Investigation of Toxoplasma gondii and Neospora caninum in different tissues of aborted foetuses of sheep in Van Province, Türkiye: Analysis by nested PCR, histopathological and immunohistochemical methods

Toxoplasma gondii and Neospora caninum are protozoon parasites from the intracellular apicomplexan family. Toxoplasma gondii is the cause of health and economic problems in the sheep industry worldwide. Neospora caninum is usually reported in cows and leads to infections causing abortions; however, its prevalence in sheep is not clear. The present study aimed to investigate the prevalence and pathology of T. gondii and N. caninum by PCR , histopathological and immune-histochemical methods in aborted sheep foetuses collected at different sheep flocks in the Van Province, Türkiye, in 2021. Firstly, the DNA of T. gondii and N. caninum were investigated by PCR in the brain, heart, and peritoneal fluid samples from 42 sheep foetuses. Toxoplasma gondii DNA was proved in 35.7% (15/42) of foetuses whereas N. caninum DNA was not determined in any of the samples. Histopathologically, all T. gondii positive brain tissue samples showed lymphohistiocytic multifocal encephalomyelitis and additional findings included necrotizing myocarditis in the positive heart samples. Toxoplasma gondii tachyzoites were identified in the lesions (diffuse or focal mononuclear cell infiltration in the meninges, and microglia proliferation, myocarditis with oedema) by anti-T. gondii antibodies by the immunohistochemical method. Based on our results, we can conclude that T. gondii is an important agent in sheep abortions and the PCR method is a suitable method for diagnosis which can also be used in heart tissue in pathological studies.


Abstract
Toxoplasma gondii and Neospora caninum are protozoon parasites from the intracellular apicomplexan family.Toxoplasma gondii is the cause of health and economic problems in the sheep industry worldwide.Neospora caninum is usually reported in cows and leads to infections causing abortions; however, its prevalence in sheep is not clear.The present study aimed to investigate the prevalence and pathology of T. gondii and N. caninum by PCR, histopathological and immunehistochemical methods in aborted sheep foetuses collected at different sheep flocks in the Van Province, Türkiye, in 2021.Firstly, the DNA of T. gondii and N. caninum were investigated by PCR in the brain, heart, and peritoneal fluid samples from 42 sheep foetuses.Toxoplasma gondii DNA was proved in 35.7% (15/42) of foetuses whereas N. caninum DNA was not determined in any of the samples.Histopathologically, all T. gondii positive brain tissue samples showed lymphohistiocytic multifocal encephalomyelitis and additional findings included necrotizing myocarditis in the positive heart samples.Toxoplasma gondii tachyzoites were identified in the lesions (diffuse or focal mononuclear cell infiltration in the meninges, and microglia proliferation, myocarditis with oedema) by anti-T.gondii antibodies by the immunohistochemical method.Based on our results, we can conclude that T. gondii is an important agent in sheep abortions and the PCR method is a suitable method for diagnosis which can also be used in heart tissue in pathological studies.

Ovine, abortion, protozoon parasites
Toxoplasma gondii and Neospora caninum are obligate intracellular protozoan parasites from the Apicomplexa phylum (Arraes-Santos et al. 2016).Toxoplasma gondii is a protozoan causing toxoplasmosis, which is among the most common parasitic diseases in humans and animals in the world (Tenter et al. 2000;Wang et al. 2011).Infected cats, the definitive host of T. gondii, may spread millions of oocysts in their faeces and thus contaminate the environment that can become the source of infection for herbivorous animals during grazing (Mor and Arslan 2007;Innes et al. 2009;Can 2010).Toxoplasma gondii infection can lead to abortions and reproductive disorders in sheep.When pregnant sheep develop acute toxoplasmosis, their placenta is invaded by tachyzoids followed by the infection of the foetus (Moraes et al. 2011;Ibrahim et al. 2017).Neosporosis is a parasitic disease of great importance in livestock caused by the obligate intracellular parasite N. caninum that was first isolated in puppies with congenital encephalomyelitis in Norway in 1984 (Dubey et al. 2007;Uzêda et al. 2007).Domestic and wild canids are the definitive host of N. caninum.Intermediate hosts, like herbivores (cattle, sheep, goat, horse, bison, and deer) become infected by ingesting infected oocysts spreading in the faeces of definitive hosts (Nath-Sharma et al. 2015;Gharekhani et al. 2016).In many hosts, contamination occurs by the transplacental route.This disease, which causes neuromuscular disorders, paralysis, and death in dogs, causes abortion and neonatal death in sheep and goats.Since T. gondii and N. caninum are very similar in structure, it has been suggested that misdiagnoses have been made for years, especially in terms of N. caninum (Dubey 2003;Figliuolo et al. 2004;Uzêda et al. 2007;Filho et al. 2017).Although N. caninum causes infections, especially in cattle and dogs, its presence has been reported in many warm-blooded animals including goats, sheep, buffalo, equids, and other domestic and wild carnivores.Bovine neosporosis is an important cause of abortion in cattle.It also causes reproductive problems in cattle.Abortions due to N. caninum in sheep have been rarely reported (Bártová et al. 2009;Ueno et al. 2009).
Abortions caused by T. gondii cause serious economic losses in sheep breeding (Ahmed et al. 2008;Moreno et al. 2012).Although N. caninum-related abortions have been reported in sheep (Pereira-Bueno et al. 2004;Ueno et al. 2009;Ezatpour et al. 2015), the number of studies on the subject is insufficient.Careful diagnosis of T. gondii and N. caninum in sheep abortions is of great importance in understanding reproductive disorders in flocks, and preventing abortions and economic losses.For the diagnosis of these two agents, serological tests and histopathological tests are frequently used.However, the morphological similarity of these two agents increases the possibility of error in histopathological tests.Molecular methods used for detection of the DNA of these two agents and its further genetic characterizations have been reported as the best method for the diagnosis of T. gondii and N. caninum from ovine foetuses and placenta (Moraes et al. 2011;Moreno et al. 2012;Ezatpour et al. 2015).
The aim of the study was to detect T. gondii and N. caninum by PCR in tissues of 42 aborted sheep foetuses in the Van Province in Türkiye, and to study the pathology of these two parasites using histopathological and immunohistochemical methods.In addition, this study aimed to determine the the usability of the peritoneal fluid and heart tissue as an alternative to brain tissue for diagnosis of T. gondii and N. caninum in pathology studies.

