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Alloantibodies against A and B blood types in cats
Veterinary Immunology and lmmunopathology, 38 ( 1993 ) 283-295 0165-2427/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved
283
Alloantibodies against A and B blood types in cats 1 JSrg Bficheler 2, Urs Giger* Section of Medical Genetics, Department of Clinical Studies, School of VeterinaryMedicine, University of Pennsylvania, 3850 Spruce Street, Philadelphia, PA 19104-6010, USA (Accepted 11 December 1992)
Abstract
This study characterizes the naturally occurring feline alloantibodies against A and B blood type. All examined type-A and type-B cats had naturally occurring antibodies against erythrocytes of the opposite blood type. In order to determine the class ofimmunoglobulins,sera from cats were analyzed using incubation with 2-mercaptoethanol (2-ME), immunoprecipitation, and gel filtration. Type-A cats had weak agglutinins of the IgM class and weak hemolysins which consisted of approximately equal parts of IgG and IgM class. Type-B cats had strong hemagglutinins and hemolysins mostly of the IgM class. Colostral antibodies were detectable in newborns as early as 4 h after birth and their own alloantibody production started at 6-8 weeks of age. The presence of naturally occurring alloantibodies, in particular the anti-A alloantibodies, renders cats susceptible to clinical incompatibility reactions. Abbreviations
NI, neonatal isoerythrolysis; PBS, phosphate buffered saline; 2-ME, 2-mercaptoethanol.
Introduction Thus far, only one feline blood group system has been described, which contains three blood types, type-A, type-B, and the very rare type-AB (Holmes, 1953; Auer and Bell, 1981 ). The frequency of blood types in domestic shorthair and longhair cats varies geographically. Even larger variations in blood type frequencies have been observed among purebred cats with type-B frequencies ranging from 0-59% (Giger et al., 1989, 1991 a, b). Type-A and typeB blood types are inherited as a simple autosomal mendellian trait with A *Corresponding author. ~Supported in part by grants from the National Institutes of Health (HL02355) and Robert H. Winn Foundation. 2present address: Department of Medicine, School of Veterinary Medicine, Tufts University, 200 Westboro Road, North Grafton, MA 01536, USA.
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being completely dominant over B (Giger et al., 1991a). The extremely rare type-AB cats could be explained by the presence of a third allele (Griot-Wenk and Giger, 1991 ). In contrast to many other animal species, cats appear to have naturally occurring alloantibodies against the other blood type (Ingebringsten, 1912, Eyquem et al., 1962, Auer and Bell, 1981; Giger et al., 1989, 1991 a, b; Wilkerson et al., 1991a). 'Naturally occurring' refers to the fact that these alloantibodies develop without prior sensitization by transfusion or pregnancy. In Australia, 96% of examined type-B plasma samples had strong agglutinins and hemolysins, with titers of > 1:64 for the agglutinin and up to 1:512 for the hemolysin (Auer and Bell, 1981 ). However, only 35% of the examined type-A plasma samples had agglutinating and hemolytic activity. Severe transfusion reactions in type-B cats receiving type-A blood (Auer et al., 1982; Giger and Akol, 1990; Giger and Biicheler, 1991; Wilkerson et al., 1991 a) and neonatal isoerythrolysis (NI) caused by colostral anti-A antibodies of type-B cats transferred to type-A newborns (Cain and Suzuki, 1985; Hubler et al., 1987; Gandolfi, 1988; Jonsson et al., 1990; Giger, 1991 ) have been described. The present study was undertaken to further characterize the agglutinins and hemolysins in type-A and type-B cats. Materials and methods
Plasma was separated from EDTA-anticoagulated feline blood samples that were submitted for blood typing (Giger et al., 1991a, b ). Additional serum for antibody characterization was obtained from 30 type-A and 30 type-B cats in the Philadelphia area for further studies. Specimens were stored in plastic tubes at - 7 0 ° C before analysis. Phosphate buffered saline pH 7.4 (PBS) containing 150 mmol 1-1 sodium chloride and 10 mmol 1-' sodium phosphate was used for all red blood cell suspensions and antibody dilutions. Rabbit complement, Drabkin's solution, 2-mercaptoethanol, ammonium sulfate, and iodoacetate were purchased from Sigma Chemicals (St. Louis, MO). Coombs reagent, goat anti-cat IgG (7-chain specific) and goat anti-cat IgM (/t-chain specific) antibodies were obtained from Kirkegaard and Perry Lab. (Gaithersburg, MD). Sephadex G 200 was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden).
Tests for agglutinins and hemolysins Plasma samples from various domestic shorthair and purebred cats were used for an agglutination screening assay as previously described (Giger et al., 1989). Briefly, 60 #l of undiluted sample plasma and 30 pl of a PBS-washed 3-5% type-A or type-B red blood cell suspension were mixed and centrifuged for 15 s at 1000 g (3500 rpm). Agglutination was graded from 0 to 4+, with 0 being no agglutination and 4 + being a single pellet-like agglutinate. Agglu-
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tination and hemolysis assays were performed as previously described (Auer and Bell, 1981; Giger et al., 1989 ). Serial two-fold dilutions of 60/tl of sample and 30/tl of a washed 3-5% type-A or type-B erythrocyte suspension were mixed and centrifuged for 15 s at 1000 g. The agglutination titer was recorded as the highest dilution which still produced a 1 + macroscopic agglutination. Some samples (n = 30) with negative macroscopic agglutination were examined for microscopic agglutination at 10 X magnification or used in a direct antiglobulin test (Coombs test ) as previously described (Coombs et al., 1945 ). Two-fold serial dilutions of heat inactivated serum (30 min at 56 °C) and 30 ~1 red blood cells combined with 30 #1 of undiluted rabbit complement, which previously had been adsorbed with an equal volume of feline erythrocytes at 20 °C for 5 min to remove naturally occurring heteroagglutinins (Giger et al., 1989), were used in the hemolysis assay. The mixtures were incubated in a water bath at 37°C for 30 min, mixed and further incubated at 20°C for 2 h. The tubes were centrifuged for 20 s at 1000 g and 50/zl of the supernatant was transferred into a tube containing 100 #1 of Drabkin's solution. After 15 min incubation, the light absorption was determined spectrophotometrically at 580 n m and compared with a completely hemolyzed control sample prepared by repeated freezing of the erythrocytes. The hemolysis titer was expressed as the highest dilution in which hemolysis reached the 10% level of the control. Some agglutination and hemolysis assays were performed at different temperatures (4, 20, 37°C).
Fractionation of immunoglobulins Immunoglobulins were separated by gel filtration according to standard methods (James and Stanworth, 1964). Briefly, the serum globulin fractions from two type-A and two type-B cats were separately prepared by precipitation of 10 ml of serum with equal amounts of 5.4 M a m m o n i u m sulfate at 4°C and centrifugation for 10 min at 1000 g. The precipitates were resuspended in 10 ml of PBS and applied to a 150 cm × 5 cm column of Sephadex G 200 in PBS. The flow rate was 35 ml h-1. Five ml samples were collected and the optical density at 280 n m was determined spectrophotometrically. The central fraction of the first protein elution containing the bulk of macroglobulins and the subsequent elution containing most of the IgG were harvested and concentrated by vacuum dialysis. These fractions were utilized for agglutination, hemolysis and immunoprecipitation assays, as previously described.
