Equine Neonatal Isoerythrolysis

Howard P. Bouchelle, III, DVM; Perry J. Bain, DVM, PhD; Paula M. Krimer, DVM, DVSc; and Kenneth S. Latimer, DVM, PhD

Class of 2003, University of Florida College of Veterinary Medicine, Gainesville FL (Bouchelle) and Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388 (Bain, Krimer, Latimer)

Introduction

Equine neonatal isoerythrolysis (NI) is a condition of foals that are born healthy, but develop a possibly life-threatening hemolytic anemia within hours to a few days after the ingestion of their mare’s colostrum. This condition occurs as a result of a hypersensitivity reaction between the mare’s antibodies in the colostrum and inherited antigens from the sire that are present on the foal's red blood cells.

Erythrocyte Antigens

Antigens that are present on the red blood cells define the blood group system(s) to which the horse belongs. According to the International Society for Animal Genetics, there are seven blood groups: A, C, D, K, P, Q, and U. Each group corresponds to a specific gene that contains two or more alleles that vary in combination. These blood group genes code for surface molecules that contain antigenic sites known as "factors." Each factor is specific within each blood group. There are variable numbers of factors for each group.

Red blood cell (RBC) antigens are clinically detected by three main tests: (1) agglutination, (2) complement-mediated lysis of test cells by antibodies directed against RBC alloantigens, or (3) antiglobulin tests.¹ A genetic marker report can be created for an individual horse that will include the following information: (1) Sample identification of horse including name, registration number, color, sex, year of birth, breed, sire name, sire registration number, dam name, and dam registration number; (2) Blood group factors; (3) Protein variants (although these factors have no known medical or performance significance); and (4) Markers recognized by various testing techniques applied. These genetic marker reports may provide information to help manage neonatal isoerythrolysis.²

Because these inherited blood factors are involved in the NI hypersensitivity reaction, NI is a genetic disease. These blood factors vary in structure and in their antigenicity (potency of antigenic response). NI occurs at different frequencies in horse foals versus mule foals, and in standardbreds versus thoroughbreds.

Pathophysiology

Neonatal isoerythrolysis is a type II hypersensitivity reaction (Fig 1). In this type of reaction, initial exposure to an antigen from a non-native cell will induce B lymphocytes to produce antibodies against offending "foreign" antigens that are present on the non-native cells. Antibody production will decrease as the offending antigens are removed from circulation. Upon re-exposure to the same cells containing these offending antigens, a greatly intensified secondary immune response to these antigens will occur. In one study, the following antigens were identified as having produced an immunologic response leading to the production of anti-RBC antibodies (see highlighted factors in Table 1): Ca, Aa, Ab, Da, Dc, Df, Ka, Pa, Ua, Qrs, Qb, Qc, and Qa.^3,7 Specifically, the Qa and Aa antigens are named historically as causing the most severe immunologic reactions.³

Mares can become sensitized (immunologically stimulated) to the offending foreign RBC antigens of the sire or foal if an event occurs which exposes the mare to these antigens (Fig. 2). These events include exposure to offending RBC antigens via blood leakage through the placenta during pregnancy or delivery, previous blood transfusions, or the administration of vaccines containing equine tissue products. The exact mechanism of sensitization at delivery is unclear at this time.^{4, 5} With pregnancy-related sensitization, the mare is sensitized to the stallion’s RBC antigens that differ from her own RBC antigens. Once the mare has been exposed to these antigens, she will respond immunologically by producing an alloantibody (usually IgM antibodies initially, then IgG antibodies). Subsequent immunologic memory can persist for many years. This sensitization after initial exposure (usually after the first pregnancy) is usually minimal. However, if repeated exposure to the same offending RBC antigens occurs with subsequent pregnancies, then alloantibody production will increase considerably.⁵

