The use and efficacy of horse blood typing tests

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THE USE A N D E F F I C A C Y OF H O R S E B L O O D T Y P I N G TESTS

A.T. Bowling, P h D

SUMMARY Horse blood typing, an assay of red blood cell and serum genetic markers, is a useful tool for horse breeders and horse registries, it has applications to veterinary medicine as well. For recognizing incorrect paternity (or maternity) a 20-system test battery is calculated to be about 96 percent effective in Thoroughbreds and Arabians and as high as 98 percent in other US breeds such as Standardbreds, Morgans, Quarter Horses, Paso Finos and Peruvian Pasos. In addition to paternity testing, horse blood typing tests can be applied to p r o b l e m s of horse i d e n t i f i c a t i o n , diagnosis and management of neonatal isoerythrolysis and selection of blood tran,qfusion donors.

INTRODUCTION The modern era of horse blood typing starts with studies of Podliachouk in France, ~5 Stormont and colleagues in the United States 2° 22 and Hesselholt in Denmark. m Working independently, they developed techniques which are still used for effective and reliable assay of genetic markers in horse blood. Rather than using naturally occurring antibodies for red cell serology, which earlier workers had shown could not easily fit into a scheme such as the ABO human blood group,they p r e p a r e d blood g r o u p i n g reagents from p l a n n e d immunizations of horses (alloimmunization). The n u m b e r of g e n e t i c d i f f e r e n c e s w h i c h c o u l d be serologically detected f r o m b l o o d samples was Author's address: Serology Laboratory, University of California, Davis, CA 95616. Acknowledgements:The author gratefully acknowledgesthe efforts and diligenceof the SerologyLaboratorystaffthat performedthe blood typing tests and wrote the computer programs for data storage and retrieval, also support of the breed registries. Volume 5, N u m b e r 4

augmented with gel electrophoresis techniques. Use of alloimmunization and electrophoresis for horse blood typing was developed on the heels of similar tests f o r inherited characteristics developed for cattle typing, which had p r o v e d extremely useful for paternity assignment in cases of questionable pedigree, r9 In 1965 Stormont and Suzuki 2~ demonstrated that a battery of ten systems, including both serological and electrophoretic tests, was effective for detecting incorrect paternity in breeds as disparate as Shetland Ponies and Thoroughbreds: The effectiveness rates were calculated to be 77.8 percent in Shetland Ponies and 62.01 percent in Thoroughbreds, the difference being due to the number and f r e q u e n c y o f distinct types in each breed. Furthermore, a pilot study demonstrated that the tests were valid: in none of 114 simulated parentage cases did the tests exclude the incorrect sire. From its inception, research on horse blood typing has been an international effort. A unified nonmenclature has been established under the aegis of the International Society for Animal Blood Group Research (ISABR) so that types can be applied to parentage problems regardless of where the horses have been tested. Judging from participation in the lates t (1983) Horse Comparison Test organized by ISABR, at least 22 laboratories throughout the world have developed some capacity for horse blood typing. Since blood typing tests are currently at least 96 percent effective in detecting incorrect paternity it is not surprising that horse registries have blood typing r e g u l a t i o n s f o r horses u n d e r their j u r i s d i c t i o n . Regulations vary from requiring owners to allow blood typing of their horses, to requiring parentage verification of all foals prior to registration. The vast amount of inalterable phenotypjc variation detectable by b l o o d typing is also useful in the identification of horses. Some countries, e.g. France, require horses to have a passport which includes blood type information. 195

This paper describes tests that can be applied to horse blood samples for an effective system of parentage investigation and identification, and discusses their application to NI, transfusion and their possible future use as a tool to select against linked genetic diseases. MATERIALS

