Pathological changes in aborted fetuses and placental tissue in cows with neosporosis

. The failure to obtain offspring due to abortion causes considerable damage to the dairy industry worldwide, costing millions of USD. Neospora caninum , a protozoan parasite of the Apicomplexa class, is the causative agent of neosporosis, a disease that leads to abortions or the birth of weak offspring in cows. Therefore, addressing this issue is crucial. The purpose of this study is to describe the pathological changes in aborted fetuses and placental tissue in cows with neosporosis. Macroscopic examination of fetal and placental parts was performed through pathological dissection. The gestation age (days) was determined using the formula: 68+2.25×CRL (cm) and/ or from historical data. DNA of N. caninum was detected by polymerase chain reaction in all fetuses and/ or fetal parts of placentas described in the study. It was found that the majority of abortions occurred at 4-5 months of gestation. External examination revealed isolated instances of skin hyperemia and haemorrhages throughout the body. Some fetuses were mummified. Autolysis of the fetus and placental parts was observed in most cases. Placentitis and oedema of the placental tissue were also noted in some cases. Internal examination of fetuses revealed changes in the brain, skeletal muscles, heart, lungs, liver, spleen, and kidneys, in addition to characteristic autolysis. These changes, including grey foci in the brain with a light grey centre, white foci, and multiple white streaks in skeletal muscles and the tongue, can be attributed to typical pathological changes in aborted fetuses with neosporosis. These changes are rare but can impact the determination of the cause of abortion, narrowing down the factors responsible

or mummified, with no specific pathological changes typically observed.S. Donahoe et al. (2015) noted that, in some cases, pathological dissection of the fetus can reveal hydrocephaly, hypoplasia of the cerebellum and brainstem, as well as scattered white or dark foci in the brain, spinal cord, heart, and skeletal muscles.K. Uesaka et al. (2018) described that placentitis and changes in the placental cotyledons are most commonly observed in the placental tissue.The pathological-anatomical changes were more pronounced in fetuses during the first and second trimesters of intrauterine development than in the last trimester.This means that the gestational age at the time of abortion affects the degree of damage to the fetus and placental tissues.Infected calves may exhibit symptoms such as hypotrophy, ataxia, hind limb paresis, exophthalmia, scoliosis, hydrocephalus, and spinal cord atrophy.
Neosporosis causes significant economic losses.For example, the financial expenses related to N. caninum infection were estimated at 710 USD (438-1043) per dairy cow, and annual economic costs were estimated at 40.5 million USD (24.6-60.3)for Turkey.These losses comprise 67.3% due to abortion, 16.8% due to extended calving intervals, 4.6% due to milk loss, 3.5% due to additional artificial insemination, and 7.7% due to veterinary and diagnostic expenses, as described in the study (Demir et al., 2020).In Brazilian dairy herds, approximately 20% of abortions are linked to neosporosis, resulting in annual losses of approximately 51.3 million USD (Perotta et al., 2021).In Argentina, economic losses are estimated at 33 million USD per year for dairy cattle and 12 million USD for beef cattle (Campero et al., 2023).
Since reproduction is a key element in agricultural production, detecting pathological changes in this context can contribute to the development of effective strategies for neosporosis control and prevention.This, in turn, will preserve herd health and have a positive impact on the economic indicators of the farming industry.The purpose of this study is to describe the pathological-anatomical changes in aborted fetuses and placental parts with detected N. caninum DNA in Ukraine.

MATERIALS AND METHODS
Between 2019 and 2022, 126 cases of abortion were investigated.Of these, 43 cases were attributed to N. caninum.A case was defined as an aborted fetus and/or placental part from a cow, resulting in the examination of 36 fetuses and 34 placental parts.Fetuses and placentas were submitted to the laboratory by veterinary practitioners from dairy and dairy-meat cattle farms in various regions of the country.
The dissections and pathological-anatomical examinations of fetuses and placental parts were conducted in the dissection room of "Centre Veterinary Diagnostics" LLC (Kyiv), following the methodology described by J.F. Mee (2016).This methodology encompasses both external and internal examinations, involving the investigation of all organs and organ systems at the