Once the mare has become sensitized to specific RBC antigen(s), subsequent foals are at risk for development for NI if they are sired by the same stallion. Adverse reactions can occur with one or more antigen types simultaneously. Because of the type of placentation In horses, the alloantibodies responsible for NI do not cross the placenta, but are secreted into the colostrum. Foals will develop to term and be born without any side effects from the mare’s immunologic response to these offending antigens. When the young foal ingests its mare’s colostrum, the colostral antibodies will be absorbed into the circulation of the foal during the first few hours after birth until "gut closure" occurs and macromolecules cannot be directly absorbed into the blood from the intestinal tract. Absorption of maternal colostral antibodies is important for the foal’s immune system function (passive transfer); however, harmful antibodies against the foal’s erythrocyte antigens also are absorbed. These harmful alloantibodies bind to offending antigen(s) on the foal’s RBCs, causing hemagglutination and extravascular or intravascular hemolysis. The higher the mare’s antibody titer to the offending RBC antigen at parturition, the higher the risk will be for development of NI. Resulting signs of NI may be subclinical or clinical.

Figure 1. Illustration representing the attachment of alloantibody to incompatible antigens on the surface of the foal's erythrocytes.

Pathogenesis of Equine Neonatal Isoerythrolysis:

Figure 2A, 2B: Flow charts depicting the development of neonatal isoerythrolysis in foals. A- initial sensitization, B- subsequent breedings.

Major Erythrocyte Antigens Involved in Neonatal Isoerythrolysis

Five major erythrocyte antigens are involved in the development of NI in foals. These antigens include:

1. Qa antigen: This antigen and the Aa antigen (below) are responsible for 90% of all cases of NI in horses;⁷ however, the Qa antigen is extremely rare in Standardbreds (Table 3). Mares that do not possess the Qa and/or Aa antigens (~19% of Thoroughbreds and ~17% of Standardbreds) are at the greatest risk for development of NI.³

2. Aa antigen: This RBC antigen has been found in the sera of both Standardbreds and Thoroughbreds (Table 3). It is commonly involved with NI as is the Qa antigen (above).

3. Ca antigen: Approximately 20% of Standardbreds and 10% of Thoroughbreds produce antibodies to this RBC antigen. However, antibodies that are reactive with this blood group are found in other species, suggesting that Ca may be a common environmental antigen. It is also hypothesized that antibodies to this blood group are natural antibodies that may occur without exposure to a RBC containing this antigen. This blood antigen may play an important role in a type of antibody-mediated immunosuppression in horses that possess it. These antibodies appear to attack fetal RBCs that cross over to the mare before the mare is able to mount an immune response to other RBC antigens such as the Aa group.⁸

4. Qc (lysin) and Db (agglutin) antigens: One study has indicated that these antigens are involved in some cases of NI. Antibody titers to the Qc antigen were elevated at parturition, but decreased over the next 4 months.⁷

5. Donkey RBC antigen: In one experimental study, the risk of an incompatible mating between a horse and a donkey (or the chance of a mare becoming sensitized to this antigen) was 100%. This high rate of sensitization may be due to a naturally occurring antibody that horses possess to this factor, differences in placentation in mule pregnancies, or differences in the antigenicity of this factor. Because clinical NI in mule foals only occurs ~8-10% of the time, it is suggested that many mule foals may have subclinical NI because the concentration of colostral antibodies against the foal’s RBCs that is required to cause overt clinical signs may differ between horse and mule foals.⁶

Clinical Presentation of Neonatal Isoerythrolysis

Clinical signs of NI may be subclinical or clinical. Foals appear healthy at birth and the onset of clinical signs occurs from several hours to as late as seven days after ingestion of colostrum. Clinical signs may vary depending upon the antigen involved, the concentration of alloantibodies in the colostrum, and the timing of colostrum administration.⁵ The major clinical signs also depend upon the degree of hemolysis.

Foals with NI usually become progressively lethargic, weak, and depressed. Mucous membranes may become pale and later icteric (Fig. 3). The degree of icterus is dependent upon time and the amount of hemolysis that occurs (Fig. 4). In cases where severe anemia is present, there will be a marked hemoglobinemia and hemoglobinuria (Fig. 5). Because of the reduced oxygen carrying capacity of the anemic blood, breathing may become shallow, rapid, and labored. Tachycardia also may develop. Foals with severe hypoxia may convulse or become comatose and die. Foals that are severely affected may develop shock and die quickly (within 6-8 hours postpartum) before icterus can occur. Generally, death may occur if NI is not recognized and treated quickly.