AND

METHODS

Assayed characteristics meet the properties cited by Rendel and Gahne ~6 as critical for use in parentage testing, namely: a) the characters are simply inherited and their mode of inheritance is fully known; b) the characters are developed at or shortly after birth and once developed remain unchanged throughout life; and c) the detection of the characters is reliable and objective. Two 10cc blood samples are needed from each horse, one in ACD anticoagulant to use as a source of red cells and one in a dry tube to use as a serum source. Blood samples are sent to the laboratory via mail or commercial courier at ambient temperature, then refrigerated until tests are completed. Red cell alloantigenic specificities are detected at seven internationally recognized blood group loci (A,C,D, K,P,Q and U) using standard immunological procedures involving hemagglutination and complement mediated hemolysis.20 22 Reagents for antigen detection are produced by alloimmunizations, followed by extensive testing and absorption of the immune sera until each behaves as monospecific. Assignment of alleles is based on reagent reaction patterns and is in accord with internationally agreed terminology (for an excellent review see reference 4). Those specificities detected by reagents primarily behave as codominants with a few exceptions (e.g D and Q locus Subtypes). Undetectable alleles act as recessives to those specificities detected by reagents and thus one phenotype can be determined by m o r e t h a n one g e n o t y p e . R e a g e n t s for red cell alloantigens, their alleles and p h e n o t y p e / g e n o t y p e considerations are shown in Table 1. TABLE 1

Alloantigenic red cell blood group variants. No. of Locus Alleles

A a, adf, adq . . . . . . . . . . . . b, be, bee, be . . . . . . . . . c, ce, e, -* . . . . . . . . . . . . C a,-* ................. D a d , bc, cq, ceqi . . . . . . . . cefq, cf, d, de . . . . . . . . . . def, dek, dqh . . . . . . . . . . dk, -* . . . . . . . . . . . . . . . . K a,-* .................. P a, b, -* . . . . . . . . . . . . . . . Q abc, ac, b, c, -* . . . . . . . U a;-* .................

Reagents

7

No. of Genotypes

66

No. of Phenotypes

33

1 10

3 78

2 72**

1 2 3 1

3 6 15 3

2 4 6 2

*Allele detectable by absence of reactions to reagents. D system negative allele not included in calculations since it is extremely infrequent. **Additional reagents, not yet internationally defined, needed to distinguish some combinations. 196

Standard methods of starch 8 17 and polyacrylamidO 2 gel e l e c t o r p h o r e s i s are used t o i d e n t i f y seven internationally recognized loci of inherited variants of enzymes and other proteins: albumin (AI), transferrin (Tf), esterase (Es), Xk, Go, protease inhibitor (Pi), 6phosphogluconate dehydrogenase (PGD), phosphoglucomutase (PG M), phosphohexose isomerase ( P H 1), catalase (Cat), carbonic anhydrase (CA) and acid p h o s p h a t a s e (AP). P o l y a c r y l a m i d e gel isoelectric focusing is used for the detection of hemoglobin (Hb) variants. 12 (Table 2).

TABLE 2 Blood protein variants. Locus

AI Tf Pi Xk Es Gc PGD PGM PHI CA Cat AP Hb

No. of Alleles

3 12 9 3 6 2 3 3 3 5 2 2 4

No. of Phenot~cpes

Variants Detected

A,I,B . . . . . . . . . . . . . . . . . . . . . D,E*,FI,F2,F3,G . . . . . . . . . . . . H t*,H2*,J,M,O,R . . . . . . . . . . . F,G, 1,L,N,O,S,U,W . . . . . . . . . F,K,S . . . . . . . . . . . . . . . . . . . . . F,G,H,I,O,R,S . . . . . . . . . . . . . F,S . . . . . . . . . . . . . . . . . . . . . . . D,F,S . . . . . . . . . . . . . . . . . . . . . F,S,V . . . . . . . . . . . . . . . . . . . . . F,I,S . . . . . . . . . . . . . . . . . . . . . . F,1,L,O,S . . . . . . . . . . . . . . . . . . F,S . . . . . . . . . . . . . . . . . . . . . . . F,S . . . . . . . . . . . . . . . . . . . . . . . A,AII,B1,BI1 . . . . . . . . . . . . . . .

6 78 45 6 22 3 6 6 6 15 3 3 10

*Terminology not internationally defined.

