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Postmortem Examination of the Equine Placenta, Fetus, and Neonate: Methods and Interpretation of Findings
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When presented with an equine abortion, stillbirth, or dead foal, it is in not always possible to rely on a state or private diagnostic laboratory to perform the required gross necropsy examination. This paper will review the gross anatomy of the equine placenta, present basic steps for doing a thorough placental examination, and describe a simple technique for performing fetal or neonatal necropsy.
2. Normal Gross Anatomic Features of the Equine Placenta: "Non-Lesions"
Performance of a thorough placental examination requires a basic understanding of placental anatomy and some familiarity with the gross features found in a normal placenta. Two essentials of a thorough gross placental examination are (1) that one can distinguish normal features [1-9] and common artifacts from lesions that may have diagnostic significance and (2) that one knows how to collect an appropriate set of samples for submission to a diagnostic laboratory. Normal features of the equine umbilical cord and the fetal membranes (the amnion and chorioallantois) are slightly more complex than in other domestic animals (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8) and will be reviewed. Routine examination of placentas, fetuses, and neonates should be encouraged and done whenever possible as these provide a knowledge base for use in future cases. Additionally, involvement of farm managers and owners during a veterinarian’s examination of a placenta after normal foaling can be a very effective opportunity for client education.
Figure 1. Placental orientation for efficient examination. Arrangement of the chorioallantoic membrane in a Y shape (A) leaves extra tissue from the ventral body "bulge" that can be appreciated by looking at the inflated equine placenta (B). This is overcome by laying the placenta flat on its side in an F shape, allowing the entire surface on one side to be evenly exposed.
Figure 2. Appreciation for the gross appearance of the two sides of the chorioallantois (maternal chorionic and fetal allantoic) is best gained by understanding the histology. In this cross-section of the uterine wall of a pregnant mare, the chorioallantois microcotyledonary villi are seen inter-digitating with microcaruncular crypts of the endometrium. The smooth "inner" allantoic surface is also shown.
Figure 3. Photomicrograph of a higher magnification of an attached equine placenta (top, chorioallantois; bottom, attached chorioallantois). Top right: a single layer of allantoic cells line the allantoic surface. Bottom right: a higher magnification of a horizontal plan through the tuft of villi embedded within endometrial caruncular crypts.
Figure 4. Because the outer, or chorionic, surface is covered with small tufts of villi (microcotyledons), the outer chorionic surface of the chorioallantois has a uniform velvety appearance as shown in this view of the chorionic surface of the chorioallantois. Radiating bands where villi are not formed (the "cervical star") correspond to the crease in the folds of the inner cervical mucosa.
Figure 5. A placenta from normal delivery will have ruptured at the cervical star, as shown in this photograph.
Figure 6. The placenta has been arranged to allow efficient examination. One-half of the allantoic surface is exposed for examination. The placenta would be flipped to exam the other allanotic side and turned inside-out to likewise examine the chorionic surfaces.
Figure 7. A line drawing depicting the principle features of a term Thoroughbred placenta.
Figure 8. The umbilical cord is relatively long in the horse and is divided into an amniotic and allantoic segment by the attachment of the amnion. The amniotic segment contains a urachus, two arteries, and one vein in these sections cut near the attachment to the fetus (left) and approximately midway between the amnion and fetus (right).
There is significant variation in the length of the umbilical cord in normal healthy pregnancies. The average length of the umbilical cord in a term Thoroughbred fetus is about 55 cm. (95% confidence interval = 36 - 83 cm) . The umbilical cord is divided into amniotic and allantoic segments. The vessels of the cord normally gently spiral several times [4,5] along its length. This normal anatomic spiraling must be distinguished from pathological twisting, or torsion of the cord, which causes restriction of blood flow. Cord torsion is common and will be discussed in one of the following sections.
The urachus, present within the loose stromal core of the amniotic section of the cord, extends from the bladder to about midway "down" the cord. The urachus opens just distal to the attachment of the amnion where urine is emptied into the allantoic cavity. Small segmental dilations of the urachus are common and protrude as thin-walled sacs extending from the surface of the cord.
The pattern of umbilical vessels is unique in the horse. Depending on where along the cord one looks, there are two arteries and one or two veins. This is because the two umbilical veins returning oxygenated blood from the chorioallantois to the fetus fuse before reaching the fetus. This anastomosis occurs within the distal amniotic segment of the cord, proximal to the area of the attachment of the amnion. A single vein is present in the proximal one-fourth of the cord (Fig. 8). When the cord breaks at delivery, short segments of the two umbilical arteries and one umbilical vein will protrude from the end of the cord. The umbilical vessels break at a point internal to the fetal abdominal wall, which, when combined with the marked vasospasm of the umbilical vessels that occurs during delivery, prevents excessive hemorrhage.
