Introduction

Neospora caninum is the causative agent of polysystemic parasitic disorder and a coccidial protozoan parasite with worldwide distribution (Dubey 2003). This disease was first identified in puppies in Norway (Bjerkås et al. 1984). Other canine hosts, including domestic dogs (Canis familiaris), coyotes (C. latrans), dingoes (C. domesticus), and gray wolves (C. lupus) are definitive hosts for N. caninum (Dubey et al. 2005). Oocysts, tachyzoites, and tissue cysts were all thought to be infectious stages in the life cycle of the parasite (Dubey 2003). Neospora caninum can be transmitted to intermediate hosts following ingestion of oocysts through contaminated food or water or can be transferred vertically from mothers to offspring via the placenta (McAllister et al. 1998). Primary intermediate hosts, such as goats, sheep, and cattle may experience stillbirths, abortions, and neonatal mortalities (Nie et al. 2018).

Neospora caninum was first detected in sheep in England in a congenitally infected lamb (Dubey et al. 1990). In Japan, South America, and Switzerland, ovine neosporosis has been documented as naturally occurring (Kobayashi et al. 2001; Koyama et al. 2001; Hässig et al. 2003a; Moore 2005), and seroprevalence reports suggest that the parasite is present worldwide (Liu et al. 2015; Villagra-Blanco et al. 2017; Dahourou et al. 2019).

It has been demonstrated that experimentally infecting pregnant ewes with tachyzoites can cause congenital infection or abortion (McAllister et al. 1996; Howe et al. 2012), and the parasite could cause mortality in newborn lambs and induce congenital infection in naturally exposed sheep (Dubey et al. 1990; Buxton et al. 1998; Hurtado et al. 2001; Helmick et al. 2002). Sheep can also be infected by ingesting oocysts shed in the feces of the definitive carnivore hosts, demonstrating the possibility of horizontal infection (O’Handley et al. 2002).

Sheep breeding industry has an important economic role in Iran. Sheep farming has become increasingly favored because of its higher production levels. Unfortunately, these kinds of farms are regularly not supported by proper feeding and management, and they are not able to avoid stressful situations, and animals have lower resistance to opportunistic pathogens like N. caninum (Selim et al. 2021).

In Iran, N. caninum was documented for the first time in dairy cattle in the Mashhad region, the northeast of the country (Sadrebazzaz et al. 2004), and from that time, many different researches have been carried out on some species of mammals and birds (Gharekhani et al. 2020).

Certain scientists have suggested that neosporosis could be responsible for inducing abortion in sheep as N. caninum DNA was detected in 9.9% and 8.5% of brain samples obtained from aborted fetuses in Mashhad and Tabriz regions, respectively (Asadpour et al. 2013; Sasani et al. 2013; Razmi and Naseri 2017). During the scrutiny, genetic material of N. caninum was identified in 6.7% and 0.7% of cardiac and cerebral specimens, correspondingly, in asymptomatic animals (Arbabi et al. 2016).

In the present study, we attempted to provide information on the role that this parasite is playing in ovine reproductive failure by establishing the prevalence of N. caninum DNA in ovine fetal material present in sheep farms with ongoing unexplained abortion problems.

Material and methods

Study duration and sample preparation

From June 2019 to February 2022, tissue samples were collected following the necropsies of aborted ovine fetuses from private farms situated in Alborz and Qazvin provinces (Fig. 1). These two provinces are located in the north of the central region of Iran at the skirts of the Alborz Mountain ranges. The Alborz Mountains have an impact on these provinces’ weather, which has cold winters and mild summers. The south of these provinces has hot and dry weather because of being adjacent to the desert, and on one side, it reaches Alborz Mountain, and on the other side, it ends in one of the driest deserts of Iran. Sheep and goat breeding in Iran, also in these two provinces, is mostly by traditional methods, and flocks under these conditions often have direct contact with stray dogs, but, despite all the above points, the clinical and economic reality of neosporosis in sheep and goats is unclear (Gharekhani et al. 2020). Tissue samples (brain, placenta, embryonic membrane, cotyledon, umbilical cord, heart, liver, spleen, and digestive track) were sent to the Protozoology Laboratory, Parasitology Department, Razi Vaccine and Serum Research Institute. In the laboratory, tissue samples were stored under freezing conditions (−20 °C) until they were used for molecular analysis.