Specificities with detectable protein products behave as codominants. The AI, Tf, Xk, Gc, Pi and PGD loci are considered closed, with each phenotype defining a unique genotype. In the Es and Tf systems, extremely rare silent alleles occur which have little or no detectable protein product. Silent alleles behave as recessives to those for which protein products can be detected. RESULTS

AND

DISCUSSION

Gene Frequencies Analysis of test results from nearly 120,000 horses tested in this laboratory from 1973 to 1983 shows that each breed has a unique profile of gene frequencies. 6 No genetic markers are exclusively confined to one breed, although certain specificities may be quite rare in a particular breed. The most common blood type for each breed can be calculated from gene frequency data and is given in Table 3. The "frequencies of these types are given in Table 4: Such c h a r t s show clearly that breed g r o u p s are genetically differentiated from each other in f a c t o r profiles. The frequency of the most common blood type for each breed provides a measure for comparison of complexity of the genetic base. For example, the rather high frequency of the most common blood type for Thoroughbreds (0.00031 or l/3226) in comparison to the EQUINE VETERINARY SCIENCE

TABLE 3 M o s t frequently occurring b l o o d type p h e n o t y p e for each o f seven breeds. Locus

TB

AR

Breed* ST MH

QH

PF

PP

A C D

adf adf adf/b adf adf adf adf a a a a a a a cg/dk bc/dk cg/dk cg/dk bc/dk bc/dk dk K a . . . . P a a a O a . . . . . c U a a a AI B AB AB AB AB AB AB -If DF~ D F 2 F2 F2 D F 2 DF~ DF~ Es 1 I 1 FI 1 1 I Xk K K K K K K K Pi L S-U L-U L-U L-S L-S L-** PGD FS FS F F F F F Gc F F F F F F F *Breeds: TB=Thoroughbred, Ar=Arabian, ST:Standardbred, M H=Morgan, QH=Quarter Horse, PF=Paso Fino, PP=Peruvian Paso. **Unnamed allele.

TABLE 4 Frequencies o f the most c o m m o n b l o o d type by breed. Breed

TB . . . . . . . . . . . . . AR ............. ST ............. MH ............ QH ............. PF ............. PP .............

RBC type

Gel type

Combined

0.039 0.029 0.009 0.012 0.009 0.001 0.004

0.008 0.005 0.008 0.003 0.002 0.003 0.001

0.00031 0.00015 0.000072 0.000036 0.000018 0.000003 0.000004

Q u a r t e r Horse (0.000018 or I / 55,555), a T h o r o u g h b r e d derived breed, is consistent with historical accounts of the two breeds. Efficacy o f B l o o d T y p i n g Tests

The most c o m m o n use of equine b l o o d t y p i n g t o d a y is the investigation o f q u e s t i o n a b l e parentage. A t y p i c a l case is one in which a m a r e has been bred to m o r e t h a n one stallion in a breeding season. In other cases, the foal o w n e r suspects t h a t the d a m is incorrect, either due to foals switching d a m s soon after birth, or to switching o f foals after weaning. The l a t t e r can occur when sex, m a r k i n g s a n d c o l o r are similar. Rarely, it is suggested that horses may have been deliberately switched (substitution of a "ringer") p a r t i c u l a r l y in order to affect the o u t c o m e o f a race. O f p a r a m o u n t concern to breed registries and owners is the effectiveness of these b l o o d t y p i n g tests in such situations. T h e effectiveness of the tests is calculated as the p r o b a b i l i t y of exclusion (PE) for detecting incorrect p a t e r n i t y when b l o o d samples a r e tested from sire, d a m and offspring. 16 ~ The PE for the 20 loci described here is a b o u t 96 percent in T h o r o u g h b r e d s and A r a b i a n s a n d as high as 98 percent in o t h e r US Volume 5, N u m b e r 4

