Ovine Abortion by Neospora caninum: First Case Reported in Argentina

The aim of this study was to describe for the first time a natural case of ovine abortion associated with Neospora caninum in a flock with reproductive losses in Argentina. The analyzed flock consisted of 256 Texel sheep, of which 134 had been mated. A single blood sample was obtained by jugular vein puncture from 220 ewes (116 adult ewes, 104 yearling ewes) and 93 lambs. Serum samples and fetal fluid were tested using the indirect fluorescence antibody test to detect antibodies against N. caninum and T. gondii. Fetal and placental tissues from aborted fetus were examined by standard gross pathology procedures and were tested using histopathology and immunohistochemistry. Moreover, DNA from fetal and placental tissues was isolated and a PCR assay to detect N. caninum, T. gondii and Chlamydia spp. was used. The pregnancy rate was 89% (119/134), the abortion rate was 8.4% (10/119) whereas the perinatal mortality rate was 15% (16/109). Out of 116 adult ewes sampled, 34.48% presented anti-N. caninum antibodies. Ten ewes had aborted, and one fetus was directly submitted to the diagnostic laboratory for further processing. Antibodies against N. caninum were detected in fetal fluid and in the aborted dam. Histopathological analysis in fetal tissues showed multifocal lymphohistiocytic glossitis, diffuse mild lymphohistiocytic endocarditis, pericarditis and focally extensive myocarditis. Severe multifocal necrotizing placentitis and diffuse mild lymphohistiocytic placentitis with the presence of lymphohistiocytic vasculitis were observed in placenta. N. caninum was immunolabeled in the placenta and fetal tongue. In addition, N. caninum DNA was detected in placenta, central neural system, lung and heart of the aborted fetus. There was no evidence of other infectious abortifacients in the aborted fetus. The present study described for the first time an ovine abortion caused by N. caninum in Argentina. Further investigations at a larger scale are required to establish the role of N. caninum as an important cause of reproductive losses in sheep flocks from the region.


Introduction
Sheep industry has an important socio-economic impact in Argentina. There are 14,746,566 sheep heads in Argentina and 16% of them are located in the Humid Pampa region [27]. The productivity of the sheep flocks depends on their reproductive efficiency. Ovine abortion is a major cause of economic losses; nevertheless, research on the etiology of ovine abortion is scarce in Argentina. Only few works have reported other abortifacient agents such as Brucella ovis, Leptospira spp., Campylobacter fetus and Toxoplasma gondii in Argentinean flocks [12,15,25].
Neospora caninum is an obligate intracellular protozoan parasite similar, as regards structure and development, to T. gondii [10]. N. caninum and T. gondii cause abortions and due to their similarity in the morphology and in the lesions induced, histopathological techniques do not enable accurate differentiation between both parasites [10]. T. gondii is one of the most common causes of reproductive failure in sheep worldwide and ovine abortions are often associated with this parasite [8]. Clinical manifestation of ovine neosporosis is like ovine toxoplasmosis although N. caninum has shown to be a more common cause of abortion in cattle [8]. For this reason, the economic, clinical, and epidemiologic importance of N. caninum infection in sheep remains under study [9,10]. Bishop et al. [3] found N. caninum DNA in the brain of a ewe with encephalitis suggesting that the parasite might cause clinical neosporosis in adult sheep. However, few studies have reported the potential involvement of N. caninum as cause of reproductive failure in sheep flocks [1,13,14,16,[19][20][21]24]. At the present, there are no reports in Latin America that confirm N. caninum as causal of ovine abortions. This work describes for the first time a natural case of ovine abortion associated with N. caninum in a flock with reproductive losses in Argentina.

Description of the Flock
The analyzed flock consisted of 256 Texel sheep, of which 134 had been mated. The farm was located in Ranchos, Buenos Aires province, Argentina (35°31ʹ24.96˝S 58°44.16ʺW). The farmer had observed a reduction in the reproductive performance during the previous lambing season. Animals were grazing native grasslands, supplemented with conserved forage (hay and silage) and concentrate during the breeding season. The affected flock was brucellosis-and tuberculosis-free. The mating period extended from March to May 2017. Ewes were synchronized for estrus and artificially inseminated. Non-pregnant ewes were resynchronized followed by natural breeding using one ram.

Sampling and Data Collection from the Animals
On March 2018, a single blood sample was obtained by jugular vein puncture from 220 ewes (116 adult ewes, 104 yearling ewes) and 93 lambs. Furthermore, there were three adult dogs and 11 cats in the farm co-habiting with the ewes. Serum samples were also obtained from the three dogs (identified as dog A, B and C), but unfortunately, it was not possible to obtain serum samples from the cats. The sera were separated by centrifugation and stored frozen (− 20 °C) until analysis for the detection of antibodies to N. caninum and T. gondii.
During the study period, there were recorded data about reproductive history of the flock. One aborted fetus was recovered and submitted to the diagnostic laboratory for diagnosis purposes. Animal procedures were performed according to standard protocols and guidelines from the Animal Ethics Committee (CICUAE#008/2015) at INTA, Argentina.