Amnion and Chorioallantois
Some of the more commonly encountered normal structures in the amnion and chorioallantois that should be appreciated include the following:
- Amniotic Plaques (on inner surface of amnion and on the outer surface of the amniotic segment of the umbilical cord)
- small, evenly dispersed focal, slightly raised, 1- to 3-mm keratinized small pinpoint plaques or pointed short papillae
- more on the cord than on the free amniotic membrane (these are less prominent than in cattle)
- they have no recognized functional significance
- microscopically they are composed of amniotic epithelium that has undergone squamous metaplasia
- Vascularization of the Amnion
- the normal equine amnion is normally markedly vascularized and the vessels are tortuous and prominent
- vessels are raised on the amniotic side of the allanto-amnion
- unknown significance, but a normal feature
- also called "allantoic calculi"
- free floating and located in the allantoic cavity
- first appear as small whitish bodies at ~90 days of gestation
- grossly, they are tan to brown, soft flattened oval shaped masses of 5 - 10 cm in diameter
- unknown significance, one hippomane is usually present
- microscopically, these laminate concretions are composed of lipids, cellular debris, degenerated blood cells, and irregular mineralized material
- Allantoic Vesicles
- clear walled, polypoid structures attached to the allantoic surface
- contain a gelatinous stroma
- microscopically, these polypoid masses are covered by allantoic epithelium
- usually small, but can be multiple and up to several centimeters in length
- frequently located along the course of large vesicles
- inconsistently present, unknown significance
- Allantochorionic Pouches
- also called "chorionic pouches"
- these are pedunculated invaginations of the chorioallantoic membrane that protrude into the allantoic cavity
- they contain sloughed necrotic endometrial cup material
- are only found near the area where the umbilical cord attaches where the endometrial cups had developed, then sloughed
- sloughing of the cups occurs between days 70 and 100 and is completed by day 130 when a targeted maternal immune response destroys cup cells
- allantochorionic pouches are also found in placentas from older fetuses and are present in one-half to two-thirds of all placentas
- occasionally, allantoic calculi (hippomanes) start to develop on the surface of these pouches, but more commonly they form on niduses within the allantoic fluid
- Cervical Star
- that area of the chorioallantois that contains radiating patterns of avillous chorion corresponding to opposing folds of the internal cervical os
- these radiating bands of pale to white, raised slightly thickened linear ridges tend to be more prominent in older mares
- the chorioallantois is normally torn in this area when the fetus is delivered
- the cervical star area is commonly the area affected by ascending bacterial or mycotic infections and therefore has significant importance during diagnostic work-ups
- Other Areas Devoid of Microcotyledonary Villi
- Other areas that are also devoid of chorioallantoic microvilli include areas of chorioallantois over the endometrial cups, at the openings of the uterine tubes, over the attachment of the yolk sac, along invaginated folds of the allantochorion caused by tension created by the presence of large allantoic vessels, and where the chorioallantois opposes tissues other than healthy glandular endometrium (eg, contact areas where placentas of twins meet).
3. Examination "in the Field" or Submit to a Diagnostic Laboratory?
There are pros and cons regarding the decision to perform the diagnostic examination and sampling yourself or arranging to have a placenta, aborted foal, and/or neonate submitted to a diagnostic laboratory. Diagnostic facilities usually have higher success rates of arriving at a definitive diagnosis if they do the examination and collection than submissions of only tissues and samples by practitioners. It is also difficult to charge appropriately for your professional time, yet there are important advantages to doing this work yourself. First and foremost is that you will be directly involved, one’s clinical acuity will be enhanced, you may arrive at a diagnosis quickly, and if not, at least the samples will be taken in a much fresher state. By taking advantage of the owner’s and/or farm manager’s concerns and interests, a professionally conducted necropsy examination can be used as a practice builder.