Figure 3. Icterus of the sclera and mucous membranes in a foal may indicate hemolysis of several hours duration.

Figure 4. The yellow discoloration of plasma from a neonatal foal indicates icterus.	Figure 5. Red-brown discoloration of the urine (left) suggests hemoglobinuria, hematuria, or myoglobinuria; however, red discoloration of the plasma (right) indicates that hemoglobinemia is present and this probably produced the hemoglobinuria.

Diagnosis

The presence of anemia in a foal can be documented quickly, easily, and economically by performing a packed cell volume (PCV). Once the presence of anemia is verified, several causes of blood loss, including NI, should be considered in the differential diagnosis (Table 4).

To determine if the foal has nursed and absorbed colostral antibodies, a measurement of the foal’s IgG concentration should be performed. Various methods can be used to measure antibody concentration in serum, plasma, or whole blood, including latex agglutination tests, zinc sulfate turbidity, and enzyme immunoassay (CITE® or SNAP® tests). Foal IgG levels greater than 800 mg/dl are generally considered indicative of adequate passive transfer.¹⁰

Foals with NI, anemia will have a decreased packed cell volume (PCV, hematocrit) and RBC count. PCV values may be <20%. The hemoglobin concentration may be increased (with intravascular hemolysis) or decreased, depending upon the time course of disease and blood sample procurement. Hemoglobinemia or icterus may be observed in the plasma of the PCV specimen following centrifugation. Mule foals also may be thrombocytopenic. Routine biochemical abnormalities may include hyperbilirubinemia (mainly unconjugated bilirubin) and possible electrolyte disturbances (e.g, hyperkalemia) from hemolysis. Urinalysis may reveal hemoglobinuria.

Definitive diagnosis of NI requires the demonstration of immunoglobulin on the surface of the foal’s RBCs. The detection of maternal antibody can be confirmed via screening the mare’s serum, plasma, or colostrum for reactivity with the sire’s RBCs. If the sire’s RBCs are not available, a panel of RBC bearing different blood groups may be used instead. High titers of alloantibody from the dam will result in agglutination. Lower titers may require the addition of a source of complement (such as fresh normal rabbit serum) to induce hemolysis. These lytic tests are believed to be a somewhat more reliable indicator for the presence of alloantibody directed toward the foal’s RBC.^{1, 4, 5}

Treatment of NI

Prognosis of the disease is dependent upon the severity of the hemolysis, when the condition is diagnosed and when therapy is instituted. Once diagnosed as NI, it is important to immediately stop the further ingestion of colostrum by the foal. This will prevent the intake of more alloantibodies against the foal’s erythrocytes from the mare. Due to the anemic and consequent hypoxic state, the foals are usually exercise-intolerant, so stress should be minimized. Foals should be provided with warmth and appropriate antimicrobial therapy, as they may not have adequate levels of maternal antibody. Supplemental oxygen may be necessary in hypoxic foals.

Supportive care is needed until the foal recovers. Intravenous fluid therapy helps promote diuresis to reduce the potentially harmful levels of hemoglobin in the kidney. Acid-base disturbances should also be corrected. A blood count of less than 3 x 10⁶ RBCs/mL or PCV less than 10-15% warrants a transfusion to provide the foal with needed RBCs. However, it is important to ensure that the donated blood does not possess antibodies to the foal’s RBCs. Finding an appropriate donor may be a difficult task, however, as there is a high prevalence of Qa and Aa in the normal equine population. Because the dam’s RBCs do not possess these offending antigens on their surface, the mare may represent a convenient donor choice if the mare’s RBCs are washed to remove the plasma along with its alloantibodies to the foal’s RBCs. The same applies for mule foals; washed RBCs from the dam may be the most convenient choice for a transfusion. If the mare’s blood is unavailable, then a crossmatched donor’s blood that does not have an immune response with the antibodies present in the mother’s serum can be used.