INTRODUCTION
Abortion remains one of the main challenges in the dairy industry, as reproductive efficiency is crucial, and the failure to obtain offspring can result in significant losses.In many studies, abortion rates range from 5 to 12%, representing a substantial loss of calves and future economic losses (Moore et al., 2021).Infections continue to be the most important cause of abortion in cattle.The infectious agents causing abortions can vary depending on the country in which they occur, with Neospora caninum often being referred to as the most common pathogen on an international level (Mee, 2020).
Neosporosis is a disease caused by an obligate intracellular protozoan parasite called Neospora caninum (Apicomplexa, Sarcocystidae), which is known to cause abortions and stillbirths in cattle, as described by M. Reichel  In the study by J. Perotta et al. (2021), it is described that N. caninum infection in cattle can occur through various routes: horizontal (ingestion of oocysts), exogenous transplacental (ingestion of oocysts by pregnant cows), and endogenous transplacental (reactivation of latent infection in the fetus within the pregnant cow).Transplacental transmission of N. caninum is the most efficient method and leads to endemic infections.The endogenous infection cycle in the host population is continuous since infected cows give birth to infected calves, which, in turn, enter the breeding herd and continue to spread the infection.
If a cow becomes infected within the first 100 days of pregnancy when lymphoid tissues are developing, the survival of the fetus is unlikely because infectious agents directly kill fetal cells, leading to resorption or fetal mummification (Donahoe et al., 2015).The paper by M. Reichel et al. (2018) noted that during the second and third trimesters, the fetus develops an increasing ability to mount an immune response (IgG antibody production).However, in many cases, this response is inadequate, as abortion is the most common outcome of infection during mid-pregnancy.Infections occurring in the third trimester, after the fetal immune system is nearly fully developed, are more likely to result in the birth of infected but clinically healthy calves.Nevertheless, the birth of uninfected calves can occur very rarely, and fetal death is also a possible outcome.
P. Melendez et al. (2020) reported that neosporosis-related abortions in cattle typically occur between the 3rd and 9th months of gestation, with the majority of cases happening during the 4th to 6th months of pregnancy.Aborted fetuses are often found in an autolytic state with the accumulation of serosanguinous fluid in body cavities, but they may also be fresh macroscopic level, for both fetuses and placental parts.The gestational age at which the abortions occurred was determined based on data provided by the owners of the aborted animals, in the absence of information, using the formula (1): duration (days) = 68 + 2.25×CRL (cm), where CRL represents the length from the crown to the tail root of the fetus (Mee, 2020).DNA from N. caninum was detected in all cases from fetal organs (including the brain, cerebellum, thymus, heart, lungs, liver, spleen, kidneys, adrenal glands, abomasum, skeletal muscles) and placental parts using the polymerase chain reaction (PCR) method.Amplification reactions were carried out using a commercial test kit from a European manufacturer on the QuantStudio 5 Real Time PCR System (Thermo Fisher Scientific, USA), following the manufacturer's recommendations.Thus, N. caninum was determined as the cause of abortion in each of the cases.
Sample collection for genetic-molecular research (PCR method) was performed directly in the dissection room of "Centre Veterinary Diagnostics" LLC during the pathological-anatomical examination.All manipulations with aborted fetuses and placental parts were performed in accordance with the provisions of the Order for scientific institutions conducting research and experiments on animals (Law of Ukraine No. 249, 2012) and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (European convention..., 1986).

RESULTS AND DISCUSSION
Based on the medical history and calculations using the formula for gestational age, it was determined that abortions caused by N. caninum occurred at the following gestational months: at 4 months in twenty cases, at 5 months in thirteen cases, at 6 months in six cases, at 7 months in two cases, and at 8 months in two cases.Upon external examination of the fetuses, it was found that out of thirty-six fetuses, twenty-nine showed signs of autolysis (Fig. 1), while two were fresh.

Figure 1. Autolysis of the fetus and fetal part of the placenta in a cow Source: photographed by the authors
Five fetuses with fetal membranes were mummified (Fig. 2).Moreover, individual findings included skin hyperemia and focal haemorrhages on the skin throughout the body, accumulation of red-coloured fluid under the skin throughout the body in twelve fetuses, and accumulation of gelatinous-like masses under the skin and in skeletal muscles throughout the body in nine fetuses.Corneal opacification was observed in twenty-nine fetuses.