The location should be well lit, with a large flat surface that is elevated to make the work less stressful. One must always be concerned with the possibility of contaminating the farm environment, or otherwise spreading infectious diseases. Care and caution should be taken; appropriate protective clothing for everyone present, with proper cleaning and disinfection of the area and all instruments and equipment. If the mare aborted in a stall and has been removed from that stall, this would be an ideal place because the environment is probably heavily contaminated already and would require cleaning and disinfection anyway. Only a few instruments are needed because 95% of the work can be done with a sharp knife. Ten percent-buffered formalin should be available, and care should be taken to insure that no spills occur. Syringes with large-bore needles are useful in collecting fetal blood. Tissues can be collected in whirl-top bags or clean plastic jars. Unfixed tissues will be divided at the diagnostic laboratory for distribution to laboratories, so duplicate samples for virology or bacteriology are not required. (Many labs provide "abortion kits" that contain necessary forms, containers, and disposable instruments; contact your laboratory and ask about these.)
Gross contamination of the placenta by straw, wood shavings, grass, etc., should be removed so that lesions are not missed. This can be done by briefly rinsing the surface with cold running water. By lifting and holding the placenta by one end and briskly, but gently, directing a stream of cold water to flow from the upper end over its surface, most debris can be quickly removed. The placenta should be weighed and laid out as flatly as possible.
For placental examination, it is important to examine all surfaces of the placenta. During delivery, most equine placentas become everted, resulting in the smooth allantoic surface being outermost. (After the fetus breaks through the amnion and chorioallantois as the cervix dilates and for a short time after the fetus is delivered, the cord remains attached to the chorioallantois, which is still attached to the endometrium. Tension on the umbilical cord slowly pulls the chorioallantois "inside-out" as it is freed from its endometrial attachment.)
Initial orientation of the placenta for examination is simplified by first finding the cord and carefully unfolding and flattening the three "arms" of the Y-shaped chorioallantoic membrane. (Those areas of the chorioallantois that fill the two horns of the uterus and the larger uterine body are by convention referred to as "horns" and "body" of the chorioallantois.) One of the first concerns is to check for completeness; retention of pieces of the tips of the horns is relatively common and can have serious consequences.
The gross and microscopic pictures in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, and Fig. 7 show the normal appearance of the equine placenta and help to explain some of the more important structural features. The two surfaces of the chorioallantois have very different appearances (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6). Both must be examined carefully for lesions. This requires examining the entire surface and then turning the placenta inside out, as one would do when rolling up a sock to continue examination of the other surface. This is facilitated by arranging the chorioallantois in an F shape.
If fresh, the chorionic surface is red, and its surface has a fine velvet-like surface. The appearance is caused by microcotyledons that are small tufts of branching villi that pulled away from their interdigitation with the endometrium as the placenta is freed (Fig. 2 and Fig. 3). Villi on the surface of the non-gravid horn will be thick and lush, whereas the villi over the larger gravid uterine horn and the uterine body will be shorter, and the chorioallantoic membrane will appear thinner because of stretching. Delivered placentas contain a significant amount of fetal blood. Uneven pressures on the placenta during even a short storage period can force blood to move within vessels and capillary beds, leaving dramatic color patterns that can be mistaken as a pathologic change (Fig. 9).
Figure 9. Color is an important gross feature; however, uneven expulsion of blood, as shown in this placenta, which is caused by pressure, can easily be mistaken as congestion or placentitis. This is a common artifact.
When the placenta is laid out with the allantoic surface outermost, the surface is smooth, allantoic vessels are prominent, and the attachment of the umbilical cord to the chorioallantois near the base of the horn is evident (Fig. 6 and Fig. 7). The amnion is not attached to the chorioallantois in the mare, an anatomic feature that contributes to the occurrence of cord torsion. Excessive twisting of the cord is relatively common in the equine but rare in other species in which the amnion is adhered, and therefore, anchored to the chorioallantois. The amnion is attached to the umbilical cord near its middle. The cord should be extended, examined, and measured, the amnion should be laid out, and both surfaces should be examined.
`The chorioallantoic cervical star deserves special attention because it is that part of the placenta that is physically against the inner os of the cervix. Because ascending infections through the cervix are common in the pregnant mare, the cervical star area must be carefully examined and routinely sampled. During normal delivery, the foal ruptures through the amnion and then through the chorioallantois at the "cervical star," which is in direct apposition to the cervix (Fig. 5). If the placenta is prematurely separated from the endometrium, the fetus can rupture the chorioallantois in an area that is not the cervical star. This finding and the presence of a markedly congested chorioallantoic membrane in the distal body near the star are important diagnostic features of premature placental separation.