2-Mercaptoethanol treatment Destruction of the IgM-disulfide bonds with 2-ME was performed as previously described (Adler and Frank, 1965) to remove IgM-activity from
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serum. Serum and 2-ME (0.1-0.4 M) in equal parts and serum and PBS (control) were incubated at 37°C for 2 h. Subsequently, the mixture was dialyzed against PBS with the addition of 0.02 M iodoacetate for 3 days at 4 °C with a four-fold dialysate change every day. 2-ME treated serum was then examined for antibody activity using agglutination and hemolysis tests as described above.
Immunoprecipitation Specific removal of immunoglobulins by immunoprecipitation was performed according to standard techniques (Kabat and Maier, 1961 ). Briefly, 20/tl of serum and 40/tl of anti-IgM or anti-IgG were mixed in a glass tube and incubated at 37°C for 30 min. After centrifugation (14 000Xg for 10 min) the supernatant, separated from the precipitate pellet, was examined for antibody activity using agglutination and hemolysis tests as described above.
Alloantibody titer in newborn kittens Sequential plasma samples were obtained from type-B kittens born to typeA and -B queens prior to, and after colostrum uptake, at 30 min and 4 h post partum. Additional plasma samples were collected every 3-5 days until kittens were 15 weeks of age. The samples were analyzed for agglutinating and hemolytic activities as previously described. Results
A total of 2180 undiluted plasma samples from the blood typing service were screened for agglutinating alloantibodies. Thirty-eight (2%) of the 1933 examined plasma samples from type-A cats agglutinated type-B erythrocytes strongly (3 + to 4 + ), 36% of the plasma samples led to a weak agglutination Table 1 Agglutination of feline erythrocytes with undiluted plasma Cat blood type
A B AB
Allo-antibodies
anti-B 1 anti-A 2 anti-A or-B 3
Number of plasma samples
1933 239 8
tTested against type-B erythrocytes. 2Tested against type-A erythrocytes. STested against type-A and type-B erythrocytes.
Agglutination None
1-2+
3-4+
1199 0 8
696 0 0
38 239 0
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( 1 + to 2 + ) oftype-B red blood cells and 62% did not show macroscopically detectable agglutination (Table 1 ). However, when the plasma samples from 30 type-A cats with negative macroscopic agglutination were further examined, type-B erythrocyte microagglutinates were found. These incubated cells were also positive for the presence of IgG and IgM in a Coombs test. All plasma samples of the 239 tested type-B cats strongly ( 4 + ) agglutinated type-A erythrocytes. The plasma of the eight tested type-AB cats did not agglutinate or hemolyze type-A or type-B red cells. The agglutinin and hemolysin titer were determined in serum from 30 typeA and 30 type-B cats (Table 2). Serum of type-A cats had a weak anti-B agglutinin titer ( l - 16 ) and a slightly stronger hemolysin titer (4-32). Type-B cats had a strong agglutinin titer (64-512 ) as well as a strong hemolysin titer Table 2 Agglutinating (in °C) Number
and hemolyzing
of cats
antibody
Antibody 1
titer in type-A and type-B cats and their thermal
dependancy
titer
2
4
8
16
32
64
128
256
512
1024
Type-A cats (n = 30) Agglutinin
10
7
8
4
1
-
-
Hemolysin
-
-
5
8
10
7
-
4°
-
-
2
8
1
20 °
-
4
7
1
-
37 °
-
5
8
-
-
4°
8
2
-
20 °
-
1
6
3
-
37 °
-
-
-
2
4
Agglutinin
Hemolysin
( n = 10)
(n = I0) 4
Type-B cats (n = 30) Agglutinin
4
12
9
5
-
Hemolysin
2
9
11
6
2
Agglutinin 4°
( n = 10) _
_
-
-
2
6
2
20 °
_
_
-
2
4
4
-
37 °
_
_
-
3
4
3
-
4 o
_
_
-
4
4
2
-
20 °
_
_
-
-
3
4
3
37 °
_
_
-
1
4
5
Hemolysin
(n = 10)
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( 6 4 - 1 0 2 4 ) . S e r u m f r o m t y p e - A a n d t y p e - B cats h a d a slightly stronger agglut i n a t i n g a c t i v i t y at 4 ° C c o m p a r e d with 20 ° C a n d 37 ° C. T h e o p t i m a l r e a c t i o n t e m p e r a t u r e for h e m o l y s i s was 3 7 ° C ( T a b l e 2). S e r u m o f t h r e e t y p e - A a n d f o u r type-B cats t r e a t e d with 2 - M E ( T a b l e 3 ) s h o w e d r e d u c e d agglutinating a n d h e m o l y z i n g activities. T h e agglutinating activity was d e c r e a s e d m o r e severely t h a n the h e m o l y t i c activity. T h u s , 2 - M E did not abolish all agglutinating a n d h e m o l y z i n g alloantibodies. I m m u n o p r e c i p i t a t i o n o f s e r u m o f t y p e - A a n d t y p e - B cats with anti-feline IgM led to a m a r k e d r e d u c t i o n o f the agglutinating activity ( T a b l e 4 ), while i m m u n o p r e c i p i t a t i o n with anti-feline IgG o n l y slightly d i m i n i s h e d the seru m ' s agglutination. I m m u n o p r e c i p i t a t i o n with anti-IgM a n d anti-IgG, respectively, resulted in an equally strong i n h i b i t i o n o f hemolysis in type-A cats. In contrast, i m m u n o p r e c i p i t a t i o n o f t y p e - B s e r u m with anti-IgM led to a strong i n h i b i t i o n a n d i m m u n o p r e c i p i t a t i o n with a n t i - I g G to a w e a k i n h i b i t i o n o f hemolysis. Table 3 Alloantibody titer after 2-Mercaptoethanol treatment (geometric mean + SD) 2-Mercaptoethanol (Molarity) 0
0.1
0.2
0.4
Type-A cats (n = 3)
Anti-B Agglutinin Anti-B Hemolysin
6 +_2.8 13 _+11.5
0 5 _+1.9
0 2 _+0.5
0 2 _+0.5
22 +_10.4 30_+21.4
10 +_5.9 11 _+5.2
5 _+1.7 7 _+1.7
Type-B cats (n = 4)
Anti-A Agglutinin Anti-A Hemolysin
240 _+171 320 +_192
Table 4 Immunoprecipitation of the blood group antibodies (titer, geometric mean _+SD ) Immunoprecipitated with PBS (control)
Anti-IgG
Anti-IgM
Anti-lgG + anti-lgM
Type-A cats (n = 4)
Anti-B Agglutinin Anti-B Hemolysin
6 _+2.6 15 _+10.7
5 +_2.6 4 _+2.4
256 _+140 359 _+125
230 _+140
0 4 + 2.4
0 1 +_0.5
8 _+0.8
2 +_0.8
14 + 10.0
1 + 0.8
Type-B cats (n = 5)
Anti-A Agglutinin Anti-A Hemolysin
256
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The first fraction after gel filtration of serum from type-A cats (IgM-fraction) had weak agglutinin and hemolysin activities (anti-B), and the IgMfraction of anti-A serum from type-B cats strongly agglutinated and hemolyzed type-A cells (Table 5). Both activities of this first fraction could be Table 5 Immunoprecipitation of serum and its antibody fractions after gel filtration Cat No.
Immunoprecipitation with PBS (control) aggl. 1
Anti-IgG hem.
2
1
Anti-IgM
aggl. 2
1
hem. 2
1
aggl. 2
hem.
