 2 out of 102 slaughtered goats (1.92%) in Brazil. Interestingly, both positive samples were isolated from brain samples, whereas all heart and tongue samples were negative (Silva et al. 2009). Santos et al. (2010) detected N. caninum DNA in 5 out of 100 brain samples of beef cattle in Brazil, whereas none of the heart samples were positive (Santos et al. 2010). These results are dissimilar to our report, in which most of the positive samples were detected in the heart samples rather than in the brain samples (6.7% versus 0.7%). Our results also indicated that in sheep the heart is more susceptible to N. caninum infection than the brain. In the current study, we used the Nc-5 gene for detection and phylogenetic analysis of N. caninum. This gene is repeated in the N. caninum sequence (Al-Qassab et al. 2010); hence, it is presented as a highly sensitive and specific gene for detection of neosporosis (Kaufmann et al. 1996;Yamage et al. 1996). The earlier study in this regard was conducted by Yamage et al. (1996), who compared the sensitivity and specificity of different primers for diagnosis of N. caninum. In this study, five forward (Np1, Np3, Np5, Np7, Np21) and four reverse (Np2, Np4, Np6, Np8) oligonucleotide primers that derived from the Nc-5 genes were compared for the detection of An asterisk represents an exact match between all sequences; a dot (•) represents a mismatch in at least one sequence.
We also sequenced four positive samples for phylogenetic analysis. We found that our sequences displayed similarity levels of 96% -99% with each other (Figure 3) and 95% -100% with N. caninum sequences deposited in GenBank (Appendix Figure 1). In comparison with molecular diagnosis, few studies have been conducted on the phylogenetic analysis of N. caninum with the Nc-5 gene (Auriemma et al. 2014;Č obádiová et al. 2013;Hughes et al. 2006). BLAST analyses indicated greater than 94% (Č obádiová et al. 2013), 96% (Auriemma et al. 2014), and 97% (Hughes et al. 2006) similarities between their sequences and other N. caninum sequences deposited in GenBank. It therefore seems that the Nc-5 gene is not a suitable biomarker for phylogenetic analysis and discrimination of genetic diversity for N. caninum. Instead, this gene is rather a highly sensitive and specific biomarker for the diagnosis of neosporosis. The use of ribosomal DNA, ITS-1, and recently microsatellites have been recommended for discriminating between N. caninum isolates (Al-Qassab et al. 2010).
Taken together, the results of this study provide molecular and epidemiological information about latent N. caninum infection in sheep in Iran. It can be expected that in future these results will contribute to revealing the role of latent N. caninum infection in the epidemiology of neosporosis in sheep.