Molecular Identification of Neospora caninum Infection in Aborted Fetuses of Sheep, Cattle, and Goats in Mazandaran Province, Northern Iran.

BACKGROUND: We aimed to identify Neospora caninum DNA in the brain samples of aborted fetuses of cattle, goats, and sheep in Mazandaran, northern Iran, using PCR.
METHODS: In total, 133 aborted fetuses (51 sheep, 78 cattle, and 4 goats) were randomly collected from different stages of gestation in various regions of Mazandaran, Iran, from Mar 2016 to May 2017. The DNA was extracted from all the brain samples using phenol chloroform isoamyl alcohol instructions. The Nc-5 gene was used for the detection of N. caninum DNA by nested-PCR assay.
RESULTS: The detection of N. caninum DNA was confirmed by the observation of a 227 bp band in 24 samples of 133 aborted fetuses (18.1%). The highest prevalence rate of N. caninum was detected in the cattle (20.5%) followed by the sheep (15.6%); however, no positive cases were reported in the goats. The highest and lowest prevalence rates of the infection were reported as 23.8% and 8.6% in Qaemshahr, and Behshahr, respectively. The prevalence rate of infection (32%) in the early gestational period was higher than those in the middle (15%) and late (3.8%) gestational periods.
CONCLUSION: The obtained data of the present study indicated that N. caninum infection may partly be responsible for abortion and economic loss in livestock farming in Mazandaran Province.

Introduction

Neospora caninum, an apicomplexan unicellular parasite, is a major and important cause of bovine abortion with worldwide distribution. Dogs and coyotes are considered definitive hosts; however, a wide range of warm-blooded animals, such as cattle, sheep, buffalos, horses, goats, rodents, and rhinoceros, are the intermediated hosts to N. caninum (1, 2). This intracellular parasite can be a cause of abortion at any stages of pregnancy in cattle; however, it usually occurs at 5–6 months of gestation and may happen more than once in the reproductive seasons (3).

Neosporosis can be horizontally and vertically transmitted in the herds. Congenital transmission is the main route (50–95%) of abortion due to N. caninum and plays an important role in the maintenance of the parasite in farms and herds (4). Nonetheless, due to the relatively limited number of studies, the clinical, epidemiological, and economic importance of neosporosis in sheep, especially in goats is still not clear (5).

There are several methods for the detection of N. caninum in aborted fetuses, including histopathology (6), immunohistochemistry (7), and PCR (8). Brain tissue, as well as the heart and liver, are the best sample for the diagnosis of neosporosis in fetuses. Among the PCR methods for the detection of N. caninum DNA, nested PCR has higher sensitivity and specificity than the other methods because it is able to amplify the small amounts of Neospora DNA in infected tissue (9).

The results of numerous studies performed on the seroprevalence of N. caninum in aborted dairy cattle in Iran indicated neosporosis should be considered a cause of economic and health problems in Iran (10–12). There is no information regarding the molecular detection of N. caninum in aborted fetuses in northern Iran.

Therefore, we aimed to identify N. caninum DNA in ruminant’s aborted fetuses (sheep, goats, and cattle) using nested PCR to evaluate the association of N. caninum with fetus abortion in northern Iran.

Materials and Methods

Ethical approval

Ethical approval was obtained from the Ethics Committee of Mazandaran University of Medical Sciences (no. 10217).

Sample Collection

A total of 133 brain samples obtained from 51 aborted sheep, 78 aborted cattle, and 4 aborted goats were collected from several industrial farms in Mazandaran province, northern Iran, from Mar 2016 to May 2017. The brain of each aborted animal was rinsed in distilled water, packaged, and transferred to the Parasitology Laboratory of Mazandaran University of Medical Sciences for the laboratory examination.

DNA Extraction

Approximately, 5 g of brain from different segments were homogenized with 70% ethanol, and DNA was extracted using phenol-chloroform extraction method. Then, 500 μl of lysis buffer (50 mM Tris-HCL, pH (8.0); 25 mM EDTA and 400 mM NaCl), 50 μl 10% sodium dodecyl sulfate, and 35 μl proteinase K (20 μg/μl) were added to 200 μl homogenized brain samples in 1.5 ml microtubes. The suspension was incubated at 56 °C overnight. For the precipitation of debris and proteins, 200 μl of sodium chloride (6M) was added to the suspension, and it was kept at 4 °C for 30 min. After centrifugation at 14,000 rpm for 15 min, the supernatants were transferred to new microtubes for extraction with phenol chloroform isoamyl alcohol (24:24:1, v/v).

The DNA was precipitated by adding 500 μl of 100% cold ethanol and 20 μl of sodium acetate solution (3M), followed by centrifugation at 14,000 rpm for 10 min and finally kept at -20 °C for 24 h (13). The pellet was washed twice by 70% ethanol and resuspended in 60 μl of Tris-EDTA buffer. The concentration of DNA was determined by NanoDrop ND100 (Thermo Scientific). The extracted DNA was stored at −20 °C until usage.