Sampling of the placenta is simplified by spreading the tissues out as described earlier. Tissue samples from seven sites as shown in Fig. 10 should be taken for histopathology, and both normal features and lesions described. It is important to label tissues so that the pathologist can more precisely evaluate the case and render a meaningful diagnosis. Placental tissues collected from different locations can be identified using different methods: cutting tissues in different shapes, attaching pins or clips, or placing tissues in small labeled containers. Figure 11 shows several labeled linear pieces of chorioallantois collected from the placenta shown in Fig. 10.
Figure 10. The chorioallantoic, cord, and amnion should routinely be samples at seven sites, and each tissue should be clearly labeled.
Figure 11. Cassettes clipped to tissue samples work well to label different samples. Clips, string, or cutting pieces into different sizes or shapes also can be used to designate where different samples are taken.
A standard report form should be filled out and submitted with the samples. A copy of a report form prepared and used by the author is included at the end of this paper. It can be copied and modified to suit one’s needs.
5. Common Artifacts Easily Confused as Being Lesions
In addition to appreciating normal anatomical structures that might be confused as lesions, it is also necessary to appreciate common artifacts. Some tissues containing changes of questionable significance may require histopathology to differentiate artifacts from diagnostic lesions.
Edema occurs both as a manifestation of altered vascular permeability in inflamed tissue (edema is a common feature in placentitis cause by many different agents) and as an artifact. Significant edema can develop during prolonged delivery. Placental tissues do not have lymphatics to drain fluids. Excessive exposure to water will also lead to fluid imbibition that grossly looks like edema. Regardless of cause, accumulated fluids will cause the placenta to weigh more than normal. Placental weight is used as a convenient screening parameter to identify abnormal placentas, so understanding why edema is present is important.
Congestion of blood vessels can result in dramatic changes in the appearance of the placenta, but vascular congestion can also be part of an inflammatory process, or can be an artifact. Congestion is one of many conditions that lead to discoloration. As noted earlier, compression with associated movement of blood from one area to another commonly occurs in placental tissues as shown in Fig. 9. The picture shows a fresh placenta just removed from a plastic bag shortly after delivery. The straight edges lining the areas of discoloration are additional clues that the color change resulted from artifactual "passive congestion." "Active vascular congestion," induced by inflammatory mediators, can sometimes be distinguished from passive congestion by turning the placenta to view the allantoic surface and examining the allantoic vessels. Focal or localized placentitis is usually associated with corresponding inflammatory mediator induced engorgement of vessels, especially smaller capillaries and veins. Neovascularization occurs in chronically inflamed placental tissues.
Exposure to air will quickly dry the delicate surface of the placenta and also lead to loss of color and irregular areas of discoloration. Autolyzed placental tissues assume a tan to grey appearance and must be differentiated from necrosis that usually is accompanied by inflammation (and the accompanying edema, vascular congestion, and accumulation of exudates). Autolysis also can lead to tissue sloughing, which may leave the impression that there is inflammatory exudates on the surface. Autolysis and necrosis can be difficult to differentiate grossly.
6. Placental Lesions That Have Diagnostic Significance
Non-infectious causes of equine fetal loss include failure of normal placental development that can result from primary lesions in the mare’s endometrium. Mares with marked endometrial lesions, such as fibrosis, are not able to support normal development of chorionic microcotyledons (chorionic villus hypoplasia) and frequently abort during the first trimester because of placental insufficiency. Inadequate placental development also occurs during pregnancy in mares with twins. Areas of placental apposition appear as large whitish avillous areas that seem fibrotic (Fig. 12).
Figure 12. Color (pale to white) and smoothness of the chorionic surface are important features found on the chorionic surface in areas of apposition of twin placentas as shown in this photograph.
Loss of functional placental exchange also occurs in body pregnancies (the horns appear rather small and that part of the placenta occupying the body is relatively large) and in the acquired condition, premature placental separation. In the latter, separation most frequently occurs near the cervical star. The parts of the placenta that separated from the endometrium become congested, resulting in dramatic reddening (Fig. 13). The absence of thickening, necrosis, or inflammatory exudate helps differentiate these conditions from placentitis. Delicate microcotyledonary villi and the vessels that feed them can be distorted or compressed. Degenerative changes may develop if the allantoic stroma becomes edematous for a prolonged period. Extensive chorioallantoic edema is a common feature in hydroallantois and can lead to loss of function because of perfusion and/or direct tissue compromise.
Figure 13. Premature placental separation. Dramatic color differences clearly define the tissues that prematurely became separated. This is also called "red-bag." The chorioallantois becomes very congested when it is detached from the endometrium.