Serology
All serum samples and fetal fluid were evaluated for anti-N. caninum and anti-T. gondii specific IgG by IFAT using slides prepared with whole N. caninum and T. gondii tachyzoites, respectively. Cut-off titers of ≥ 1:50 and 1 ≥ 10 were defined for serum and fetal fluid, respectively [17]. The highest serological dilution with complete peripheral positive reactions was considered the end-point titer. Positive control sera were obtained from N. caninum and T. gondii experimentally infected ewes. Negative control sera were obtained from T. gondii and N. caninum free ewes [28]. Dogs sera were tested by IFAT as previously mentioned Basso et al. [2] using a cut-off titer of ≥ 1:50.

Necropsy and Sampling from the Aborted Fetus and Placenta
Fetal and placental tissues were examined by standard gross pathology procedures [5]. Fetal tissue samples including lung, spleen and abomasal content were extracted to determine the presence of aerobic and microaerophilic bacteria according to the methods described by Campero et al. [5]. Fetal cavity fluids were collected and stored at − 20 °C until assessment of antibodies to N. caninum and T. gondii by IFAT. Placental and fetal tissue samples (central neural system, heart, lung, liver, tongue, forelimb and hind limbs muscles) were collected for DNA extraction and histopathological analysis according to Hecker et al. [18].

Histopathology and Immunohistochemistry
Five µm-thick sections of each fetal tissue were cut, mounted on glass microscope slides and stained with hematoxylin and eosin (H&E). Tissues that had compatible lesions with N. caninum and T. gondii infection were selected and analyzed by immunohistochemistry (IHC) using polyclonal antibodies against N. caninum (kindly provided by Dr. M. Anderson, UCDavis, USA) and T. gondii (kindly provided by Dr. M.C. Venturini, inmunoparasitology laboratory (LAINPA), FCV, UNLP, Argentina) and the Dako Envision kit (Carpinteria, CA, USA), employed according to the manufacturer's instructions.

DNA Extraction and PCR
DNA was isolated using a commercially available kit according to the manufacturer's recommendations (DNeasy Tissue Kit, QIAGEN Group, Germany). DNA concentration was measured using an Epoch micro-volume spectrophotometer system (Epoc, Bioteck ® Instruments, Inc., Vermont, USA). The concentration of DNA for all samples was adjusted to 50-100 ng/μL.
For the detection of Neospora DNA, an adapted singletube nested-PCR on the internal transcribed spacer (ITS1) region of N. caninum was carried out with four oligonucleotides as described by Buxton et al. [4]. In addition, a PCR assay on the B1 gene [23] and on omp 1 gene [26] was used to detect T. gondii and Chlamydia spp., respectively. Purified N. caninum, T. gondii and Chlamydia spp. DNA were used as positive controls. Secondary amplification products were visualized in a 1.8% agarose gel electrophoresis and SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA, USA) under UV light.

Statistical Analysis
The association between serological status and reproductive performance was evaluated by applying Chi-square test or Fisher's exact test. P values < 0.05 were required to demonstrate statistical significance. All statistical analyses were performed using Graph-Pad Prism 5 v.5.01 (GraphPad Software, San Diego, CA, USA).

Description of the Flock
The pregnancy rate was 89% (119/134), the abortion rate was 8.4% (10/119) whereas the perinatal mortality rate was 15% (16/109). At the end of the lambing season (September 2017 to November 2017), 93 ewes delivered 143 lambs: 50 ewes delivered twins and 43 delivered single lambs. During the study period, 10 ewes aborted but only one fetus was recovered and submitted to the diagnostic laboratory for diagnosis purposes. The aborted dam was 1.5 years old and it was its first gestation.

Serological Study
Neospora caninum and T. gondii global seroprevalence was 26.8% and 59.1%, respectively. Detailed information on prevalence for each category is provided in Table 1. Ewes (adult and yearling) were more likely to be N. caninum and T. gondii seropositive in comparison with lambs (P < 0.05). Furthermore, adult ewes had more likely higher antibody´s levels against T. gondii than yearling ewes (P < 0.05).
The serum of the aborted dam had high antibody titers against N. caninum (1:12,800) and was T. gondii-seronegative. Antibody titers against N. caninum were higher in seropositive aborted ewes when compared with those seropositive non-aborted ewes (P < 0.05), although there was no association between reproductive losses and seropositivity to N. caninum or T. gondii (P > 0.05). There was a positive correlation between the levels of antibodies against both protozoa in lambs, indicating that as levels of antibodies to N. caninum increased, also antibodies levels to T. gondii increased (P < 0.001). Moreover, there was a significant association between dams and lambs T. gondii serostatus, but this effect was not observed with N. caninum serostatus.