Nested Polymerase Chain Reaction

Nested PCR was performed to detect the DNA of N. caninum using Nc-5 gene with external primers, including Np21plus (5′-CCCAGTGCGTCCAATCCTGTAAC-3′) and Np6plus (5′ CTCGCCAGTCCAACCTACGTCTTCT-3′), as well as internal primers, including Np6 (5′ CAGTCAACCTACGTCTTCT-3′) and Np7 (5′- GGGTGAACCGAGGGAGTTG-3′) (14, 15). The first step of PCR was carried out to amplify the fragment of 337bp in a volume of 25 μl containing 3 μl of genomic DNA, 0.75 μl of each external primer (10 pmol/μl) (BioNeer, Korea), 12.5 μl of commercial premix (Ampliqon, Denmark), and 8 μl of molecular biology grade H2O. The PCR conditions include initial denaturation for 7 min at 94 °C, 30 cycles of denaturing at 94 °C for 30 sec, annealing at 60 °C for 30 sec, extension at 72 °C for 30 sec, and final extension at 72 °C for 7 min (BioRad C1000, USA).

As the second step of PCR, a fragment of 227 bp was amplified by 1 μl primary PCR product, 0.5 μl of each internal primer (10 pmol/μl) (BioNeer, Korea), 12.5 μl of commercial premix (Ampliqon, Denmark), and 10.5 μl of molecular biology grade H2O in a volume of 25 μl. The cycling parameters were considered the third stage, including preheating at 94 °C for 5 min, followed by 35 cycles of 20 sec at 94 °C, 20 sec at 60 °C, 20 sec at 72 °C, and final extension for 5 min at 72 °C (16). Nested PCR product was subjected to electrophoresis on a 1.5% agarose gel in a Tris-Borate-EDTA buffer at 90 V for 20 min and visualized with ultraviolet transilluminator after staining with SYBR Safe (Life Technologies Corporation, USA).

Sequence Alignments and Phylogenetic Analyses

Some positive nested PCR products were directly sequenced by targeting Nc-5 gene marker in both directions using the aforementioned primers by Genetic Analyzer automated sequencer. The sequences of the samples were aligned and edited in consensus positions, compared to the GenBank sequences of all regional species using Sequencher Tmv.4.1.4 software. The similarity between the present sequenced isolates and sequences of other countries was determined using MegAlign software. In addition, MEGA 6 software and neighbor-joining algorithm were used in order to perform phylogenetic analysis.

Results

The brain samples of 133 aborted fetuses, including 51 sheep, 78 cattle, and 4 goats, were surveyed by nested PCR for the detection of N. caninum; out of 133 brains samples, 24 samples (18.04%) were positive in this regard. As shown in Table 1, the prevalence rate of N. caninum in the aborted cattle fetuses (20.5%) was higher than that of the aborted sheep fetuses (15.6%); however, no positive case was observed in the goat fetuses. Therefore, there was no relationship between the type of livestock and prevalence of N. caninum. Furthermore, the results of the present study indicated that N. caninum caused was considerably more in the early trimester (32%) than others (P<0.05). The highest prevalence of this parasite was reported in Qaemshahr, Iran, followed by Sari, Babol, Amol, and Behshahr, Iran. The results of the statistical analysis did not show a significant relationship between living area and N. caninum caused abortion.

Table 1:

Association between the types of animals, gestational period, and area with the presence of Neospora caninum DNA from ruminants that aborted

Variable		Number of samples	Number of Positive (%)	Odds Ratio	Confidence Interval 95%	P value
Type of animals	Sheep	51	8 (15.6)
	Cattle	78	16 (20.5)	0.7	(0.2–1.9)	0.6
	Goat	4	0 (0)	-	-	-
Stage of pregnancy	Early	46	15 (32)	1
	Middle	60	9 (15)	2.7	(1.1–7.2)	0.03
	The Late	27	1 (3.7)	12.5	(1.9–276)	0.003
Area/city	Sari	56	13 (23.2)
	Babol	15	2 (13.3)	1.9	(0.3–20)	0.5
	Behshahr	23	2 (8.6)	3.1	(0.7–22)	0.1
	Amol	18	2 (11.1)	2.4	(0.5–17)	0.2
	Qaemshahr	21	5 (23.8)	0.9	(0.2–3.4)	0.9

The sequencing of nested PCR products obtained from three brain samples of the aborted fetuses (i.e., 1 sheep and 2 cattle) showed that the amplified sequence was N. caninum specific. All the data sequences of the present study were deposited in the GenBank with accession numbers of MH841974, MH795879, and MH752687. The results demonstrated our sequences shared 96% to 99% similarity with each other and 96% to 100% similarity with N. caninum deposited in GenBank. Phylogenetic trees showed intraspecific variations between our isolates and other N. caninum specimens deposited in GenBank (Fig. 1). Analysis of our sequences showed high similarity with N. caninum isolated from cattle in Iran (MH410658), Spain (AV494944), and China (JN634858).