Normal anatomic spiraling must be distinguished from twisting, or torsion of the cord, which is pathologic and causes restriction of blood flow (Fig. 14 and Fig. 15). Slight cord torsion may have no detectible deleterious effect, but after it reaches a certain degree, torsion can cause either acute fetal compromise (with fetal death and abortion), or subacute to chronic cord compression with resultant chronic placental insufficiency. Torsion of the amniotic segment of the cord can lead to constriction of urine flow through the urachus, resulting in urachal and/or bladder distention. Edema and hemorrhage are present if there are many acute twists, but in cases with only a few twists, changes in the cord may be minimal or not grossly evident.
Figure 14. The umbilical cord normally is spiraled a few times but can become twisted causing either partial or complete obstruction to blood and/or urine flow. Twists of the cord can involve just the amniotic or allantoic segments.
Figure 15. Torsed cord from an aborted foal. The cord in edematous and the urachus slightly distended. Commonly there is also hemorrhage.
Abnormalities of cord length are also common, with excessive length being more common than a pathologically short cord. Excessively long cords (arbitrarily suggested as cords >85 cm at term) are associated with degenerative changes in the microcotyledons and placental insufficiency that can lead to compromised fetal growth or abortion. Although such compromise obviously has effects on fetal health, no studies have been published that assess effects on neonatal or adult growth or performance of fetuses that survive. Recently, there is a much broader appreciation for how common different forms of placental insufficiency are. In human medicine, the association between compromised fetal growth and diseases that develop in adult life in humans has been firmly established for a number of diseases. This relationship is referred to as the "Barker Hypothesis" after the British epidemiologist whose work first drew attention to the causal relationship of abnormal placental size and development of coronary heart disease in later life. It is hoped that growth and performance data from the horse will be published that addresses the question if there is a similar association between life in utero for a foal and long-term effects on its adult health and performance.
Ossification of Remnants of the Umbilical Cord
Occasionally remnants of the yolk sac ossify and the associated tissues undergo hyperplasia and hypertrophy . These can either be small and incidental or large. Larger ones are usually pedunculated masses attached to the allantoic segment of the umbilical cord by a stalk of tissue. Larger ossified yolk sac masses are commonly misdiagnosed as being a blighted twin fetus. There are two reasons for this. A condition exists in cattle in which a small abnormally developed fetal mass and its placenta are found attached to the placenta of its normal twin (the abnormal fetal mass is called an "amorphous globosus"). Second, the gross appearance of the equine ossified yolk sac remnant that looks like a fluid-filled sphere with bony plates forming part of the outer shell, resembles a malformed head. It is difficult to explain why ossification of the yolk sac develops into the form of a spherical cystic mass. Although the confusion is understandable, now that a series of cases have been studied, it is clear that they develop from yolk sac tissue. It is also interesting that they occur only in the horse. They are relevant because they sometimes mechanically interfere with cord blood flow to placenta and lead to abortion .
Infectious Conditions: Lesions of Diagnostic Significance
Amnionitis is most commonly encountered in severe cases of bacterial or mycotic placentitis, but it is rarely observed in the absence of significant inflammation of the chorioallantois. It usually is accompanied by funisitis (inflammation of the cord).
During the initial stages of the 2001 Mare Reproductive Loss Syndrome (MRLS) abortion losses in Kentucky, clinicians and pathologists encountered an unusual syndrome involving early fetal death and late-term abortion in which there was prominent amnionitis and funisitis without accompanying significant chorioallantoic involvement. The unusual finding became a key diagnostic feature commonly observed in aborted late-term fetuses in the MRLS that so devastated the equine industry in 2001 and 2002 . Uncommon bacteria were also frequently isolated from these cases, but the complete pathogenesis for this disease is still not known. Experimental studies have now implicated exposure to the Eastern Tent Caterpillar .
Amnionitis is recognized grossly by tan to pale appearance of a roughened irregular amniotic membrane covering the amniotic segment of the cord. In more severe cases, adherent necrotic debris is attached, and necrosis and/or congestion and edema of the amnion and cord stroma can be seen.
Meconium staining is considered to be a fetal response to severe distress in utero. The fetal skin, amniotic fluids, and amniotic membrane are stained yellow. It accompanies fetal hypoxia and is found in a variety of fetal and placental infections and when the fetus becomes hypoxic from cord compression or placental separation.