1
2
1
2
Type-A (n = 2)
Serum IgG-fraction IgM-fraction
4 0 2
8 1 8
8 4 4
16 8 8
4 0 2
8 0 8
4 0 4
8 I 8
0 0 0
2 1 0
4 4 0
8 8 1
128 2 64
256 2 128
128 8 64
512 16 256
128 0 64
128 0 128
64 0 64
128 2 256
8 2 0
4 2 0
16 4 4
16 16 16
Type-B (n = 2)
Serum IgG-fraction IgM-fraction
aggl., agglutinin-titer; hem, hemolysin-titer. 600
400
!
500 "
.o ~
300
= .¢
200 /
,
loo
ii
b
0
23
46
69
92
115
Days after Birth Fig. 1. D e v e l o p m e n t o f blood type alloantibodies in n e w b o r n cats. ( • ) type-B kittens after ingestion of type-B queen's colostrum (n = 14). ( • ) type-B kittens that were not allowed to nurse (n = 4 ). ( ,, ) type-A kittens that were not allowed to nurse, or h a d signs o f N I (n = 12 ).
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specifically inhibited by immunoprecipitation with anti-IgM, but not with anti-IgG. Only weak agglutinating activity was found in the second fraction (IgG-fraction). The second fraction Of type-A cat serum showed weak hemolysis of type-B red cells. The IgG-fraction of serum from type-B cats led to weak agglutination and moderate hemolysis of type-A cells. These reactions were weaker than with the IgM-fraction of anti-A serum, but still stronger than the IgG-fraction of anti-B serum. Both agglutinating and hemolytic activities of the IgG fractions could be inhibited by immunoprecipitation with anti-IgG, but not with anti-IgM. Anti-A alloantibodies were assessed in serum from newborn to 15-weekold kittens (Fig. 1 ). No alloantibodies were detected at birth in any plasma from kittens prior to colostrum intake. However, at the first time point after colostrum ingestion (4 h after birth) anti-A alloantibodies were present in type-B kittens born to type-B queens and highest serum antibody titers were reached after 24 h. These antibodies were agglutinins and hemolysins. The kittens' antibody titers which were as high as their type-B mother's serum titers, slowly declined over the next few weeks with an apparent half life of 8 days. No anti-A alloantibodies were detected in the plasma of type-A kittens born to type-B queens, although these antibodies are immediately bound to type-A erythrocytes and may cause mild to severe signs of NI as well as a positive Coombs test in type-A kittens (Giger, 1991 ). Furthermore, no antiA alloantibodies were found in type-B kittens that were not allowed to nurse from their type-B queen, but were foster nursed, and in type-B kittens born to type-A queens. However, at 6-8 weeks of age all type-B kittens developed their own anti-A alloantibodies without exposure to type-A erythrocytes. At 12 weeks of age their plasma anti-A titer reached levels comparable with that found in adult type-B cats. In type-A kittens the anti-B antibody titers detected were only 1:2 at 12 weeks of age. Discussion Feline iso- or alloantibodies were first detected at the beginning of this century, and recently, their clinical importance has been exemplified by reports of feline neonatal isoerythrolysis (Cain and Suzuki, 1985; Hubler et al., 1987; Gandolfi, 1988; Jonsson et al., 1990; Giger, 1991 ) and hemolytic transfusion reactions (Auer et al., 1982; Giger and Akol, 1990; Giger and Biicheler, 1991; Wilkerson et al., 1991 a). The studies described here further characterize these alloantibodies and their development. Based on this large survey of 2180 cats, all type-A and type-B cats older than 2 months of age have alloantibodies. In particular, all type-B cats have high plasma titers of anti-A agglutinins and hemolysins, with titers all exceeding 1:64. No adult type-B cat without anti-A antibodies was encountered. These data support previous results from Australia (Auer and Bell, 1981 )
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and Japan (Ejima et al., 1986), although these investigators reported on a few type-B cats lacking alloantibodies. As these type-B cats were not further characterized, these animals may have been young kittens, either born to typeA queens or born to type-B queens, but not allowed to nurse. Furthermore, all type-A cats had anti-B agglutinins, although microscopic examination was necessary for the detection of the typically weak agglutinin reactions. The antibody involvement in the weak agglutinin reactions was confirmed by a positive direct antiglobulin test. Only one third of all examined type-A cats had macroscopic hemagglutinins and hemolysins with very few of them reaching agglutinin and hemolysin titers of 1:16 and 1:32, respectively. Similar observations were made by Auer and Bell in Australia ( 1981 ). As expected, plasma from tested type-AB cats did not contain any alloantibodies. Based upon the early development and universal occurrence of alloantibodies without prior exposure to blood group antigens either by transfusion or pregnancy, these anti-A and anti-B alloantibodies are considered 'naturally occurring' (Springer et al., 19.62; Tizard, 1982; H6gmann, 1988). At birth, type-B kittens have no detectable alloantibodies in their plasma, but acquire high anti-A alloantibody titers during the first 2 days, through passive transfer via colostrum from type-B queens. These titers slowly decrease during the first few weeks of life with a half life similar to other maternal antibodies. Type-B kittens born to both type-A or -B queens will develop anti-A alloantibodies between 6-8 weeks of age. The reason for the occurrence of these alloantibodies is not known, but may be induced by exposure to similar or identical antigenic determinants that commonly are present in nature, such as food or bacterial antigens (Springer et al., 1962; Tizard, 1982; H6gmann, 1988). As the feline AB blood group antigens have been determined to be carbohydrate containing determinants (Andrews et al., 1992 ), their antibody induction may be similar to the situation with human blood groups like the ABO-system. Since most of the agglutinating activity in feline plasma could be removed after exposure to 2-ME and immunoprecipitation with anti-IgM, the agglutinin in type-A and type-B cats is predominantly of IgM-type. Hemolytic activity was found in both IgM and IgG fractions after gel filtration. Hemolysis could still be observed after 2-ME treatment and immunoprecipitation. Therefore, the hemolysins consist of both IgM and IgG, although the hemolytic activity in type-B cats appears to be mostly owing to IgM. Our data confirms and extends results from two previous reports that studied the hemagglutinins in type-B cats. Ikemoto et al. ( 1981 ) detected agglutination in the IgM-fraction of gel-filtered type-B sera, but no agglutinins were found in the IgG-fraction. Wilkerson et al. ( 1991 b) confirmed the predominant role of IgM for agglutination, but additionally found some weak agglu-
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tinin activity in the IgG-fraction after gel filtration, as observed in this study. This IgG-fraction may be important in neonatal isoerythrolysis. Based on the universal presence of naturally occurring feline alloantibodies, cats are at risk for a transfusion reaction-even when reeeivingtheflrst mismatched blood. Owing to the strong anti-A antibodies, severe transfusion reactions have been reported only in type~B cats recei~ingtype,A blood (Auer et al., 1982; Giger and Akol, 1990; Wilkerson et al., 199 l a). In mismatched transfusions, all type-B cats destroy transfused type-A erythrocytes via complement-mediated intravascular hemolysis within minutes to a few hours (Giger and Biicheler, 1991 ). Since all type-B cats have high IgM-alloantibody titer, transfused incompatible type-A erythrocytes get heavily coated with IgM which will activate the lytic C5-9 complex resulting in intravascular hemolysis (Schreiber and Frank, 1972 ). In contrast, type-A cats receiving type-B blood ......... S igiiS. .l.. t.a.u.~ t u ~~----~ .typ~,-L~ . . . . n ~__~. did not snow any m aj o~- Clllllk~l,l vu x ) , ~ v.~.,.