b r e e d s such as S t a n d a r d b r e d s , M o r g a n s , Q u a r t e r Horses, P a s o F i n o s and P e r u v i a n Pasos. 6 In a c t u a l practice the effectiveness c o u l d be slightly l o w e r when a m a r e has been b r e d to two stallions, since owners tend to breed mares to related stallions (e.g., a s t a l l i o n a n d his son) r a t h e r t h a n t w o stallions selected at r a n d o m f r o m the breed. It is e x p e c t e d t h a t the tests w o u l d be slightly m o r e effective t h a n 96 percent if b o t h p a r e n t s were incorrect. The efficacy o f a single system o r locus d e p e n d s on t h e n u m b e r of alleles, their frequencies a n d whether the genotypes can be directly determined from the phenotypes.16 T h e m o s t effective a r e closed loci h a v i n g five o r m o r e alleles with a p p r e c i a b l e frequencies, n a m e l y D, Tf, a n d Pi. T a k e n t o g e t h e r t h e three loci have a theoretical P E between 0.864 a n d 0.978 in each of the seven breeds. T h e a d d i t i o n a l 17 loci c o n t r i b u t e o n l y slightly to the c u m u l a t i v e t o t a l PE. Since the PE o f the s t a n d a r d test is so high, it obviously has b e c o m e increasingly difficult to a u g m e n t it. The m o s t i m p o r t a n t use o f a d d i t i o n a l loci might be to p r o v i d e solutions based on m o r e t h a n one locus. F r o m a legal s t a n d p o i n t , one locus is sufficient, b u t any case is t h a t much more c o n v i n c i n g if m o r e t h a n one locus can be shown to be significant to the solution. O t h e r s e r u m (plasma) and red cell loci have been described (e.g. references 24, 25). Tests have been d e v e l o p e d as well for genetic m a r k e r s of l y m p h o c y t e s a n d u n d o u b t e d l y the m o s t useful single locus by which the test battery can be a u g m e n t e d is that for Equine L y m p h o c y t e A n t i g e n (ELA). E L A c u r r e n t l y has ten specificities b e h a v i n g as alleles at a single locus, which have been recognized and accepted by i n t e r n a t i o n a l E L A workshops. 3 Identifications o f Individuals

O b v i o u s l y b l o o d t y p i n g provides a vast a r r a y o f inalterable differences that can be used for identification, but it does have the d r a w b a c k that the tests require a blood sample, special reagents, skilled laboratory personnel and time. Sex and external marks of signalment ( n a t u r a l signalment such as color, hair whorls, chestnuts a n d white m a r k i n g s a n d a p p l i e d signalment such as brands, electronic i m p l a n t s , t a t t o o s a n d freczemarks) a r e a d d i t i o n a l differences that can be used. 12 These d o not require l a b o r a t o r y tests a n d so they can be rapidly defined. S t a n d a r d p r o c e d u r e s have been established for signalment definition, including description of p h e n o t y p e with eight c o a t c o l o r loci: A, C, D, E, G, Rn, Rn, To and W (see reference 23). Unfortunately, s o m e red cell factor t e r m i n o l o g y overlaps assignments p r e v i o u s l y in use for c o a t color. Even bearing in m i n d possible a l t e r a t i o n of sex (stallion to gelding) and external marks of signalment, the c o m b i n a t i o n of i n a l t e r a b l e b l o o d t y p e genetic m a r k e r s with external c h a r a c t e r s would be e x p e c t e d to p r o v i d e a unique identity for every horse. Clearly, it is e x t r e m e l y unlikely that a horse's battery of genetic markers, including b o t h c o a t c o l o r and b l o o d type, could be m a t c h e d except by a m o n o z y g o t i c twin. Ringer detection is virtually a s s u r e d when horses have been t h o r o u g h l y identified at birth. 197

T o illustrate the virtual i m p o s s i b i l i t y o f u n d e t e c t e d s u b s t i t u t i o n of a horse, c o n s i d e r the following e x a m p l e . S u p p o s e s o m e o n e wished to substitute one A r a b i a n f o r a n o t h e r with the most f r e q u e n t l y e n c o u n t e r e d b l o o d t y p e for an A R , which would be the most difficult test f o r ringer detection. The f r e q u e n c y of that type is 0.00015, one in 6667 (See Table 4), T h e substitute must of c o u r s e not only share that type, but also coat c o l o r must be the same. F o r A R s the most f r e q u e u t coat color is grey a n d its p r o b a b i l i t y is 0.42 ( c a l c u l a t e d from gene frequencies, G--0.24:E=0.31). 7 T h u s the p r o b a b i l i t y of occurrence o f the most c o m m o n a r r a y Of genetic markers for b l o o d type and coat c o l o r for an A R is 0.000063 (one in 15,873). The A r a b i a n H o r s e Registry of A m e r i c a has over 300,000 horses since its first r e g i s t r a t i o n in 1908. O n l y 19 of those horses would be expected to meet criteria for the most c o m m o n color and b l o o d t y p e p h e n o t y p e . Of course the s u b s t i t u t e must match by sex as well, so only half that n u m b e r w o u l d qualify. It is e x c e e d i n g l y unlikely that any two of the 10 meeting sex, c o a t c o l o r and blood t y p e requirements w o u l d be c o n t e m p o r a r i e s and so s i m i l a r in m a r k i n g s t h a t a s u b s t i t u t i o n of one for a n o t h e r would have been a t t e m p t e d . Clearly, if the m o s t c o m m o n p h e n o t y p e is so difficult to match, the u n d e t e c t e d substitution of any horse for a n o t h e r is virtually impossible. If a horse has not been identified by b l o o d t y p i n g then d e t e c t i o n of its substitute becomes a p r o b l e m of p a r e n t a g e v e r i f i c a t i o n . R i n g e r d e t e c t i o n w o u l d be slightly more effective t h a n p a t e r n i t y testing b e c a u s e b o t h p a r e n t s are likely to be incorrect.