There are several large retrospective studies that address causes of equine abortion [11-17]. Infectious disease caused by viruses, bacteria, and fungi produce lesions that are sometimes suggestive of their etiology, but laboratory confirmation is commonly required. Few "pathognomonic" (unique to only one cause) lesions exist, thus requiring routine collection of tissues for additional examination by specialized laboratories in diagnostic centers. Regional differences in the causes of abortion do occur. Some conditions, such as abortion caused by twinning and equine herpes abortions are occurring less commonly in some areas. This is attributed to better reproductive management (use of ultrasound) and widespread use of vaccines. The corollary to this is that the relative percentage of abortions directly attributable to the umbilical cord have risen. In a recent retrospective study by Smith et al  of 1252 equine fetal and neonatal submissions to their diagnostic laboratory, problems associated with the umbilical cord were the most common diagnosis. In that study, only about 10% of the cases had placentitis.
As noted previously, placentitis is usually associated with heavy placentas. Normal placental weight at term is about 11% of the weight of the foal; edema is responsible for much of the increased weight. Because placentitis in the mare is commonly associated with ascending infections, lesions frequently are found in the chorioallantois near the cervical star (Fig. 16). The most prominent changes will be on the chorionic surface because infections tend to spread from the cervix, extending between the endometrium and chorioallantoic membranes. Maternal inflammatory cells from the endometrium accumulate on the endometrial surface and invade fetal tissues in areas where microorganisms are present (Fig. 17). Comparison of the three sections taken from a normal, a congested, and a markedly inflamed and necrotic placenta revealed the destructive effects that microbial infections and the associated inflammation have on the delicate villous interface.
Figure 16. Chronic inflammation and necrosis at the cervical star area of the chorioallantois from a fetus with mycotic placentitis.
Figure 17. Subgross pictures of tissue sections taken from three equine placentas photographed at the same magnification. A section from a normal placenta (top; allantoic surface top; chorion villi on the lower side). The middle section is from a placenta with a torsed cord; note the marked vascular congestion. Contrast these to the massively necrotic, thickened section (bottom) with extensive inflammation and reactive allantoic cystic change associated with chronic mycotic placentitis.
Areas of inflammation and necrosis of the chorioallantois appear grossly as tan to brown, thickened areas with or without fibrin necrotic exudate on the chorionic surface. Cytology is sometimes useful and may reveal inflammatory cells and sometimes causative microorganisms (fungal hyphae, bacteria, etc.) can be recognized. Ascending infections sometimes extend only 10 - 20 cm from the star area, and in other cases, may extend along the ventral aspect of the chorioallantois. Notable exceptions to this ascending pattern are found in placentitis caused by nocardioform bacteria (Crossiella equi sp) , which typically is not an ascending placentitis but instead typically causes placentitis in a locally extensive pattern near the cranial ventral uterine body and attachment of the horns. Leptospirosis [14,19] and candida infections, on the other hand, cause diffuse placentitis. Affected placentas may appear to be redder than normal and may have small irregular areas of pallor. Occasionally significant vascular involvement can lead to development of areas of placental infarction, which will be tan to grey and have a clear line demarcating the necrotic and viable tissues.
Although equine herpes viruses do not commonly cause gross lesions of diagnostic significance in the placenta, there have been a number of papers that address the value of examining placental tissues in the establishment of a diagnosis of herpes abortion [20-24].
7. Sample Collection and Submission
An important part of a complete gross examination is collection of a standardized set of tissues for submission for ancillary testing. Many laboratories publish guidelines for collection and submission of samples. Clinicians are encouraged to contact the laboratories for advice and help.
Multiple samples taken from different parts of the placenta are important, especially for histopathology. Inclusion of placental tissue from the cervical star area for microbiology and histopathology is essential. Suggested areas for routine sample collection are shown in Fig. 10, and a map is included in the pathology report form. Samples collected for bacteriology and virology should be shipped refrigerated, not frozen. Use of special transport media should be considered in consultation with laboratory personnel. Samples for histopathology should be fixed in 10% buffered formalin. Any placental or tissues with suspicious lesions should also be submitted. Clearly label all samples and submit them with a detailed description. Use of digital or conventional photography is encouraged and is very helpful when pictures are included with case submissions.
8. Postmortem Examination of the Fetus and Neonate
As noted above, attention must be paid to potential environmental contamination and protection of personnel. The fetus should be weighed, and the crown-rump length should be taken with the fetus slightly bent in a natural position. It is examined carefully for congenital anomalies. Limbs should be evaluated for evidence of contracture or other congenital anomalies. If the placenta is still attached to the fetus, it should be removed a few centimeters from the umbilicus. The fetus is laid on its left side and the body cavities opened.