~. a. . . W g l ' e steadily cleared from circulation within 5 days, thus rendering the transfusion inefficacious. Coating oftype-B erythrocytes with anti-B alloantibodies of the IgG and IgM class mostly results in macrophage-mediated destruction of transfused cells. The hemolyzing IgM-alloantibody titer in type-A cats is apparently too low to cause severe intravascular hemolysis. Neonatal isoerythrolysis has been described in blood type-A kittens born to blood type-B queens (Cain and Suzuki, 1985; Hubler et al., 1987; Gandolfi, 1988; Jonsson et al., 1990; Giger, 1991 ). Kittens will acquire maternal alloantibodies of the IgG- and to a lesser extend of the IgM-class via colostrum during the first 2 days of life (U. Giger and J. Biicheler, unpublished results, 1991 ). Cotostrat anti-A atloantibodies have been demonstrated in the type-B kitten's plasma as early as 4 h after birth. Iftype-A or type,AB kittens are born to a type-B queen, colostral antibodies will bind to and lyse erythrocytes in the newborn. The hemolysis may occur intravascularly as well as extravascularly and result in anemia, hemoglobinuria, jaundice and death (Giger, 1991 ). Since all adult type-B cats have high alloantibody titers, type-A kittens from primiparous type-B queens are at risk for NI. Clinical signs of NI may be peracute, acute or subclinical. The authors identified several type-A kittens that had laboratory abnormalities such as anemia and positive Coombs tests, but no clinical evidence of NI (Giger, 1991 ). The factors that determine the severity of hemolysis have not been identified. Furthermore, erythrocytes from kittens with subclinical NI were Coombs-positive for IgM as well as IgG (U. Giger and J. B~icheler, unpublished results, 1991 ). In preliminary studies very high IgG titers were found in colostrum from type-B cats, whereas the colostral IgM concentrations were low. Interestingly, the authors observed that surviving kittens may rarely develop tail-tip necrosis between 1 and 3 weeks post partum, which was also observed by Gandolfi (1988) and Jonsson (1990). Histologic examination showed thromboembolism and ischemic necrosis. The present study shows, that the in vitro agglutinating activity of feline IgM al'
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loantibodies is inversely correlated to temperature. Erythrocyte-bound IgM in these kittens may have caused a cold agglutinin reaction with microagglutination and vascular stasis in areas of lower body temperature which may result in tail tip necrosis. Important variables for the development of clinical signs of NI might be the amount and time of colostrum uptake, IgG-titer or subclasses, intestinal permeability for alloantibodies and the activity of the macrophage system in the neonate. In humans two different groups of antisera composition can be distinguished. Blood group antigens such as the Rhesus antigens have a protein nature and are usually recognized by IgG antibodies, which first have to be stimulated by immunogenic contact to erythrocytes of the other blood type, e.g. after a mismatched transfusion or exposure to incompatible fetal blood during pregnancy. In contrast, carbohydrate epitopes bound to membrane proteins or lipids on erythrocytes, i.e. antigens of the ABO-system, are recognized by naturally occurring antibodies (H~gmann, 1988; Anderson and Kelten, 1989 ). Molecular analysis of the feline blood group antigens (Andrews et al., 1991; U. Giger and J. BiJcheler, unpublished results, 1991 ) indicates, that the antigenic determinants of the feline AB-blood group system contain carbohydrate moieties similar to the human ABO-blood group system. The composition of the naturally occurring antibodies in type-A and type-B cats resembles that in humans with antigens of the ABO-system. Type-A cats have a low alloantibody titer with a greater proportion of IgG, as has been found in people with blood type-O. Type-B cats resemble people with blood type-A or -B having high alloantibody titers of the IgM class (Garratty, 1989 ). TypeAB cats, as well as humans with blood type-AB, are probably immunotolerant against either antigen and cannot develop alloantibodies against the A- or Bantigen. The antibody characterization in combination with the observed patterns of hemolysis in transfusion experiments suggest that cats and humans have similar mechanisms for immune mediated destruction of incompatible red cells suggesting that the cat may serve as an animal model for human transfusion reactions and hemolysis of the newborn.
References Adler, D. and Frank, L., 1965. Dissociation of macroglobutins with mercaptoethanot. J. Immun., 95: 39-42. Anderson, D.R. and Kelten, J.G., 1989. Mechanisms ofintravascular and extravaskular red cell destruction. In: F.T. Nance (Editor), Immune Destruction Of Red Blood Cells. Am. Assoc. Blood Banks, Arlington, VA, pp. 1-52. Andrews, GIAI, Chavey, PIS., Smith, J.E. and Rich, C., 1992. N-Glycolylneuraminic Acid and N-Acetylneuraminic Acid define feline blood group A and B antigens. Blood, 79: 2484-2491. Auer, L. and Bell, K., 1981. The AB blood group system in cats. Anim. Blood Groups Biochem. Genet., 12: 287-297.
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U, G~geWVeterrnary Immunology
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Auer, L. and Bell, K., 1983. Transfusion reactions in cats due to AB blood group incompatibility. Res. Vet. Sci., 35: 145-152. Auer, L., Bell, K. and Coates, S., 1982. Transfusion reactions in the cat. J. Am. Vet. Med. Assoc., 180: 129-130. Cain, G.R. and Suzuki, Y., 1985. Presumptive neonatal isoerythrolysis in cats. J. Am. Vet. Med. Assoc., 187: 46-48. Coombs, R.R.A., Mourant, A.E. and Race, R.R., 1945. A new test for the detection of weak and incomplete Rh-agglutinins. Brit. J. Exper. Path., 26: 255-261. Ejima, H., Kurokawa, K. and Ikemoto, S., 1986. Feline red blood cell groups detected by naturally occurring isoantibody. Jpn. J. Vet. Sci., 48: 97 l-976. Eyquem, A., Podliachovk, L. and Milot, P., 1962. Blood groups in chimpanzees, horses, sheep, pigs and other mammals. Ann. NY Acad. Sci., 97: 320-328. Gandolfi, R.C., 1988. Feline neonatal isoerythrolysis: a case report. Calif. Vet., 3: 9-l 1. Garratty, G., 1989. Factors influencing the pathogenicity of red cell auto- and alloantibodies. In: F.T. Nance (Editor), Immune Destruction Of Red Blood Cells. Am. Assoc. Blood Banks, Arlington, VA, pp. 109- 170. Giger, U., 199 1. Feline neonatal isoerythrolysis: A major cause of the fading kitten syndrome. Proc. Am. Coll. Vet. Intern. Med., 9: 347-350. Giger, U. and Akol, K.G., 1990. Acute hemolytic transfusion reaction in an Abyssinian cat with type-B blood. J. Vet. Int. Med., 4: 3 15-3 16. Giger, U. and Biicheler, J., 199 1. Transfusion of type-A and type-B blood to cats. J. Am. Vet. Med. Assoc., 198: 41 l-418. Giger, U., Kilrain, C.G., Filippich, L.J. and Bell, K., 1989, Frequencies of feline blood groups in the UnitedStates. J. Am. Vet. Vet. Assoc., 195: 1230-1232. Giger, U., Biicheler, J. and Patterson, D.F., 199 1a. Frequency and inheritance of A and B blood types in feline breeds of the United States. J. Heredity, 82: 15-20. Giger, U., Griot-Wenk, M., Biicheler, J., Smith, S., Diserens, D., Hale, A. and Patterson, D., 199 lb. Geographical variation of the feline blood type frequencies in the United States. Feline Practice, 19: 21-27. Griot-Wenk, M. and Giger, U., 1991. Cats with type AB blood in the United States. Proc. Am. Coll. Vet. Intern. Med., 9: 894. Hogmann, C.F., 1988. Immunologic transfusion reactions. Acta Anaest. Stand., 32, (suppl.) 89: 4-12. Holmes, R., 1953. The occurrence of blood groups in cats. J. Exper. Biol., 30: 350-357. Hubler, M., Kaelin, S., Hagen, A. and Ruesch, P., 1987. Feline neonatal isoerythrolysis in two litters. J. Small Anim. Praet., 28: 833-838. Ingebringsten, R., 1912. The influence of isoagglutinins on the final results of homoplastic transpIantation of arteries. J. Exp. Med., 16: 169-177. Ikemoto, S., Sakuria, Y. and Fukui, M., 198 i. individual difference within the cat blood group detected by isoagglutinin. Jpn. J. Vet. Sci., 43: 433-434. James, K. and Stanworth, D.R., 1964. Gel filtration of immunoglobulins. J. Chromatogr., 15: 325. Jonsson, N.N., Pullen, C. and Watson, A.D.J., 1990. Neunatal isoerythrolysis in Himalayan kittens. Austr. Vet. J., 67: 4 16-4 17. Kabat, E.A. and Maier, M.M., 1961. Immunoprecipitation. In: Experimental Immunochemis try, 2nd edn. Springfield, IL, pp. 22-25. Schreiber, A.D. and Frank, M.M., 1972. Role of antibody and complement in the immune clearance and destruction of erythrocytes. I. in vivo effects of IgG and IgM complementfixing sites. J. Clin. Invest., 5 1: 575-582. Springer, G-F., Williamson, P. and Readler, B.L., 1962. Blood group active gram-negative bacteria and higher plants. Ann. NY Acad. Sci., 97: 104-l 15.