Fig. 1
figure 1

A map showing the sampling site’s geographical location. Alborz and Qazvin provinces are marked with a red circle

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Questionnaire data collection of risk factors

A questionnaire asking about the history of abortion in livestock has been completed by participating farm owners. The data obtained from the owners included fetal age (the fetus died prior to or during labor), size of flock, type of farming (traditional or industrial), type of flocks (containing sheep, goat, cow, or a combination of them), source of feeding (in the stable, possibility to graze or both), source of water (stagnant water source or municipal water), type of habitat (mountainous or plain), and presence of the dog and cat at the farm.

DNA extraction

A portion of the tissues from the aborted fetus including brain, placenta, embryonic membrane, cotyledon, umbilical cord, and a mixture of various tissues (heart, liver, spleen, and digestive track) were removed, homogenized, and utilized for DNA extraction by the phenol-chloroform extraction procedure, as described previously (Biase et al. 2002; Jameie et al. 2022; Nasiri and Jameie 2023). The resulting DNA was kept at −20 °C for following PCR assessments.

PCR analyses

The Nc-5 gene that has been shown to be highly specific to N. caninum was chosen for detection of this parasite as the target sequence by using combinations of four pairs of primer in two nested PCR (Hughes et al. 2006).

Procedure I

Briefly, as described previously (Hughes et al. 2006; Nasiri and Jameie 2023), first round of PCR (337b) was done using a pair of N. caninum Nc-5 gene specific primers: Np21 PLUS and Np6 PLUS (Liddell et al. 1999), and then the second round of PCR(225b) were performed with a pair of primers: Np7 and Np6 (Yamage et al. 1996; Baszler et al. 1999). Amplification of the first round reaction was performed with initial denaturation for 5 min at 95 °C, followed by 40 cycles of 94 °C for 40 s, 63 °C for 40 s, and 72 °C for 70 s, and final extension at 72 °C for 10 min. The conditions of the reaction were identical to the first round, with the exception of annealing temperature of 56 °C.

Procedure II

As described previously (Nasiri and Jameie 2023), in the preliminary PCR (275bp), detection was executed using Np4 and Np7 primers. The amplification involved an initial denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 57 °C for 30 s, 72 °C for 60 s, and a final extension at 72 °C for 10 min. The second set of primers, Np6 and Np7, were used for conducting semi-nested PCR, which targeted a fragment of 225 base pairs. The conditions of the second reaction was identical to the first PCR, except that the annealing temperature was decreased to 56 °C (Baszler et al. 1999).

A single-tube nested PCR for ITS1 gene (249 bp)

As described previously (Nasiri and Jameie 2023), samples that were NC-5 gene positive, were analyzed to validate the existence of the ITS1 gene. Single-tube nested-ITS1 PCR using the external primers (ITS-1-N-F1 and ITS-1-N-R1) and the internal primers (ITS-1-N-F2 and ITS-1-N-R2) was used to find parasite ITS1 gene. The amplification conditions involved 1 cycle of 94 °C for 3 min, 15 cycles of 94 °C for 30 s, 65 °C for 45 s, and 72 °C for 1 min, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and a final cycle lasting 5 min at 72 °C. For all PCR reactions, a positive control (DNA extracted from the brain of the experimentally infected inbred BALB/c mouse and DNA extracted from cultures of NC1 strain of N. caninum) and a negative control (DNA extracted from the brain of inbred BALB/c mice (confirmed to be serologically and molecularly negative for neosporosis) and double distilled water) were included (Nasiri and Jameie 2023; Buxton et al. 1998; Hurtado et al. 2001; Regidor-Cerrillo et al. 2014; García-Sánchez et al. 2020).