The initial steps in the necropsy involve making cuts that allow the right fore and hind limbs to be laid back and the abdominal and thoracic cavities to be opened and their organs clearly exposed (Fig. 18). To do this, tissues under the right fore and right hind limbs should be cut, and the legs should be flipped back so that the carcass continues to lie flat on its left side. An incision is made from the tip of the mandible, continuing ventrally down the midline extending down the ventral abdomen to the anus. The skin is dissected laterally over the right side of the fetus. The abdominal wall is removed by making an incision just caudal to the ribs and extending the incision ventrally along the rib cage to the mid-line dorsally across the dorsal paralumbar fossa, and caudally along the caudal area of the abdominal cavity back to the middle at the pubis. This will free a flap of tissue that can either be removed by extending the incision cranially along the ventral midline or by simply laying the lateral abdominal wall "flap" ventrally thus exposing the abdominal cavity.
Figure 18. Necropsy of the equine fetus is done by initially making a skin incision along the midline, opening the axilla and femoral joints, and removing the right abdominal and right thoracic wall to allow gross assessment sampling of fetal organs. Figure was prepared by Mr. Michael Simmons of the Office of Educational Development at Cornell University.
The rib cage is removed by cutting along the costral-chondral junctions to the thoracic inlet, followed by cutting the dorsal ends of the ribs a few centimeters from their attachment to the vertebral column. Both cuts are most easily done from a caudal to cranial direction. Alternatively, two or three ribs can be freed at a time by cutting the inter-costal muscles and manually bending them dorsally enough to break them near their attachment to the vertebral column. Care must be taken because this technique produces sharp fragmented ends. By doing two or three ribs at a time, the entire thoracic wall can be laid back quickly. The thoracic and abdominal cavities are exposed for examination and sampling of viscera as shown in Fig. 18. Care should be taken to collect tissue for culture and heart blood for culture and/or serology without contaminating them. By changing gloves and using clean instruments, one can avoid confounding culture efforts. Swabs or samples of tissues can be collected by first searing the surface of larger organs using a propane torch to heat a flat spatula.
Pleural and peritoneal fluids may be used for serology if blood is not available. This is because vascular permeability changes occur quickly and immunoglobulins will pass into these fluids. A full set of tissues should be collected from the placenta and fetus for every submission. For histopathology, fixation is important; a thin flat piece of tissue (<1 cm in thickness) should be collected as gently as possible and placed in buffered formalin solution. The total amount of formalin should be 10 times the volume of the tissues being fixed. Once thoroughly fixed, most of the formalin can be discarded before shipping.
In addition to fetal heart blood, the following tissues should be sampled.
- Adrenal Gland
- Placenta (chorioallantois, amnion, and cord; see report form)
- Brain (entire brain)
- Small Intestine
- Any other tissue you wish to submit
The necropsy is continued by removing the heart, lungs, trachea, and esophagus. The tongue, trachea, and esophagus are dissected free as gentle traction is used to lift and pull the tissues caudally. The tissues (referred to as the "pluck"), are removed together. The trachea and esophagus are opened longitudinally, starting from the pharyngeal end. Each lung lobe is sectioned, and all cut surfaces are examined. The heart is examined by making two incisions in the ventricles. The right heart is opened by cutting the lateral ventricle wall just lateral to the septum. By extending the cut from the coronary grove down to the apex and continuing up along the other side, the lateral wall can be laid back and the chamber examined. The right atrium is exposed by making a cut through the right atrial ventricular (AV) valve. The left heart is usually opened by making a single cut through the middle of the lateral ventricular wall. After the valves and ventricle are examined, the knife is placed under the AV valve into the aortic valve and a cut made dorsally to expose both lining of the base of the aorta and the left atria.