Materials and Methods
The study area and sample collection The present study was conducted in the Van Province in Türkiye, located in the Eastern Anatolian Region and bordering with Iran (Fig. 1).The samples consisted of 42 sheep foetuses aborted during the first 1-3 months of pregnancy in different sheep flocks bred in different locations of the Van Province in 2021.All the sheep from which the samples were taken were of the Morkaraman breed.A total of 126 tissue samples (42 brain, 42 heart, and 42 peritoneal fluid samples) were obtained from 42 foetuses.The obtained samples were stored at −20 °C until the PCR analyses.Tissue fragments from the brain and heart tissue, except frozen samples, were fixed in 10% buffered formalin for histopathological and immunohistochemical analysis.

DNA extraction, PCR amplification, and sequence analysis
The DNA extraction was carried from the brain, heart, and peritoneal fluid of 42 ovine foetuses by the Pure Link™ Genomic DNA Mini Kit (Thermo Fisher, Carlsbad, CA, USA).

Histopathology
PCR-positive tissue samples were washed under running water for removing formalin.Afterward, routine pathological tissue tracing was performed and passed from graded alcohol (50%, 75%, 96%, 100%) and xylol series, and embedded in paraffin blocks.Tissue sections of a size of 5 µm were placed onto slides with Leica RM 2125 RT (the first 3 sections and each 10 th section).The preparations were treated with alcohol and xylol series and stained with haematoxylin-eosin (HE).All samples were examined under a high-resolution light microscope (Olympus DP-73 camera, Olympus BX53-DIC microscope, Tokyo, Japan).

Immunohistochemistry
For the immunohistochemical examination, 4 μm thick sections were obtained from the paraffin-embedded tissue blocks and placed on poly-L-lysine-coated glass slides that were stained with the streptavidin-biotin-Fig. 1.The area in Türkiye where aborted sheep foetuses were collected for this study.peroxidase complex (ABC) technique after routine deparaffinization and rehydration procedures.Antigen retrieval was performed in a microwave oven with citrate buffer (pH 6.0) (700 W, 20 min).Endogenous peroxidase activation in the tissues was blocked for 15 min with 0.3% hydrogen peroxide (H 2 O 2 ) in 0.01 mol/l PBS in methanol at room temperature.Before applying the primary antibody, the tissues were incubated for 20 min with 5% goat blocking serum for protein blocking.Then, the sections were incubated with primary anti-T.gondii antibodies for 1 h at room temperature.Tissues were kept in rabbit anti-mouse biontinylated secondary antibody for 30 min after removing the unbound primary antibody.Then, the sections were made to react with horseradish peroxidase streptavidin for 30 min.After washing with PBS, the sections were treated and incubated with DAB (3,3'-Diaminobenzidine, Dako, Glostrup, Denmark) for 5 min.Finally, the background of the tissue sections was stained with haematoxylin.For negative controls, PBS was used instead of the primary antibody.All staining steps were carried out at 37 °C and in humidity cabinets.PBS solution was used as a wash-away solution during all the staining steps.