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Tizard, R., 1982. An introduction to immunology, 2nd edn., Saunders, Philadelphia, PA, pp. 165-177. Wilkerson, M.J., Wardrop K.J., Meyers, K.M. and Giger, U., 1991a. Two cat colonies with A and B blood types and a clinical transfusion reaction. Feline Practice, 19 (2): 22-26. Wilkerson, M.J., Meyers, K.M. and Wardrop, K.J., 1991b. Anti-A isoagglutinins in two blood type B cats are IgG and IgM. Vet. Clin. Path., 20: 10-14.
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Alloantibodies against A and B blood types in cats 1 JSrg Bficheler 2, Urs Giger* Section of Medical Genetics, Department of Clinical Studies, School of VeterinaryMedicine, University of Pennsylvania, 3850 Spruce Street, Philadelphia, PA 19104-6010, USA (Accepted 11 December 1992)
Abstract
This study characterizes the naturally occurring feline alloantibodies against A and B blood type. All examined type-A and type-B cats had naturally occurring antibodies against erythrocytes of the opposite blood type. In order to determine the class ofimmunoglobulins,sera from cats were analyzed using incubation with 2-mercaptoethanol (2-ME), immunoprecipitation, and gel filtration. Type-A cats had weak agglutinins of the IgM class and weak hemolysins which consisted of approximately equal parts of IgG and IgM class. Type-B cats had strong hemagglutinins and hemolysins mostly of the IgM class. Colostral antibodies were detectable in newborns as early as 4 h after birth and their own alloantibody production started at 6-8 weeks of age. The presence of naturally occurring alloantibodies, in particular the anti-A alloantibodies, renders cats susceptible to clinical incompatibility reactions. Abbreviations
NI, neonatal isoerythrolysis; PBS, phosphate buffered saline; 2-ME, 2-mercaptoethanol.
Introduction Thus far, only one feline blood group system has been described, which contains three blood types, type-A, type-B, and the very rare type-AB (Holmes, 1953; Auer and Bell, 1981 ). The frequency of blood types in domestic shorthair and longhair cats varies geographically. Even larger variations in blood type frequencies have been observed among purebred cats with type-B frequencies ranging from 0-59% (Giger et al., 1989, 1991 a, b). Type-A and typeB blood types are inherited as a simple autosomal mendellian trait with A *Corresponding author. ~Supported in part by grants from the National Institutes of Health (HL02355) and Robert H. Winn Foundation. 2present address: Department of Medicine, School of Veterinary Medicine, Tufts University, 200 Westboro Road, North Grafton, MA 01536, USA.
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being completely dominant over B (Giger et al., 1991a). The extremely rare type-AB cats could be explained by the presence of a third allele (Griot-Wenk and Giger, 1991 ). In contrast to many other animal species, cats appear to have naturally occurring alloantibodies against the other blood type (Ingebringsten, 1912, Eyquem et al., 1962, Auer and Bell, 1981; Giger et al., 1989, 1991 a, b; Wilkerson et al., 1991a). 'Naturally occurring' refers to the fact that these alloantibodies develop without prior sensitization by transfusion or pregnancy. In Australia, 96% of examined type-B plasma samples had strong agglutinins and hemolysins, with titers of > 1:64 for the agglutinin and up to 1:512 for the hemolysin (Auer and Bell, 1981 ). However, only 35% of the examined type-A plasma samples had agglutinating and hemolytic activity. Severe transfusion reactions in type-B cats receiving type-A blood (Auer et al., 1982; Giger and Akol, 1990; Giger and Biicheler, 1991; Wilkerson et al., 1991 a) and neonatal isoerythrolysis (NI) caused by colostral anti-A antibodies of type-B cats transferred to type-A newborns (Cain and Suzuki, 1985; Hubler et al., 1987; Gandolfi, 1988; Jonsson et al., 1990; Giger, 1991 ) have been described. The present study was undertaken to further characterize the agglutinins and hemolysins in type-A and type-B cats. Materials and methods
Plasma was separated from EDTA-anticoagulated feline blood samples that were submitted for blood typing (Giger et al., 1991a, b ). Additional serum for antibody characterization was obtained from 30 type-A and 30 type-B cats in the Philadelphia area for further studies. Specimens were stored in plastic tubes at - 7 0 ° C before analysis. Phosphate buffered saline pH 7.4 (PBS) containing 150 mmol 1-1 sodium chloride and 10 mmol 1-' sodium phosphate was used for all red blood cell suspensions and antibody dilutions. Rabbit complement, Drabkin's solution, 2-mercaptoethanol, ammonium sulfate, and iodoacetate were purchased from Sigma Chemicals (St. Louis, MO). Coombs reagent, goat anti-cat IgG (7-chain specific) and goat anti-cat IgM (/t-chain specific) antibodies were obtained from Kirkegaard and Perry Lab. (Gaithersburg, MD). Sephadex G 200 was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden).