A complete necropsy would normally include examination and removal of the brain. Lesions found in the fetal brain include cerebral degeneration associated with hypoxia seem in severe placentitis or chronic cord torsion. Meningitis is commonly present in foals with septicemia. The brain is removed by making a T-shaped incision through the skin on the dorsum of the cranium starting between the eyes, extending along the dorsal midline, and joining it over the occipital crest with a cut that extends across the back of the head. The two skin flaps are reflected cranial-laterally. The head is removed by making a deep cut ventrally through the muscles at the back of the neck. The cut extends parallel with the back of the head to the foramen magnum. Then the head is turned and bent slightly laterally, and a lateral cut from the side of the neck is made, resulting in further exposure of the occipital joint. Gentle lateral pressure opens the joint further, and the head is removed. The brain is exposed by removing a rhomboid piece of dorsal calvarium using rongeurs, heavy scissors, or a saw. Three cuts through the calvarium are required. The first is a cut just in back of the orbital bones, extending across the skull. Two roughly parallel cuts are then made in a sagittal plane starting in the dorsal-lateral corners of the foramen magnum, extending dorsal-laterally (45° angle to the midline) rostrally. These cuts extend rostrally to the cross-sectional cut. They extend between the eyes to the nasal area. Extending the two sagittal cuts beyond the eyes is helpful to ensure release of the deeper aspects of the bones. Alternatively, rongeurs can be used to break small pieces of the calvarium away, starting at the foramen magnum, until the brain is sufficiently exposed that it can be removed.
Gross Lesions with Diagnostic Significance
When bacterial infections involving the placenta extend into the fetus, there may be evidence of fetal septicemia, fibrinous pleuritis, and/or peritonitis, and occasionally evidence of fetal hepatitis or pneumonia. Lesions become evident when there is tissue necrosis, vascular congestion, or permeability change leading to fluid fluxes, hemorrhage, or inflammatory cellular infiltrates or exudates. Focal lesions frequently produce color differences that make them readily apparent if the fetus is fresh. Autolysis leads to fluids moving into the abdominal or thoracic cavities. These fluids will be red or tan when erythrocytes lyse. The volume of fluids found in the abdomen or thorax can be quite large.
Many different microbes are capable of causing infection of the equine fetus and placenta. Most are not recognized until bacterial, viral, or serological methods are performed. Microbes can occasionally produce unusual lesions. For example, mycobacteria cause granulomatous lesions in fetal organs, and colitis is found in fetuses experimentally infected with Ehrlichia (Potomac Horse Fever) .
The most common and important viral cause of abortion around the world is equine herpes virus 1 (and occasionally herpesvirus 4). As is typical for herpes viruses, the virus kills cells, and small areas of cytolytic destruction are visible grossly as small white foci of necrosis. Although focal necrosis can occur in nearly any tissue, equine herpes virus infection of the lung and liver are most readily recognized. They are not accompanied by much inflammation, as would be the case with focal bacterial infection. The necrosis can be extensive and result in swelling of the involved organs. Swollen fetal lung can press on the inner rib cage and leave "rib impressions," which if found, suggest herpes infection (Fig. 19). Equine herpes virus preferentially replicates in the lung in the epithelial cells lining the airways. These cells undergo necrosis and slough and can result in fibrin "casts" in the trachea (Fig. 20), an uncommon, but very suggestive lesion of herpes infection. In foals born with herpes infection, the damage to their lungs can preclude survival, regardless of medical intervention.
Figure 19. There are relatively few gross fetal lesions that are pathognomonic for specific diseases; however, the focal necrosis of hepatic, splenic, and lung are typical for equine herpes virus. Massively fetal lesions lead to expansion of the fetal lung, leaving "rib impressions" as shown in this photograph.
Figure 20. Extensive viral replication leads to necrosis of pulmonary tissues. The equine herpesvirus replicating in respiratory epithelial cells causes extensive necrosis and sloughing. Additionally, vascular permeability changes results in fibrinous casts formation as seen in the trachea in this picture.
Bacterial infections acquired in utero can also be carried into the neonatal period. Acute and chronic placental insufficiency can produce sufficient fetal hypoxia to affect the central nervous system and manifest as the dummy foal syndrome. Bacterial infections initially starting as placentitis frequently extend to the fetus, and the resulting septicemia, toxemia, and associated compromise of multiple organ systems present a common major perinatal medical challenge. Lesions of fetuses and neonates with bacterial septicemia are similar and include petechial hemorrhage of serosal surfaces, lymphadenopathy, fibrinous pleuritis and/or peritonitis, foci of necrosis, and inflammation or frank abscess development. Fibrinous pericarditis, arthritis, or meningitis are not as commonly found in bacteremic fetuses as in neonatal foals. The pathogeneses of the diseases these infections produce are complex and involve initiation of extensive cytokine cascades  and multiple organ system failures are common. As noted in the previous section, identification of newborn foals at risk should include routine, systematic placental examinations.
The author thanks Dr. Walter Zent for valued clinical insights, support, and infectious enthusiasm for attempts to further our knowledge of equine placentation in health and disease.
Copy of a report form prepared and used by the author: available in PDF