Results
In this study, heart, brain, and peritoneal fluids from 42 aborted foetuses (in total 126 samples) were examined.Toxoplasma gondii was detected in 15 (35.7%) foetuses (in the brain of 3 foetuses, in the heart of 3 foetuses, and in both the brain and the heart of 9 foetuses) while peritoneal fluid was negative.All tissue samples were negative for N. caninum.The 529-bp repeat element regions of sequenced T. gondii were compared with reference genes in the GenBank (Table 1).The results of the sequences performed with the F primers were not evaluated because they did not have the quality to be analysed.The 230 bp region in the results of the sequences performed with the R primers was checked with BLAST.Except for sample 11, all other samples showed 100% overlap with T. gondii sequences.G-C transversion was observed in sample 11 at position 138.The nucleotide sequence of Toxoplasma positive samples in this study is given in Table 2.
In the histopathological and immunohistochemical analyses performed with positive brain samples, the lesions were characterized by perivascular mononuclear cell infiltration, diffuse or focal mononuclear cell infiltration in the meninges, and macrophages/microglia proliferation (Plate VII, Fig. 2a).The inflammatory scores are presented in Fig. 2a.Tissue cysts and tachyzoites were observed in all T. gondii positive animals (Plate VII, Fig. 2b, 2c).Inflammatory lesions in the brain were more pronounced at the beginning of the infection and during the established chronic infection.Anti-T.gondii immunopositivity was mainly observed in cells located in areas characterized by glial proliferation (Fig. 2a) and perivascular mononuclear cell infiltration as demonstrated by histopathology.Lymphocytic myocarditis, myocardial inflammation with oedema and mononuclear cells, destruction of the cardiomyocyte, perivascular mononuclear cell infiltration, and diffuse or focal mononuclear cell infiltration, necrosis and tachyzoites were identified in heart tissue (Plate VII, Fig. 2d).
Although N. caninum has been reported to cause abortions in sheep and congenital infections and deaths in newborn lambs, it is not considered among the main causes of abortion in sheep (Innes et al. 2001;Koyama et al. 2001;Hässig et al. 2003).The prevalence of N. caninum tested in sheep by PCR in the world is the following: 27.7% in Pakistan (Nasir et al. 2012), 8.8% (Ueno et al. 2009) and 62.2% in Brazil (Filho et al. 2017), 12% in the Czech Republic (Bártová et al. 2009), 10.1% in Spain (Panadero et al. 2010), 1.53% in Iran (Ezatpour et al. 2015) and 16.8% in Greece (Anastasia et al. 2013).In Türkiye, the prevalence of N. caninum in sheep was 0.8%, 2.7%, and 0% in Karaman, Konya, and Zonguldak, respectively (Zhou et al. 2016), 12.4% in Adana (Ekşi et al. 2018), 0% in Van (Har and Başbuğan 2019) and Elazig (Özkaraca et al. 2016) and 2.1% in Kars (Gökçe et al. 2015).In our study, N. caninum was not detected in any of the 42 foetal tissue samples, and this result is consistent with the results of the study by Özkaraca et al. (2016).
In sheep, protozoal abortions are often associated with T. gondii infection.The effect of N. caninum on sheep abortions is still obscure.The signs and lesions of toxoplasmosis and neosporosis are similar.Hence, serological tests performed in maternal blood or molecular tests performed in the tissues directly obtained from foetuses play an important role.The PCR test was reported to be the most specific test to reveal the aetiological agents.Histopathological tests performed after these tests increase the reliability (Hässig et al. 2003;Moreno et al. 2012;Irehan et al. 2022).Varying rates of T. gondii and N. caninum have been reported in previous studies conducted with PCR tests in tissue samples of aborted foetuses.In previous studies, the tissue used for PCR was the brain.Hässig et al. (2003), reported N. caninum in four of the brain tissues of 20 aborted foetuses, and T. gondii in three of them; Moreno et al. (2012) Hurtado et al. (2001) reported T. gondii in nine out of 53 sheep foetuses.In this study, T. gondii DNA was determined in 15 (35.7%) out of 42 sheep foetuses; however, N. caninum was not determined.Toxoplasma gondii DNA was detected in a total of 15 foetuses (both heart and brain tissue of nine foetuses, in only the brain tissue of 3 foetuses, and only the heart tissue of 3 foetuses) and it was concluded that heart tissue was a suitable tissue for molecular studies.We suggest that the different results among the studies may have resulted from the environmental conditions, the distribution of the definitive hosts, the susceptibility of the sheep, and the suitability of the primers to be used in PCR studies.It has been reported that the earlier the pregnancy period of pregnant sheep with toxoplasmosis, the more severe the consequences, and the more waste and re-infections (Dubey 2009).The material of this study comprised early aborted (1-3 months) foetuses as reported by Dubey (2009) indicating that T. gondii should be considered first among the aetiological factors in aborted foetuses.