Prevention of NI

The main methods used to prevent NI are as follows:

• Identify broodmares that are negative for the Qa and/or Aa erythrocyte antigens. This can be done as previously discussed via blood typing in a genetic marker report or by a simple cross match. These mares are at highest risk for developing alloantibodies to "offending" antigens on the sire's or foal's RBCs.²

• Identify sires that are positive for the Qa and Aa antigens, if they are to be bred to mares that are negative for Qa and/or Aa antigens. This will help prevent broodmares from becoming sensitized to these two main offending antigens.

• Determine the probability of NI in unintended or potentially incompatible matings. If an unintended or potentially incompatible mating results, the mare’s serum is collected two weeks prior to parturition and tested against known blood cell groups or against the sire’s red blood cells. The presence of hemolysis or agglutination suggests that NI will develop.^4,9

• Withhold the mare's colostrum from the foal until it is proven to be safe or gut closure has occurred and macromolecules cannot be absorbed. If NI is a potential problem that may develop from the ingestion of colostrum, then colostrum can be withheld from the foal until a crossmatch is performed between the mare’s serum and the offspring’s RBCs. If agglutination or hemolysis is present, then NI may occur and the mare's colostrum should be withheld from the foal. Passive transfer can still be accomplished by foster feeding the foal provided that the material (colostrum from another mare or plasma) is devoid of antibodies that could result in NI.⁹ The foal should be foster fed for 2-3 days until gut closure occurs.

• Perform a Jaundice Foal Agglutination (JFA) test. This is a field screen test to detect NI. The foal's RBCs are exposed to the mare's colostrum or serum. If the cells agglutinate, then NI may develop. Results of the JFA test have been shown to correlate well with the standard hemolytic assay. The JFA test also may be able to detect antibody that is not identified on the standard agglutination tests. Control tubes are used to ensure that the test has been performed correctly. Positive reactions at 1:16 or greater suggest incompatibility and the risk of NI.¹

  References

1. McClure JJ, Parish SM: Diseases caused by Allogenic Incompatibilities. In: Smith BP, Parish, SM, Hines, MT (Eds.): Large Animal Internal Medicine, 3^rd Edition. St. Louis, Mosby, Inc., 2002, pp. 1604-1613.

2. Bowling AT: Horse Genetics. Cambridge, UK, Cab International, University Press, 1996, pp. 95-96, 112-114, 176.

3. Bailey E: Prevalence of ant-red blood cell antibodies in the serum and colostrum of mares and its relationship to neonatal isoerythrolysis. Am J Vet Res 43:1917-1921, 1982.

4. Barker RN: Neonatal Isoerythrolysis. In: Feldmen BF, Zinkl JG, Jain NC, (Eds.): Schalm’s Veterinary Hematology, 5^th ed. Lippincott Williams & Wilkins, Baltimore, 2000, pp. 175-176.

5. Tizard IR: Veterinary Immunology: An introduction, 6^th ed. Philadelphia, W. B. Saunders Co., 2000, pp. 324-331.

6. Traub-Dargatz JL, McClure JJ, Koch C, Schlipf JW: Neonatal isoerythrolysis in mule foals. J Am Vet Med Assoc 206:67-70, 1995.

7. MacLeay JM: Neonatal Isoerythrolysis involving the Qc and Db antigens in a foal. J Am Vet Med Assoc 219:79-81, 2001.

8. Bailey E, Albright DG, Henney PJ: Equine neonatal isoerythrolysis: Evidence for prevention by maternal antibodies to the Ca blood group antigen. Am J Vet Res 49:1218-1222, 1988.

9. Meyer DJ, Harvey JW: Veterinary Laboratory Medicine: Interpretation and Diagnosis, 2^nd ed. Philadelphia, W. B. Saunders Co., 1998, p. 146.

10. Duncan JR, Prasse KW, Mahaffey EA (Eds.): Veterinary Laboratory Medicine: Clinical Pathology, 3^rd ed. Ames, Iowa State University Press, 1994, p. 114.

Acknowledgement

"Medicine Helpers" by Susan Laubmeier is from Hawkwing Art Studio; it is copyrighted and used with permission of the artist.

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