Tests for agglutinins and hemolysins Plasma samples from various domestic shorthair and purebred cats were used for an agglutination screening assay as previously described (Giger et al., 1989). Briefly, 60 #l of undiluted sample plasma and 30 pl of a PBS-washed 3-5% type-A or type-B red blood cell suspension were mixed and centrifuged for 15 s at 1000 g (3500 rpm). Agglutination was graded from 0 to 4+, with 0 being no agglutination and 4 + being a single pellet-like agglutinate. Agglu-
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tination and hemolysis assays were performed as previously described (Auer and Bell, 1981; Giger et al., 1989 ). Serial two-fold dilutions of 60/tl of sample and 30/tl of a washed 3-5% type-A or type-B erythrocyte suspension were mixed and centrifuged for 15 s at 1000 g. The agglutination titer was recorded as the highest dilution which still produced a 1 + macroscopic agglutination. Some samples (n = 30) with negative macroscopic agglutination were examined for microscopic agglutination at 10 X magnification or used in a direct antiglobulin test (Coombs test ) as previously described (Coombs et al., 1945 ). Two-fold serial dilutions of heat inactivated serum (30 min at 56 °C) and 30 ~1 red blood cells combined with 30 #1 of undiluted rabbit complement, which previously had been adsorbed with an equal volume of feline erythrocytes at 20 °C for 5 min to remove naturally occurring heteroagglutinins (Giger et al., 1989), were used in the hemolysis assay. The mixtures were incubated in a water bath at 37°C for 30 min, mixed and further incubated at 20°C for 2 h. The tubes were centrifuged for 20 s at 1000 g and 50/zl of the supernatant was transferred into a tube containing 100 #1 of Drabkin's solution. After 15 min incubation, the light absorption was determined spectrophotometrically at 580 n m and compared with a completely hemolyzed control sample prepared by repeated freezing of the erythrocytes. The hemolysis titer was expressed as the highest dilution in which hemolysis reached the 10% level of the control. Some agglutination and hemolysis assays were performed at different temperatures (4, 20, 37°C).
Fractionation of immunoglobulins Immunoglobulins were separated by gel filtration according to standard methods (James and Stanworth, 1964). Briefly, the serum globulin fractions from two type-A and two type-B cats were separately prepared by precipitation of 10 ml of serum with equal amounts of 5.4 M a m m o n i u m sulfate at 4°C and centrifugation for 10 min at 1000 g. The precipitates were resuspended in 10 ml of PBS and applied to a 150 cm × 5 cm column of Sephadex G 200 in PBS. The flow rate was 35 ml h-1. Five ml samples were collected and the optical density at 280 n m was determined spectrophotometrically. The central fraction of the first protein elution containing the bulk of macroglobulins and the subsequent elution containing most of the IgG were harvested and concentrated by vacuum dialysis. These fractions were utilized for agglutination, hemolysis and immunoprecipitation assays, as previously described.
2-Mercaptoethanol treatment Destruction of the IgM-disulfide bonds with 2-ME was performed as previously described (Adler and Frank, 1965) to remove IgM-activity from
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serum. Serum and 2-ME (0.1-0.4 M) in equal parts and serum and PBS (control) were incubated at 37°C for 2 h. Subsequently, the mixture was dialyzed against PBS with the addition of 0.02 M iodoacetate for 3 days at 4 °C with a four-fold dialysate change every day. 2-ME treated serum was then examined for antibody activity using agglutination and hemolysis tests as described above.
Immunoprecipitation Specific removal of immunoglobulins by immunoprecipitation was performed according to standard techniques (Kabat and Maier, 1961 ). Briefly, 20/tl of serum and 40/tl of anti-IgM or anti-IgG were mixed in a glass tube and incubated at 37°C for 30 min. After centrifugation (14 000Xg for 10 min) the supernatant, separated from the precipitate pellet, was examined for antibody activity using agglutination and hemolysis tests as described above.
Alloantibody titer in newborn kittens Sequential plasma samples were obtained from type-B kittens born to typeA and -B queens prior to, and after colostrum uptake, at 30 min and 4 h post partum. Additional plasma samples were collected every 3-5 days until kittens were 15 weeks of age. The samples were analyzed for agglutinating and hemolytic activities as previously described. Results
A total of 2180 undiluted plasma samples from the blood typing service were screened for agglutinating alloantibodies. Thirty-eight (2%) of the 1933 examined plasma samples from type-A cats agglutinated type-B erythrocytes strongly (3 + to 4 + ), 36% of the plasma samples led to a weak agglutination Table 1 Agglutination of feline erythrocytes with undiluted plasma Cat blood type
A B AB
Allo-antibodies
anti-B 1 anti-A 2 anti-A or-B 3
Number of plasma samples
1933 239 8
tTested against type-B erythrocytes. 2Tested against type-A erythrocytes. STested against type-A and type-B erythrocytes.
Agglutination None
1-2+
3-4+
1199 0 8
696 0 0
38 239 0
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( 1 + to 2 + ) oftype-B red blood cells and 62% did not show macroscopically detectable agglutination (Table 1 ). However, when the plasma samples from 30 type-A cats with negative macroscopic agglutination were further examined, type-B erythrocyte microagglutinates were found. These incubated cells were also positive for the presence of IgG and IgM in a Coombs test. All plasma samples of the 239 tested type-B cats strongly ( 4 + ) agglutinated type-A erythrocytes. The plasma of the eight tested type-AB cats did not agglutinate or hemolyze type-A or type-B red cells. The agglutinin and hemolysin titer were determined in serum from 30 typeA and 30 type-B cats (Table 2). Serum of type-A cats had a weak anti-B agglutinin titer ( l - 16 ) and a slightly stronger hemolysin titer (4-32). Type-B cats had a strong agglutinin titer (64-512 ) as well as a strong hemolysin titer Table 2 Agglutinating (in °C) Number
and hemolyzing
of cats
antibody
Antibody 1
titer in type-A and type-B cats and their thermal
dependancy
titer
2
4
8
16
32
64
128
256
512
1024
Type-A cats (n = 30) Agglutinin
10
7
8
4
1
-
-
Hemolysin
-
-
5
8
10
7
-
4°
-
-
2
8
1
20 °
-
4
7
1
-
37 °
-
5
8
-
-
4°
8
2
-
20 °
-
1
6
3
-
37 °
-
-
-
2
4
Agglutinin
Hemolysin
( n = 10)
(n = I0) 4
Type-B cats (n = 30) Agglutinin
4
12
9
5
-
Hemolysin
2
9
11
6
2
Agglutinin 4°
( n = 10) _
_
-
-
2
6
2
20 °
_
_
-
2
4
4
-
37 °
_
_
-
3
4
3
-
4 o
_
_
-
4
4
2
-
20 °
_
_
-
-
3
4
3
37 °
_
_
-
1
4
5
Hemolysin
(n = 10)
288