ITS1gene phylogenetic analysis

The positive PCR products of the four amplified ITS1gene were sequenced in both the reverse and forward directions. The results were aligned with the ITS1 gene partial sequences of N. caninum from previous researches that were presented in GenBank using the BioEdit Sequence Alignment Editor program (Hall 1999). The maximum-likelihood method was used to perform the phylogenetic tree and evolutionary analyses by using MEGA11 software. The bootstrap results were computed for 1000 replicates (Tamura et al. 2013).

Statistical analysis

The data about the relationship between the different risk factors and molecular results were analyzed using the non-parametric Chi-square (χ2) test (SPSS 26.0, Chicago, IL, USA) software, and the results were considered significant when the probability score was < 0.05.

Ethics statement

The Animal Care Committee of Razi Vaccine and Serum Research Institute, Alborz, Iran, approved all animal experimentation procedures. All animals used in our experiment were treated humanely and in accordance with National Research Council’s Guide for the Care and Use of Laboratory Animals.

Results

In collaboration with active veterinarians in the field and veterinary organizations, 100 samples [brain (n = 39), placenta (n = 8), embryonic membrane (n = 7), cotyledon (n = 7), umbilical cord (n = 2), and homogenate mixture of all tissues (heart, liver, spleen, and digestive track) (n = 37)] were collected. Tissue homogenates contained portions of each of the tissues submitted from the cases. These samples were obtained following the necropsies of 39 individual aborted ovine fetuses.

Analysis of risk factors associated with N. caninum infection

In total, 27 questionnaires containing epidemiological information related to the collected aborted fetus were received from investigating farms. There was no relationship between some factors like the type of farming, type of flocks, source of feeding, type of habitat, and presence of dogs or cats with the positive results of the molecular detection of N. caninum (p>0.05). This analysis indicated that there was significant variation only between the source of water and the positive molecular results (p=0.005) (Table 1).

Table 1 Statistical analysis of data obtained from questionnaires compared to molecular results
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Molecular analyses

By the molecular assessment of the NC-5 gene in different tissue samples, this gene was amplified from the 27 tissue samples belonging to the 23 aborted ovine fetuses that all 27 positives tissue samples for the NC-5 gene were also positive for the ITS1 gene.

Brain tissue was the most frequently submitted tissue, and the highest number of positive cases were observed in this tissue.

The results of molecular analysis of the presence of parasite in the different tissues are shown in Table 2.

Table 2 The number of tissues examined with the number and percentage of molecular results for each tissue
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The results of molecular analysis of the presence of the parasite by number and code of the aborted fetus sample and the type of tissue examined in each case are presented in Table 3.

Table 3 Evolutionary differences of detected Neospora caninum in the aborted fetus (p distance)
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Phylogenetic analysis

After sequencing, four positive sequences were submitted to GenBank (accession numbers OL825739, OL825740, OM484267, and OM484268). To characterize the most resembling sites, existing sequences were subjected to the basic local alignment search tool (BLAST), and after alignment with BioEdit, the findings indicated that our sequences exhibited likeness to one another and to other affirmative instances of N. caninum NC-5 gene recorded in Gen Bank (Supplementary Table S1).

The phylogeny of the N. caninum was investigated by computerized analysis of the NC-5 gene sequences of 44 N. caninum isolates that four samples obtained in the present work, plus those of 40 other studies about N. caninum and other apicomplexan parasites (Fig. 2). The phylogenetic results indicated that the grouping of the N. caninum lineages of this study could be divided into the same clades. Maximum likelihood was employed to construct phylogenetic trees (Fig. 2), which revealed resemblance between N. caninum isolates investigated in this research and other N. caninum found in animals from various regions across the globe, which were archived in the GenBank.

Fig. 2
figure 2

The infection percentage of each examined tissue

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Phylogenetic tree showed more similarity among N. caninum isolates in this study with N. caninum isolated from cattle in Iran, human umbilical cord blood from Brazil, and from coyote and sheep hosts (Fig. 3).