Neonatal lsoerythrolysis Blood typing can be used to analyze the b l o o d g r o u p i n c o m p a t i b i l i t y between m a r e a n d stallion in cases o f foal d e a t h due to n e o n a t a l isoerythrolysis (NI). M a r e s negative for factors A a or G a bred to stallions positive for those factors are the most c o m m o n i n c o m p a t i b i l i t i e s seen, a l t h o u g h other red cell factors have been i m p l i c a t e d as well. The percentage of m a t i n g s at potential risk to produce affected foals based on blood group c o n s i d e r a t i o n s alone is m u c h higher t h a n the incidence of clinical disease. No single f a c t o r has been f o u n d which c o u l d identify high risk m a r e s l a c k i n g a previous h i s t o r y o f the disease in her foals. F o r a recent review of clinical m a n a g e m e n t of this d i s e a s e see W i t h a m , C a r l s o n a n d B o w l i n g . 26 If sensitization in a m a r e has been detected a n d the specificity d e t e r m i n e d , then in some cases it m a y be possible to find a stallion l a c k i n g the factor t h a t will a v o i d the p r o b l e m in future foals from the mare. F i n d i n g a stallion that meets b l o o d t y p e as well as pedigree a n d c o n f o r m a t i o n criteria m a y be difficult. Table 5 uses gene f r e q u e n c y d a t a to calculate the incidence of stallions l a c k i n g A a or Q a in each of seven breeds. F r o m this d a t a it can be seen that for T h o r o u g h b r e d s , in which A a is in very high freguency, only 2 stallions in 100 could be selected to avoid the NI sensitization p r o b l e m for A a sensitized mares. On the o t h e r hand, a v o i d i n g an A a b r e e d i n g for a sensitized m a r e w o u l d be easier to achieve by selecting a m o n g Q H stallions. A v o i d i n g a Q a 198

sensitization might be m o r e easily m a n a g e d , even for T h o r o u g h b r e d s in which Q a is relatively frequently found.

Selection of Whole Blood Donors: Single whole b l o o d transfusion in horses can usually be p e r f o r m e d w i t h o u t b l o o d g r o u p matbhing. Because of the relative ease of t r a n s f u s i n g u n m a t c h e d horses, it might be assumed that most horses have the s a m e b l o o d groups, but clearly that is not the case. Lack o f adverse reactions to a single t r a n s f u s i o n is most likely due to the fact that naturally o c c u r r i n g anti-red-cell a n t i b o d i e s are rather infrequent in horses. Transfusions with unselected d o n o r s are unlikely to be between horses of matched b l o o d groups a n d will likely lead to sensitization of the recipient, with ensuing p r o b l e m s a n t i c i p a t e d for future transfusions or, in b r e e d i n g mares, f o r the prospect of N I in foals. T a b l e 4 Suggests that because blood group frequencies are so different between breeds, the best d o n o r in u n m a t c h e d whole blood t r a n s f u s i o n s is at least a horse of the s a m e breed as the recipient.

Breed

TABLE 5 Population frequency of horses negative for Aa and Qa. Aa negative Qa negative

TB . . . . . . . . . . . . . . . . AR ................ ST . . . . . . . . . . . . . . . . MH . . . . . . . . . . . . . . . QH . . . . . . . . . . . . . . . . PF ................ PP . . . . . . . . . . . . . . . .