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( 6 4 - 1 0 2 4 ) . S e r u m f r o m t y p e - A a n d t y p e - B cats h a d a slightly stronger agglut i n a t i n g a c t i v i t y at 4 ° C c o m p a r e d with 20 ° C a n d 37 ° C. T h e o p t i m a l r e a c t i o n t e m p e r a t u r e for h e m o l y s i s was 3 7 ° C ( T a b l e 2). S e r u m o f t h r e e t y p e - A a n d f o u r type-B cats t r e a t e d with 2 - M E ( T a b l e 3 ) s h o w e d r e d u c e d agglutinating a n d h e m o l y z i n g activities. T h e agglutinating activity was d e c r e a s e d m o r e severely t h a n the h e m o l y t i c activity. T h u s , 2 - M E did not abolish all agglutinating a n d h e m o l y z i n g alloantibodies. I m m u n o p r e c i p i t a t i o n o f s e r u m o f t y p e - A a n d t y p e - B cats with anti-feline IgM led to a m a r k e d r e d u c t i o n o f the agglutinating activity ( T a b l e 4 ), while i m m u n o p r e c i p i t a t i o n with anti-feline IgG o n l y slightly d i m i n i s h e d the seru m ' s agglutination. I m m u n o p r e c i p i t a t i o n with anti-IgM a n d anti-IgG, respectively, resulted in an equally strong i n h i b i t i o n o f hemolysis in type-A cats. In contrast, i m m u n o p r e c i p i t a t i o n o f t y p e - B s e r u m with anti-IgM led to a strong i n h i b i t i o n a n d i m m u n o p r e c i p i t a t i o n with a n t i - I g G to a w e a k i n h i b i t i o n o f hemolysis. Table 3 Alloantibody titer after 2-Mercaptoethanol treatment (geometric mean + SD) 2-Mercaptoethanol (Molarity) 0
0.1
0.2
0.4
Type-A cats (n = 3)
Anti-B Agglutinin Anti-B Hemolysin
6 +_2.8 13 _+11.5
0 5 _+1.9
0 2 _+0.5
0 2 _+0.5
22 +_10.4 30_+21.4
10 +_5.9 11 _+5.2
5 _+1.7 7 _+1.7
Type-B cats (n = 4)
Anti-A Agglutinin Anti-A Hemolysin
240 _+171 320 +_192
Table 4 Immunoprecipitation of the blood group antibodies (titer, geometric mean _+SD ) Immunoprecipitated with PBS (control)
Anti-IgG
Anti-IgM
Anti-lgG + anti-lgM
Type-A cats (n = 4)
Anti-B Agglutinin Anti-B Hemolysin
6 _+2.6 15 _+10.7
5 +_2.6 4 _+2.4
256 _+140 359 _+125
230 _+140
0 4 + 2.4
0 1 +_0.5
8 _+0.8
2 +_0.8
14 + 10.0
1 + 0.8
Type-B cats (n = 5)
Anti-A Agglutinin Anti-A Hemolysin
256
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The first fraction after gel filtration of serum from type-A cats (IgM-fraction) had weak agglutinin and hemolysin activities (anti-B), and the IgMfraction of anti-A serum from type-B cats strongly agglutinated and hemolyzed type-A cells (Table 5). Both activities of this first fraction could be Table 5 Immunoprecipitation of serum and its antibody fractions after gel filtration Cat No.
Immunoprecipitation with PBS (control) aggl. 1
Anti-IgG hem.
2
1
Anti-IgM
aggl. 2
1
hem. 2
1
aggl. 2
hem.
1
2
1
2
Type-A (n = 2)
Serum IgG-fraction IgM-fraction
4 0 2
8 1 8
8 4 4
16 8 8
4 0 2
8 0 8
4 0 4
8 I 8
0 0 0
2 1 0
4 4 0
8 8 1
128 2 64
256 2 128
128 8 64
512 16 256
128 0 64
128 0 128
64 0 64
128 2 256
8 2 0
4 2 0
16 4 4
16 16 16
Type-B (n = 2)
Serum IgG-fraction IgM-fraction
aggl., agglutinin-titer; hem, hemolysin-titer. 600
400
!
500 "
.o ~
300
= .¢
200 /
,
loo
ii
b
0
23
46
69
92
115
Days after Birth Fig. 1. D e v e l o p m e n t o f blood type alloantibodies in n e w b o r n cats. ( • ) type-B kittens after ingestion of type-B queen's colostrum (n = 14). ( • ) type-B kittens that were not allowed to nurse (n = 4 ). ( ,, ) type-A kittens that were not allowed to nurse, or h a d signs o f N I (n = 12 ).
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specifically inhibited by immunoprecipitation with anti-IgM, but not with anti-IgG. Only weak agglutinating activity was found in the second fraction (IgG-fraction). The second fraction Of type-A cat serum showed weak hemolysis of type-B red cells. The IgG-fraction of serum from type-B cats led to weak agglutination and moderate hemolysis of type-A cells. These reactions were weaker than with the IgM-fraction of anti-A serum, but still stronger than the IgG-fraction of anti-B serum. Both agglutinating and hemolytic activities of the IgG fractions could be inhibited by immunoprecipitation with anti-IgG, but not with anti-IgM. Anti-A alloantibodies were assessed in serum from newborn to 15-weekold kittens (Fig. 1 ). No alloantibodies were detected at birth in any plasma from kittens prior to colostrum intake. However, at the first time point after colostrum ingestion (4 h after birth) anti-A alloantibodies were present in type-B kittens born to type-B queens and highest serum antibody titers were reached after 24 h. These antibodies were agglutinins and hemolysins. The kittens' antibody titers which were as high as their type-B mother's serum titers, slowly declined over the next few weeks with an apparent half life of 8 days. No anti-A alloantibodies were detected in the plasma of type-A kittens born to type-B queens, although these antibodies are immediately bound to type-A erythrocytes and may cause mild to severe signs of NI as well as a positive Coombs test in type-A kittens (Giger, 1991 ). Furthermore, no antiA alloantibodies were found in type-B kittens that were not allowed to nurse from their type-B queen, but were foster nursed, and in type-B kittens born to type-A queens. However, at 6-8 weeks of age all type-B kittens developed their own anti-A alloantibodies without exposure to type-A erythrocytes. At 12 weeks of age their plasma anti-A titer reached levels comparable with that found in adult type-B cats. In type-A kittens the anti-B antibody titers detected were only 1:2 at 12 weeks of age. Discussion Feline iso- or alloantibodies were first detected at the beginning of this century, and recently, their clinical importance has been exemplified by reports of feline neonatal isoerythrolysis (Cain and Suzuki, 1985; Hubler et al., 1987; Gandolfi, 1988; Jonsson et al., 1990; Giger, 1991 ) and hemolytic transfusion reactions (Auer et al., 1982; Giger and Akol, 1990; Giger and Biicheler, 1991; Wilkerson et al., 1991 a). The studies described here further characterize these alloantibodies and their development. Based on this large survey of 2180 cats, all type-A and type-B cats older than 2 months of age have alloantibodies. In particular, all type-B cats have high plasma titers of anti-A agglutinins and hemolysins, with titers all exceeding 1:64. No adult type-B cat without anti-A antibodies was encountered. These data support previous results from Australia (Auer and Bell, 1981 )
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and Japan (Ejima et al., 1986), although these investigators reported on a few type-B cats lacking alloantibodies. As these type-B cats were not further characterized, these animals may have been young kittens, either born to typeA queens or born to type-B queens, but not allowed to nurse. Furthermore, all type-A cats had anti-B agglutinins, although microscopic examination was necessary for the detection of the typically weak agglutinin reactions. The antibody involvement in the weak agglutinin reactions was confirmed by a positive direct antiglobulin test. Only one third of all examined type-A cats had macroscopic hemagglutinins and hemolysins with very few of them reaching agglutinin and hemolysin titers of 1:16 and 1:32, respectively. Similar observations were made by Auer and Bell in Australia ( 1981 ). As expected, plasma from tested type-AB cats did not contain any alloantibodies. Based upon the early development and universal occurrence of alloantibodies without prior exposure to blood group antigens either by transfusion or pregnancy, these anti-A and anti-B alloantibodies are considered 'naturally occurring' (Springer et al., 19.62; Tizard, 1982; H6gmann, 1988). At birth, type-B kittens have no detectable alloantibodies in their plasma, but acquire high anti-A alloantibody titers during the first 2 days, through passive transfer via colostrum from type-B queens. These titers slowly decrease during the first few weeks of life with a half life similar to other maternal antibodies. Type-B kittens born to both type-A or -B queens will develop anti-A alloantibodies between 6-8 weeks of age. The reason for the occurrence of these alloantibodies is not known, but may be induced by exposure