Fetal Study
The aborted fetus recovered was a twin and had 112 days of gestation. Fetal central neural system (CNS) was liquid, therefore, it could not be processed for histopathological and immunohistochemical studies. Other fetal tissues and the placenta had a moderate state of mummification (Fig. 1a).
Histopathological analysis in fetal tissues showed multifocal lymphohistiocytic glossitis (Fig. 1b), diffuse mild lymphohistiocytic endocarditis, pericarditis and focally extensive myocarditis. Severe multifocal necrotizing placentitis and diffuse mild lymphohistiocytic placentitis with the presence of lymphohistiocytic vasculitis were observed in placenta (Fig. 1c). Positive N. caninum immunolabelling was detected in tongue (Fig. 1d) and placenta. In addition, antibodies against N. caninum were detected in fetal fluid (1:800) and Neospora DNA was detected in CNS, heart, lung and placenta. Immunohistochemistry for T. gondii was negative in placenta and all fetal tissues analyzed and no serological evidence of T. gondii was detected in the fetal fluid. No evidence of other abortigenic pathogens (Brucella spp., Campylobacter spp, Salmonella spp., Chlamydia spp.) was found in the tested tissues.

Discussion
Ovine neosporosis was first described in sheep in 1990 associated with congenital infection in a lamb [7]. Afterwards, N. caninum has been sporadically reported as cause of reproductive disorders in sheep [1,13,14,16,20,21,29]. The present study is the first description of an ovine abortion caused by N. caninum in Argentina. The characteristic fetal lesions, intralesional identification of the parasite by IHC, detection of N. caninum DNA and serology confirmed the diagnosis in the analyzed fetus. Moreover, there was no evidence of other infectious abortifacients in the aborted fetus.
Several authors have described foci of leukomalacia, isolated granulomas or necrogranulomas (some with mineralized centers) and occasional tissue cysts in CNS of ewes aborted fetuses due to N. caninum [16,22,30]. Unfortunately, neural tissues were not available for histopathological examination. However, the presence of N. caninum DNA in fetal CNS was confirmed by PCR. In addition, characteristic lesions of neosporosis were found in placenta, fetal heart and tongue; and the IHC confirmed the presence of the parasite in placenta and tongue.
West et al. [29] related N. caninum as a cause of abortion in maiden ewes of New Zealand. These authors found positive N. caninum titers in fetal fluids and compatible histopathological lesions in aborted fetuses, but they described that the aborted ewe showed low antibody titers compared with those found in aborted cows. In the present work, high N. caninum antibody titers were found in the aborted ewe and fetus, in agreement with previous reports in cattle and goats [6,9].
In the present study a higher flock N. caninum seroprevalence was found compared with previous reports in other countries [8,11,28]. Previous works of our group reported low N. caninum seroprevalence in a dairy (3%) and Texel flock (1.54%) from Humid Pampa, Argentina [17,19]. In agreement with our results, Gonzalez-Warleta et al. [13] reported a high seroprevalence in a Spanish flock where N. caninum infection was the cause of the low reproductive performance. Based on our serological findings and the presence of N. caninum in the aborted fetuses, we can hypothesize that N. caninum is an important cause of reproductive losses in this flock. However, we cannot assume that all reproductive losses in the flock were caused by N. caninum because other aborted fetuses were not available for analysis and no significant association was detected between seropositive animals to N. caninum and reproductive losses. Although the flock was brucellosis and tuberculosis free, the authors cannot rule out the presence of other infectious abortifacients.
Toxoplasma gondii seroprevalence in the studied flock was high (59.10%) compared with seroprevalences previously reported in Argentina [15,17,19] and in other countries [8]. In the analyzed farm there were nine cats cohabiting with the sheep. Unfortunately, these cats were not sampled, in order to confirm them as the possible source of infection. The high T. gondii seroprevalence is probably due to the intensive husbandry conditions of this flock and the more probable exposition to contaminated feed or water with oocysts of T. gondii. Although there was not association between T. gondii seropositivity and reproductive losses, and T. gondii was not detected in the aborted fetus, it is difficult to discard the possibility role of this protozoon as a cause of reproductive losses in this flock.
The results of this study showed that N. caninum and T. gondii seroprevalence increased with the age of animals, indicating that the horizontal transmission of both infections was occurring in this flock. This hypothesis could be strengthened for the fact that the analyzed dogs were seropositive to N. caninum and T. gondii and that these animals and cats were co-habiting with the sheep and their lambs.
The present study described for the first time an ovine abortion caused by N. caninum in Argentina. In addition, high seroprevalence were detected against N. caninum and T. gondii in the flock, indicating that the reproductive losses could be caused by these parasites. Further investigations at a larger scale are required to continue providing detailed information and establish the impact of neosporosis and toxoplasmosis on sheep reproductive losses of farms from Argentina.