Fig. 1:

Phylogenetic tree of N. caninum isolates from Mazandaran Province, Iran and the other isolates deposited in GenBank

Discussion

Neosporosis has emerged as one of the most common infections of abortion dairy cattle worldwide. The clinical symptoms of N. caninum have been observed in sheep, goats, deer, and horses. Furthermore, antibodies have been detected in the sera of camels, water buffaloes, foxes, coyotes, and felids (4).

Fetus abortion caused by N. caninum is the common reproductive problem that leads to major economic loss in cattle and sheep husbandry (17).

The PCR has been used for the detection of N. caninum since 1996, and several target genes, including 18S rDNA, 28S rDNA, ITS1, and Nc-5, have been used as target genes for the diagnosis of this parasite. Among these genes, Nc-5 has been most frequently used because not found in the genome of the parasites of this protozoan family, such as T. gondii, Hammondia hammondi, and Sarcocystiss cruzi. The PCR, especially nested PCR has high sensitivity and specificity for the detection of parasites while using a specific gene (18). In the present study, N. caninum was detected in the brains of 24 (18.04%) cases out of 133 examined aborted fetuses using nested PCR.

In Iran, neosporosis has been reported from 0.9% to 8.5% in aborted bovine fetuses by various PCR assays (19, 20). In addition, the results of previous studies in Chahar Mahal Bakhtiari and Mashhad, Iran, showed that the prevalence rates of N. caninum were 11% (21) and 33% in aborted bovine fetuses, respectively (22).

In other countries, N .caninum was detected in the brain samples of 5%, 6.8%, 8.6%, and 18.9% of cattle fetuses in Brazil (23), aborted sheep fetuses in Spain, aborted sheep fetuses in Italy, and 18.9 aborted bovine fetuses in England (14), respectively(24, 25). The main causes of various results can be due to the frequency of definitive hosts in the studied areas (e.g., dogs), uses of various diagnostic methods, as well as climate and environmental factors (26). Therefore, farmers should further become aware of maintaining hygienic conditions and keeping livestock food out of the reach of dogs.

In this study, three positive samples were sequenced for phylogenetic analysis. The sequences results displayed high similarity with N. caninum isolated from cattle in Mashhad (Iran), Spain and China that clustered with them in the phylogenetic analysis. The results of phylogenetic analysis indicted that N. caninum from different hosts and geographical area are genetically diverse and can be classified into two main clades (Fig. 1), although the two clades proposal is inadequately supported (Bf=78%, Fig. 1). A few investigations have been performed on the phylogenetic analysis of N. caninum with the Nc-5 gene. BLAST analyses of Nc-5 gene showed greater than 94% to 97% similarities between N. caninum sequences deposited in GenBank. Hence, it seems the Nc-5 gene is only a highly sensitive gene for the diagnosis of neosporosis and will be suggested to use ITS-1 and microsatellite genes for phylogenetic analyses (27).

The prevalence rate of neosporosis was significantly higher in the cattle, compared with the sheep. Such a difference in prevalence rate between these animals could be due to the high susceptibility of cattle to neosporosis infection (28) that is in line with the findings of a study in North Africa (29). As reported clinical cases associated with N. caninum infection in goats are uncommon (30), no positive effect was observed in the results of the present study regarding neosporosis among the goats. The abortion of fetuses due to N. caninum occurs from 3 months of gestation to term (3). In this study, a higher number of abortions caused by N. caninum in the early trimester of gestation (32%) was similar to the results of other studies (within the range of 6–8 months) (31, 32).

The prevalence rates of aborted fetuses infected with N. caninum were reported. This difference could be related to the livestock rearing system and number of dogs present in these cities. However, fetal mortality can be prevented in bovines by vaccination. Currently, there is no commercial vaccine for neosporosis (33); therefore, must be prevented from consuming placentas and fetuses by dogs and wild canids. Fetal tissues and placentas should be collected and disposed as far as possible.

Low sample size especially in goat samples and unavailability of aborted fetal samples in traditional farms are the limitations of the present study.

Conclusion

The detection of N. caninum DNA in the aborted fetuses of cattle and sheep showed that N. caninum could be the main agent of abortion in these ruminants. Awareness programs should be established for both farmers and veterinarians regarding the risks associated with this parasite. In addition, it is required to perform further investigations using molecular methods and larger sample sizes to confirm the above-mentioned hypothesis in ruminants’ aborted fetuses in Iran.