0.02 0.03 0.19 0.19 0.26 0.26 0.22

0.15 0.63 0.99 0.99 0.68 0.85 0.96

Gene Linkage and its Possible Use in Selective Breeding It should be possible to utilize linkage i n f o r m a t i o n as a tool for selection. F o r example, close linkage of a blood type m a r k e r to a recessive lethal trait for which no biochemical c a r r i e r test was available could be utilized within a family line to select breeding stock likely to be free of the deleterious trait. A l t h o u g h it would require a vast q u a n t i t y of d a t a in o r d e r to discern the several linkages, it might also be possible to find some m a j o r genes of p o l y g e n i c traits (such as p e r f o r m a n c e ability) linked to b l o o d t y p e markers which could then be used for selection of y o u n g stock. Eleven b l o o d type and three coat color loci have so far been identified as belonging to four a u t o s o m a l linkage groups (LGs), but specific c h r o m o s o m e a s s i g n m e n t has not yet been possible. F o r a review of h o r s e linkage groups see S a n d b e r g and Andersson. TM K and PG I) are in linkage g r o u p (LG) I; A1, Gc and Es a n d 3 coat c o l o r loci (E, R a n d To) are in LG ll; PHI and Xk are in LG IV. A is closely linked to E L A (Equine L y m p h o c y t e Antigen), considered to be the major histocompatibility l o c u s , in L G I I l . C . D , P , Q , T F , P G M , C A , A p and Hb have been shown to be i n d e p e n d e n t of the defined LGs a n d of each other. U,Pi and Cat have undefined linkage relationships. Thus the 20 loci of this r e p o r t identify at least 13 linkage groups and perhaps as m a n y as I6. A n d e r s s o n 23 calculated that 16 loci ( A ; C , D , K , P , Q , A I , T f , Xk,Es, P G D , P G M , P H I , EQUINE VETERINARY SCIENCE

CA,Hb,and

A P ) c o v e r e d a b o u t 25 p e r c e n t of t h e e q u i n e

genome.

The p r o m i n e n t a s s o c i a t i o n o f t h e Pi l o c u s w i t h s u c h h u m a n d i s e a s e s a s e m p h y s e m a has led to t h e s p e c u l a t i o n that such a relationship may also be found for certain h o r s e d i s e a s e s , j4 A t p r e s e n t s u c h a n a s s o c i a t i o n h a s n o t been clearly shown; perhaps the recent expansion of the Pi l o c u s w i t h a c r y l a m i d e t e c h n i q u e s 5 will a l l o w b e t t e r discrimination of disease association. T h e m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x ( M HC), w h i c h c a n b e a s s a y e d w i t h l y m p h o c y t e s , h a s b e e n s h o w n in h u m a n s t o h a v e s i g n i f i c a n t d i s e a s e a s s o c i a t i o n a n d it is p o s s i b l e t h a t h o r s e l y m p h o c y t e a n t i g e n s will a l s o b e u s e f u l in t h i s r e g a r d . T h e m u l t i p l e s p e c i f i c i t i e s a l r e a d y d e t e r m i n e d a t b o t h A a n d E L A o f t h e h o r s e r e d cellM HC complex provide exciting possibilities for effective studies of possible association of resistance or s u s c e p t i b i l i t y t o disease. L a z a r y et al. ~ p r o v i d e d e v i d e n c e that an antigen of the lymphocyte locus ELY-I has i n c r e a s e d f r e q u e n c y in h o r s e s w i t h c h r o n i c b r o n c h i t i s o r laminitis. At the present time no traits with positive selective v a l u e , s u c h as p e r f o r m a n c e a b i l i t y , a r e k n o w n to b e c o n d i t i o n e d by b l o o d t y p e m a r k e r s o r l i n k e d t o t h e m , b u t k n o w l e d g e o f t h e gene m a p is c l e a r l y p o t e n t i a l l y u s e f u l t o horse breeding. Exploration of the linkage relationships o f t h e o t h e r k n o w n loci as well as i d e n t i f i c a t i o n o f additional polymorphic systems should extend the g e n e t i c m a p t o t h e p o i n t t h a t it m a y i n d e e d fulfill its p o t e n t i a l u s e f u l n e s s as a s e l e c t i o n t o o l f o r b r e e d e r s .