Source: photographed by the authors
During internal examination of the fetuses, watery consistency of the brain was observed in ten cases, and hyperemia in seven cases.Individual findings in the brain included multiple grey-coloured foci with a light grey centre, ranging in diameter from 1 to 4 mm, and hyperemia of the brain's soft meninges.Dark-red fluid was observed in the thoracic and abdominal cavities in twenty-six fetuses.Generalised tissue reddening was noted in eleven fetuses.Heart hyperemia was found in three fetuses.Atelectasis of the lungs was observed in thirty-one fetuses.Interstitial oedema of the lungs -in two fetuses.Hyperemia of the lungs was noted in one fetus.The liver showed flaccid consistency in eleven cases, oedema in two cases, uneven colouration in three cases, and uneven colouration of the liver along with grey-coloured foci with diameters ranging from 2 to 8 mm in three fetuses.Individual findings included icterus of the liver, hepatomegaly, congestive hyperemia, and firm consistency of the liver (Fig. 3).

Figure 3. Unevenly coloured liver in a cow fetus (arrow) Source: photographed by the authors
Flaccid consistency of the spleen, a smeared pattern, and the presence of dirty-red fluid oozing from the spleen's cut surface were found in eleven fetuses, while oedema was found in four fetuses.Red colouration of the renal capsule and perirenal fat, softening of the renal cortical layer, were identified in eleven fetuses, hyperemia of the kidneys in five, and multiple grey-coloured foci with diameters ranging from 1 to 4 mm in the renal cortex in one fetus.Multiple white-coloured streaks with diameters ranging from 1 to 4 mm in skeletal muscles were observed in two fetuses (Fig. 4), multiple white-coloured streaks ranging from 2 to 6 mm in length in skeletal muscles in three fetuses, and in two fetuses, white-coloured streaks were observed in the tongue (Fig. 5).Autolysis of the placenta was found in nineteen samples, oedema of the placenta (Fig. 6) -in three, fibrinous placentitis -in three (Fig. 7), serous placentitis -in two, foci of chorionic necrosis -in one.Placenta without visible changes was observed in one case.