Fig. 3
figure 3

Phylogenetic relationships obtained for four Neospora caninum sequences (OL825739, OL825740, OM484267, and OM484268) (red rhombus and circle) using the maximum likelihood method based on their NC-5 gene sequences supported by 1000 bootstrap replicates. They represented the sequences obtained in this study

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As shown in Table 3, evolutionary differences of N. caninum that were collected from the current study (1–4) with other recently detected samples that were expressed as pair-wise distance (p distance) varied from 0.00 to 2.571.

Discussion

The efficiency of sheep herds relies on their ability to reproduce, but unfortunately, sheep abortion is a significant contributor to financial losses (Hecker et al. 2019). Many infectious agents may lead to ruminants’ abortions, either due to non-specific path (such as cause maternal stress or due to febrile disease), or due to fatal infection of the fetus. Some of the infectious organisms are mostly opportunistic, while others are specific ovine fetus pathogens leading to characteristic syndromes of abortion (Tirosh-Levy et al. 2022). Specific infectious agents that cause ovine abortions include some bacterial species, such as Chlamydophila abortus (enzootic abortion of ewes),Campylobacter fetus fetus, Campylobacter jejuni, Listeria monocytogenes, and Brucella medinensis; viruses including Simbu serogroup and Blue tongue; Border disease; and protozoa including Sarcocystis spp., N. caninum, and T. gondii (Buxton and Henderson 1999; Menzies 2011; Lindsay and Dubey 2020).

From different infectious agents that cause abortion in sheep, we can point to the N. caninum (Buxton and Henderson 1999). This parasite causes abortions, and clinical manifestation of ovine neosporosis is like ovine toxoplasmosis (Hecker et al. 2019). Due to the significant economic impact of neosporosis in ruminants and the absence of any cure or immunization, effective prevention and control measures represent the optimal means of minimizing N. caninum transmission (Hemphill et al. 2016; Machado 2019). Accordingly, the evaluation of related risk factors affecting its prevalence is crucial to reduce the dissemination of the disease (Wang et al. 2018; Mendonça et al. 2019; Selim and Ali 2020; Selim and Radwan 2020).

Different studies indicate that the diagnosis of neosporosis as the cause of an abortion cluster should be done with caution, due to its high seroprevalence in the general sheep population irrelevant to the abortion history. Comprehensive epidemiological investigation of the entire flock is necessary for an accurate diagnosis of reproductive failure in sheep (Tirosh-Levy et al. 2022).

Neospora caninum infection has been sporadically reported as a cause of reproductive failures such as abortion and neonatal mortality in sheep in Iran, similar to toxoplasmosis (Dubey and Schares 2011; Razmi and Naseri 2017). In the present study, no statistically significant correlation between contact with dogs and abortion was present.

In this research, N. caninum was detected in examined aborted fetuses which was higher than the results of other studies in Iran (1 to 8.5%) (Asadpour et al. 2013; Sasani et al. 2013). In other countries, N. caninum was found in Switzerland 25% (Hässig et al. 2003b), in Spain 4.7% (Moreno et al. 2012), and in Italy 2% (Masala et al. 2007).

Among the limitations of the present research, it can be pointed out that the number of aborted fetuses examined was not large enough to be statistically valid for comparisons, and sometimes it was not possible to clearly express the statistical significance of the difference in the results. Another limitation of this study is that only one cause of infectious abortion pursued which is a major limitation, and not an exhaustive evaluation of potential causes of reproductive losses in these flocks was done.

Conclusions

The above results are important regardless of the percentage and tissues involved in the presence of this parasite and show that N. caninum can be one of the main causes of abortion in sheep. But what is important is to provide a suitable solution to farmers to prevent future abortions. Sheep and goats infected with N. caninum may abort or give birth to defective lambs. In the control of the disease, it is recommended to control dogs by not having access to infected tissues, in particular embryonic membranes. Studies have shown an association between abortion epidemics and the presence of dogs in livestock, and therefore, dogs should be prevented from approaching sheep and accessing infected aborted tissues, and aborted tissues should be removed from the livestock so that dogs and other intermediate hosts cannot access them. Different control methods should be used to prevent an increase in the number of stray dogs, and every effort should be made to prevent the contamination of water and food of livestock with dog feces.