to similar or identical antigenic determinants that commonly are present in nature, such as food or bacterial antigens (Springer et al., 1962; Tizard, 1982; H6gmann, 1988). As the feline AB blood group antigens have been determined to be carbohydrate containing determinants (Andrews et al., 1992 ), their antibody induction may be similar to the situation with human blood groups like the ABO-system. Since most of the agglutinating activity in feline plasma could be removed after exposure to 2-ME and immunoprecipitation with anti-IgM, the agglutinin in type-A and type-B cats is predominantly of IgM-type. Hemolytic activity was found in both IgM and IgG fractions after gel filtration. Hemolysis could still be observed after 2-ME treatment and immunoprecipitation. Therefore, the hemolysins consist of both IgM and IgG, although the hemolytic activity in type-B cats appears to be mostly owing to IgM. Our data confirms and extends results from two previous reports that studied the hemagglutinins in type-B cats. Ikemoto et al. ( 1981 ) detected agglutination in the IgM-fraction of gel-filtered type-B sera, but no agglutinins were found in the IgG-fraction. Wilkerson et al. ( 1991 b) confirmed the predominant role of IgM for agglutination, but additionally found some weak agglu-
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tinin activity in the IgG-fraction after gel filtration, as observed in this study. This IgG-fraction may be important in neonatal isoerythrolysis. Based on the universal presence of naturally occurring feline alloantibodies, cats are at risk for a transfusion reaction-even when reeeivingtheflrst mismatched blood. Owing to the strong anti-A antibodies, severe transfusion reactions have been reported only in type~B cats recei~ingtype,A blood (Auer et al., 1982; Giger and Akol, 1990; Wilkerson et al., 199 l a). In mismatched transfusions, all type-B cats destroy transfused type-A erythrocytes via complement-mediated intravascular hemolysis within minutes to a few hours (Giger and Biicheler, 1991 ). Since all type-B cats have high IgM-alloantibody titer, transfused incompatible type-A erythrocytes get heavily coated with IgM which will activate the lytic C5-9 complex resulting in intravascular hemolysis (Schreiber and Frank, 1972 ). In contrast, type-A cats receiving type-B blood ......... S igiiS. .l.. t.a.u.~ t u ~~----~ .typ~,-L~ . . . . n ~__~. did not snow any m aj o~- Clllllk~l,l vu x ) , ~ v.~.,.~. a. . . W g l ' e steadily cleared from circulation within 5 days, thus rendering the transfusion inefficacious. Coating oftype-B erythrocytes with anti-B alloantibodies of the IgG and IgM class mostly results in macrophage-mediated destruction of transfused cells. The hemolyzing IgM-alloantibody titer in type-A cats is apparently too low to cause severe intravascular hemolysis. Neonatal isoerythrolysis has been described in blood type-A kittens born to blood type-B queens (Cain and Suzuki, 1985; Hubler et al., 1987; Gandolfi, 1988; Jonsson et al., 1990; Giger, 1991 ). Kittens will acquire maternal alloantibodies of the IgG- and to a lesser extend of the IgM-class via colostrum during the first 2 days of life (U. Giger and J. Biicheler, unpublished results, 1991 ). Cotostrat anti-A atloantibodies have been demonstrated in the type-B kitten's plasma as early as 4 h after birth. Iftype-A or type,AB kittens are born to a type-B queen, colostral antibodies will bind to and lyse erythrocytes in the newborn. The hemolysis may occur intravascularly as well as extravascularly and result in anemia, hemoglobinuria, jaundice and death (Giger, 1991 ). Since all adult type-B cats have high alloantibody titers, type-A kittens from primiparous type-B queens are at risk for NI. Clinical signs of NI may be peracute, acute or subclinical. The authors identified several type-A kittens that had laboratory abnormalities such as anemia and positive Coombs tests, but no clinical evidence of NI (Giger, 1991 ). The factors that determine the severity of hemolysis have not been identified. Furthermore, erythrocytes from kittens with subclinical NI were Coombs-positive for IgM as well as IgG (U. Giger and J. B~icheler, unpublished results, 1991 ). In preliminary studies very high IgG titers were found in colostrum from type-B cats, whereas the colostral IgM concentrations were low. Interestingly, the authors observed that surviving kittens may rarely develop tail-tip necrosis between 1 and 3 weeks post partum, which was also observed by Gandolfi (1988) and Jonsson (1990). Histologic examination showed thromboembolism and ischemic necrosis. The present study shows, that the in vitro agglutinating activity of feline IgM al'
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loantibodies is inversely correlated to temperature. Erythrocyte-bound IgM in these kittens may have caused a cold agglutinin reaction with microagglutination and vascular stasis in areas of lower body temperature which may result in tail tip necrosis. Important variables for the development of clinical signs of NI might be the amount and time of colostrum uptake, IgG-titer or subclasses, intestinal permeability for alloantibodies and the activity of the macrophage system in the neonate. In humans two different groups of antisera composition can be distinguished. Blood group antigens such as the Rhesus antigens have a protein nature and are usually recognized by IgG antibodies, which first have to be stimulated by immunogenic contact to erythrocytes of the other blood type, e.g. after a mismatched transfusion or exposure to incompatible fetal blood during pregnancy. In contrast, carbohydrate epitopes bound to membrane proteins or lipids on erythrocytes, i.e. antigens of the ABO-system, are recognized by naturally occurring antibodies (H~gmann, 1988; Anderson and Kelten, 1989 ). Molecular analysis of the feline blood group antigens (Andrews et al., 1991; U. Giger and J. BiJcheler, unpublished results, 1991 ) indicates, that the antigenic determinants of the feline AB-blood group system contain carbohydrate moieties similar to the human ABO-blood group system. The composition of the naturally occurring antibodies in type-A and type-B cats resembles that in humans with antigens of the ABO-system. Type-A cats have a low alloantibody titer with a greater proportion of IgG, as has been found in people with blood type-O. Type-B cats resemble people with blood type-A or -B having high alloantibody titers of the IgM class (Garratty, 1989 ). TypeAB cats, as well as humans with blood type-AB, are probably immunotolerant against either antigen and cannot develop alloantibodies against the A- or Bantigen. The antibody characterization in combination with the observed patterns of hemolysis in transfusion experiments suggest that cats and humans have similar mechanisms for immune mediated destruction of incompatible red cells suggesting that the cat may serve as an animal model for human transfusion reactions and hemolysis of the newborn.
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Tizard, R., 1982. An introduction to immunology, 2nd edn., Saunders, Philadelphia, PA, pp. 165-177. Wilkerson, M.J., Wardrop K.J., Meyers, K.M. and Giger, U., 1991a. Two cat colonies with A and B blood types and a clinical transfusion reaction. Feline Practice, 19 (2): 22-26. Wilkerson, M.J., Meyers, K.M. and Wardrop, K.J., 1991b. Anti-A isoagglutinins in two blood type B cats are IgG and IgM. Vet. Clin. Path., 20: 10-14.