Fibrin on and between chorion fronds (arrows) Source: photographed by the authors
Thus, in this study, autolysis was present in twenty-nine out of thirty-six fetuses (Fig. 8) and manifested as corneal opacification; accumulation of gelatinous-like masses under the skin and in skeletal muscles or red-coloured fluid under the skin throughout the body; accumulation of dark-red fluid in the thoracic and abdominal cavities; generalised tissue reddening; flaccid liver consistency; flaccid consistency, smeared pattern, and dirty-red fluid oozing from the spleen's cut surface; red colouration of the renal capsule and perirenal fat, softening of the renal cortical layer.Autolysis can obscure or distort pathological-anatomical changes and, as a result, negatively affect the determination of the direct cause of abortion.Furthermore, the results of the conducted study indicate a weak manifestation and infrequent occurrence of changes, except for signs of autolysis, in the fetuses and fetal parts of the placenta due to neosporosis.For example, a study of 655 cases of bovine abortions in California found that approximately 20% of examined fetuses showed no specific pathological anatomical changes (Clothier & Anderson, 2016), and any other changes present could have been concealed by autolysis.According to the authors who have investigated this issue, specific changes in fetuses and placentas for neosporosis were also not found (Costa et al., 2021;Nayeri et al., 2022).
Various data indicate that aborted fetuses from cows infected with N. caninum, both experimentally ( Jiménez-Pelayo et al., 2019) and naturally (Dubey et al., 2017), were predominantly in a state of autolysis of varying severity.However, cases of mummification also occur.Fetal mummification is not a characteristic feature of neosporosis because it occurs far from always and can be a consequence of other factors: viral bovine diarrhoea, leptospirosis, fungal diseases, mechanical factors such as umbilical cord compression and/or torsion, uterine torsion, placental insufficiency, genetic anomalies, abnormal hormonal backgrounds, and chromosomal anomalies (Lefebvre, 2015).Some sources J. Dubey et al. (2017) report cases of hydrocephalus, cerebellar hypoplasia, and hypoplasia of brain tissue.There were no such changes in this study.The pathological-anatomical examination of the fetuses showed that most commonly, the brain had a watery consistency, typically a result of the presence of a large amount of fluid (Njaa, 2012).Hyperemia of the brain was also often detected.Only in isolated cases were multiple grey-coloured foci with a light grey centre, ranging in diameter from 1 to 4 mm, and hyperemia of the brain's soft meninges found.These changes in the brain are histologically consistent with foci of necrosis (Donahoe et al., 2015).
In all cases, atelectasis of the lungs was noted, indicating that the fetus did not breathe, which is normal for aborted fetuses (Ancker & Agerholm, 2010).In some cases, hyperemia and oedema of the interstitial connective tissue of the lungs were found, which are nonspecific changes that can result from various factors.Multiple white-coloured foci with diameters ranging from 1 to 4 mm and multiple white-coloured streaks in skeletal muscles and the tongue, ranging from 2 to 6 mm in length, were observed in some fetuses, which are indicative of necrosis.These changes are similar to those described earlier and are very rare (Dubey et al., 2017).
Uneven colouration of the liver, as mostly observed in this study, can be a sign of dystrophic changes.In addition, grey-coloured foci with diameters ranging from 2 to 8 mm, which can sometimes also be observed in abortions caused by Listeria monocytogenes, Salmonella spp., and bovine herpesvirus-1 (BHV-1), were found (Njaa, 2012).Individual findings included oedema, congestive hyperemia, icterus, and hepatomegaly, which can be the result of various factors as they are not commonly mentioned in the context of neosporosis.In most cases, no visible changes were found in the spleen, while in some, oedema was observed.Apart from autolysis, the kidneys showed hyperemia and, in isolated cases, multiple grey-coloured foci in the renal cortex with diameters ranging from 1 to 4 mm, indicating necrosis or inflammation.
It is worth noting that abortion can be entirely triggered by an infection localized exclusively in the placenta and is often the focus of pathological changes rather than the fetus itself.Virtually all infectious agents causing abortion are transported to the pregnant uterus through the bloodstream and therefore initially localise in the placenta, possibly later spreading to the fetus (Ancker & Agerholm, 2010).In the current study, autolysis of the placenta was predominantly observed, making it difficult to evaluate changes in many cases.There were also cases of oedema, serous placentitis, and focal chorion necrosis.Similar changes in the fetal part of the placenta are described in abortions caused by bovine herpesvirus-1 (BHV-1), Campylobacter fetus, and Tritrichomonas foetus (Njaa, 2012).
In summary, most of the pathological anatomical changes identified in this study can manifest due to the involvement of other infectious agents in fetuses and placentas.Therefore, attention should be paid to this when establishing a preliminary diagnosis, and additional methods of investigation, such as histological and genetic-molecular analysis (PCR method), should be used to establish a final diagnosis.However, among all the findings, the changes in the brain, skeletal muscles, and tongue, which are rarely reported for other causes of abortion, are valuable for establishing a preliminary diagnosis.

CONCLUSIONS
Any information related to N. caninum is important for the development and implementation of measures to combat bovine neosporosis in Ukraine.Considering its impact on the reproduction of cattle worldwide, examining the prevalence of N. caninum in the country is crucial.This study established that abortions due to neosporosis occurred between 4-8 months of pregnancy, but most of them were between 4-5 months.When evaluating the condition of fetuses and the fetal part of the placenta, autolysis was observed in most cases.In addition to changes characteristic of autolysis, some fetuses exhibited pathological changes in skeletal muscles, the brain, liver, kidneys, and the fetal part of the placenta, which, based on other sources and our research, are common in aborted fetuses due to neosporosis and are considered nonspecific.Changes such as grey-coloured lesions with a light grey centre in the brain, white-coloured lesions, and multiple white stripes in skeletal muscles and the tongue can be attributed to typical pathological changes seen in aborted fetuses due to neosporosis.While these changes are rare, they can still serve as diagnostic markers for identifying the cause of abortion.
The association of some pathological changes identified in this study with neosporosis requires further clarification through additional research.Therefore, pathological examinations should be conducted whenever possible.In the future, research can delve deeper into the pathological changes associated with neosporosis in cows and their impact on aborted fetuses and the placenta.Understanding the mechanisms underlying these changes will help better comprehend the nature of the disease and develop more effective strategies for prevention and treatment.

Figure 4 .
Figure 4. Multiple white streaks in skeletal muscles of the fetus (arrows) Source: photographed by the authors

Figure 5 .
Figure 5. Multiple white streaks in the tongue of the fetus (arrows) Source: photographed by the authors

Figure 6 .Figure 7 .
Figure 6.Oedema of the fetal part of the placenta (arrow) Source: photographed by the authors