Protozoan abortion in sheep is traditionally associated with T. gondii.The characteristic necrotic lesions of toxoplasmosis are usually observed in the central nervous system (Dubey 2003;Partoandazanpoor et al. 2020).Toxoplasma gondii invades astrocytes, neurons, and other neuroglia, causing diffuse inflammatory foci, blood vessel clamping and inflammatory meningeal cell infiltrates.Previous studies have reported gliosis foci, necrosis foci, focal large non-suppurative encephalitis areas, mononuclear cell increase, encephalitis findings, tachyzoite, bradyzoite, and pseudocysts in the brain of foetuses aborted due to toxoplasmosis (Hurtado et al. 2001;Pereira-Bueno et al. 2004;Moreno et al. 2012;Castaño et al. 2016;Atmaca et al. 2019).The findings reported in previous studies were identified in 12 foetal brain tissues in the current study.
In some cases, the lesions in foetal tissues are not extensive but may cause abortion, whereas in other cases lambs with toxoplasmosis born with severely damaged placentas may appear healthy.Therefore, examination of various foetal tissues is important in understanding the mechanism of miscarriage (Dubey 2009;Castaño et al. 2016).
Toxoplasma gondii-related lesions are most intensive in the brain and the placenta, and mainly the brain, placenta, and liver are the most commonly used tissues for molecular and pathological examinations.However, the spread of lesions is time-related and autolysis in these tissues may make diagnosis impossible (Hurtado et al. 2001;Castaño et al. 2016).The number of studies investigating T. gondii in different tissues of the foetus is very insufficient.Hurtado et al. (2001) investigated T. gondii DNA by PCR in 145 tissue samples (lung, spleen, liver, placenta, foetal fluid, brain) obtained from 53 sheep foetuses and a stillborn lamb, and found positivity in nine foetuses and one stillborn lamb.These investigators detected agents in all nine histopathologically examined PCR-positive brain samples, seven of ten spleen samples, eight of nine lung samples, all three of three placental samples, five of seven liver samples, one of two kidney samples, and three of eight foetal fluid samples.The same investigators reported that lung, spleen, and liver tissues could be used in molecular and pathological examinations, following the brain and the placenta.Moreno et al. (2012) detected T. gondii and N. caninum DNA in aborted sheep foetus samples by PCR and histopathologically determined T. gondii-related lesions mainly in brain tissue, and also in the heart, lung, liver, and kidney samples.In this study, the lesions were detected in both the heart and brain tissue of nine PCR positive foetuses, only in the brain tissue of three foetuses, and only in the heart tissue of three foetuses.In the heart tissue, histopathologically and immunohistochemically, lymphocytic myocarditis, myocardial inflammation with oedema and mononuclear cells, destruction of the cardiomyocyte, perivascular mononuclear cell infiltration, necrosis and diffuse or focal mononuclear cell infiltration were determined (Fig. 2D).These results indicate that the heart tissue is suitable for use in investigations for T. gondii.Similar results may also be observed in Neospora caninum-associated abortion; therefore, it is difficult to discriminate from a pathological perspective and the PCR method is the gold standard for discrimination (McAllister et al. 1996;Hurtado et al. 2001;Moreno et al. 2012).
In conclusion, based on the results of this study, it can be stated that T. gondii is an important cause of abortion in sheep in this region.It was also concluded that the PCR method is an important tool to diagnose protozoal abortion agents and that the heart tissue may also be used for histopathological and immunohistochemical analysis when brain tissue is not available.The use of peritoneal fluid was not found to be suitable for the diagnosis of T. gondii.Although N. caninum was not detected in aborted sheep foetuses in this study, further studies are needed to determine the role of N. caninum in abortions in sheep.

Fig. 2 .
Fig. 2. A -Brain of the sheep infected with Toxoplasma gondii showing severe lymphohistiocytic encephalitis (black star), necrosis (red star), and severe hemorragia (arrow).Haematoxylin and eosin staining.B -Immunohistochemical labelling of Toxoplasma gondii tissue cysts (arrow) in the brain of the infected sheep.Immunohistochemical staining.C -Toxoplasma gondii tachyzoites (arrow) in brain.Immunohistochemical staining.D -Heart tissue of the sheep infected with Toxoplasma gondii showing cellular degeneration (arrows) with intralesional Toxoplasma gondii tachyzoites (arrow heads).Haematoxylin and eosin staining.

Table 1 .
Comparison of the sequence results of this study with the reference gene in the NCBI Nucleotide Database and similarity rates.

Table 2 .
The nucleotide sequences performed with the R primers of Toxoplasma gondii positive samples in this study.