Non-specific haemolytic alloantibody causing equine neonatal isoerythrolysis

NEONATAL isoerythrolysis (NI) is an uncommon disease in foals, with a prevalence of only 1 per cent being reported in thoroughbred horses and 2 per cent in standardbred horses (McClure 1997). The condition could be life-threatening in severe cases and may require intensive management (Snook 2001). With conventional NI, the best way to prevent disease is by identifying mares that are at risk of producing antibodies that cause NI (Snook 2001). Predicting and/or diagnosing NI requires laboratory demonstration of maternal alloantibodies binding to the foal’s red blood cells (RBCs) using either lytic or agglutination tests. Because the alloantibodies responsible for NI act more strongly as haemolysins than as agglutinins, tests that identify haemolysis are reportedly more accurate than those that demonstrate agglutination (Bailey 1982, Becht and others 1983, Whiting and David 2000). Blood group factors Aa and Qa account for the majority of NI cases reported; however, factors Ab, Ac, Db, Pa, Pb, Qc, and Ua have also been associated with cases of NI (Zaruby and others 1992, Whiting and David 2000, MacLeay 2001). Factors Aa and Qa are reported to be the most strongly antigenic, which may account for their higher incidence in NI (Whiting and David 2000). The prevalence of NI varies among breeds, and depends on the probability of the mare producing alloantibodies, the likelihood that the foal inherited the antigen, the amount of alloantibodies contained within the colostrum, the strength of antigenicity of the offending antigen, and the amount of immunoglobulin absorbed by the foal (Whiting and David 2000). In the Department of Equine Science, Utrecht University, a Friesian filly developed an unusually severe case of NI (de Graaf-Roelfsema and others 2002). The dam of the foal was blood typed and a serological test was performed to determine the presence of agglutinating or haemolytic antibodies and the responsible antigenic factor. The test revealed a haemolytic alloantibody that caused haemolysis of RBCs of all the horses in a test panel, except those from the mare itself, which could not be attributed to a specific antigenic group. This is in contrast to conventional NI in which an alloantibody is reacting with a specific antigenic factor. This short communication describes this type of NI, which was due to a non-specific haemolytic alloantibody in Friesian foals. The prevalence of the non-specific antibody was investigated by testing dams of healthy Friesian foals and dams of Friesian foals suspected of having NI for the presence of specific and non-specific haemolytic alloantibodies during the summers of 1999 to 2003. The following criteria were used to determine a combination ‘suspected of NI’: a foal with clinical signs of NI (anaemia, icterus and haemolysis), or a mare that had delivered a foal with NI in previous years and which was mated again to a stallion with an incompatible blood group factor. Combinations of Friesian dams with healthy foals (defined by the absence of the ‘suspected NI signs’) younger than one week were selected for the control group. Eleven combinations were suspected of having NI (Table 1), and five combinations were used as a control group. The two mares with foals suspected of having non-specific NI were tested repeatedly in following years. The average age of the mares was 10·5 (3·5) years, and they were all multiparous, except for one control mare. A serum sample and a heparinised blood sample were collected by venepuncture from the mares with foals suspected of having NI. The heparinised sample was used for blood typing and the serum sample was used for the detection of haemolytic or agglutinating antibodies. Only a serum sample was collected from the dams in the control group. When haemolytic alloantibodies were found in the control group, blood typing was also performed. The blood samples were taken from one month before parturition to 14 days postpartum, except for mare A; its blood was taken 28 days postpartum and one blood sample was taken 11 months later while it was not breeding. Samples were taken from control group within seven days postpartum. A colostrum sample from one mare (mare B) with a foal with non-specific NI was also tested for the presence of haemolytic alloantibodies. The blood types of the Friesian stallions were already known, due to the official registration of ‘awarded’ (selected for mating) stallions in the Netherlands. The mares’ sera were tested for the presence of haemolytic as well as agglutinating antibodies against a panel of RBCs, from horses that represented all the known blood groups for the Friesian horse. Each mare’s own RBCs were used as an additional (negative) control. All the potential lytic factors were represented in the panel, except Ka, because Ka has not been reported in the Friesian horse (van Haeringen and van Haeringen 1992). The tests were performed as described by van Haeringen (1989), based on the methods of Stormont (1975) and Sandberg (1979). RBCs were washed two or three times with 0·9 per cent sodium chloride, and a 2 per cent suspension was prepared. Microtitre plates were used to make a serial dilution of the mares’ serum and 0·9 per cent sodium chloride (1:2 to 1:512). Each well received 25 μl RBCs, 25 μl serum and 25 μl rabbit serum, which was used as a complement source. The test plates were incubated at 27°C for 45 minutes. After mixing again, the test plates were evaluated for haemolysis. Lysis at a dilution of 1:2 was considered to be negative, lysis at a dilution of 1:4 (after parturition) was considered negligible, whereas lysis at dilutions of 1:8 and above were considered to be positive. The direct agglutination test was performed in exactly the same way, except the complement source was not added and the plates were incubated at 37°C for two hours. The same procedures were also used for the colostrum sample. The blood was typed by testing RBCs from the mares with the internationally recognised specific antibodies to the Haemolytic Incompatible blood Times of Horse Year alloantibody group factor of Titre sampling the dame or sire