REFERENCES I. Anderson L: Studies on genetic" finkage in domestic animals with special ,referenee to the horse. Thesis: Swedish University of Agricultural Sciences, Uppsala, 1983. 2. Anonymous: proceedings, 2nd International Horse Identification Seminar. Tucson Washington State University, Pullman. 3. Bailey E, Antczak DF, Bernoco D, Bull RW, Fister R, Guerin G, Lazary S, Matthews S: Joint report of the second international workshop on lymphocyte atloantigens of the horse, October 3-8 1982. Anita Blood Grps Biochem Gen 15:123-132. 4.Bell K: The blood groups of domestic mammals. In: Agar NS and Board PG. (eds) Red blood cells o f domestic mammals Amsterdam, Elsevier Biomedical Press. pp. 133-164, 1983. 5. Bell K, Patterson S, Pollitt CC: The plasma protease inhibitor system (Pi) of Standardbred horses. Anita Blood Grps Biochem Gen i5:191-206, 1984.

6. Bowling AT, Clark RS: Blood group and protein polymorphism gene frequencies for seven breeds of horses in the United States. Anita Blood Grps Bioehem Gen 16: in press, 1985.

7. Bowling AT, Dodd J, Suzuki Y, Stormont C: Population and family data on Arabian horses in the United States (abstract) Anita Blood Grps Biochem Gen l l:Supp 1:19-20, 1980. 8. Braend M: Genetic variation in equine blood proteins, In: Bryans JT and Gerber H, (eds) Proceedings. 3rd International Conference on Equine Infectious Diseases Paris, Karger, Basel, 1972 pp. 394-406, 1978. 9. Braend M, Johanses KE: Haemoglobin types in Norwegian horses. Anita Blood Grps Biochem Gen 14:305-307, 1983. I0. Hesselholt M: Studies on blood and serum types of the Icelandic horses lcta Vet S t a n d 7:206-225, 1966. I I. Jamieson A: The genetics of transferrins in cattle Heredity 20:419441, 1965. 12. Juneja RK, Gahne B, Sandberg K: Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum. Anita Bh)od Grps Bioehem Gen 9: 235-251, 1978. 13. Lazary S. Gerber H, de Week AL, Arnold P: Equine leukocyte antigen system. 111. Non-MHC linked alloantigenic system in horses.J tmmun~)gen 9:327-334, 1982. 14, Matthews AG: Identification and characterization of the major antiproteases in equine serum and an investigation of their role in the onset of chronic obstructive pulmonary disease (COPD). Equine Vet J 11:177-182, 1979. 15. Podliachouk L: Les antigenes de groupes sanquins des equides e t leur transmission hereditaire Thesis. University of Paris, 1957. 16. Rendel 3, Gahne B: Parentage tests in cattle using erythrocytc antigens and serum transferrins. Anita Prod 3:307-314, 1961. 17. Sandberg K: Blood typing of horses: current status and application to identification problems. In: (ed) Proceedings. 1st World Congress ~)["Genetic.~ Applied to Livestock Proch,'tion Madrid, pp. 253-265, 1974. 18. Sandberg K, Andersson L: Genetic linkage in the horse. 1. Linkage relationships among 15 blood marker loci. Hereditas 100:199208, 1974, 19, Stormont C: The early history of cattle blood groups. hnmunogen 6:1- t 5, 1978. 20. Stormont C, Suzuki Y: Genetic systems of blood groups in horses. Genetics. 50:915-929, 1964. 21. Stormont C, Suzuki Y: Paternity tests in horses. Cornell Vet 55:365-377, 1965. 22. Stormont C, Suzuki Y, Rhode EA: Serology of horse blood groups. Cornell Vet 54:439-452, 1964. 23. Trommershausen-Smith A: Positive horse identification. Part 3: coat color genetics. Fqttine Pratt 1:24-35, 1979. 24. Weitkamp LR, Costello-Leary P, Guttormsen SA: Equine marker genes: ploymorphism for plasminogen. Anita Blood Grp,~ Biochem Gen 14:219-223, 1983. 25. Weitkamp LR, Guttormsen SA, Costello-Leary P: Equine gene mapping: close linkage between the loci for soluble malic enzyme and Xk(PAL Anita BA~od Grps Biochem Gen 13:279-284, 1982. 26. Witham CL. Carlson GP, Bowling AT: Neonatal isoerythrolysis in foals: management and prevention. Cali[~rnia Vet II :21-23,34. 1984.

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