EP0673509A1 - Methodology for developing a superior line of domesticated animals - Google Patents

Methodology for developing a superior line of domesticated animals

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Publication number
EP0673509A1
EP0673509A1 EP94901714A EP94901714A EP0673509A1 EP 0673509 A1 EP0673509 A1 EP 0673509A1 EP 94901714 A EP94901714 A EP 94901714A EP 94901714 A EP94901714 A EP 94901714A EP 0673509 A1 EP0673509 A1 EP 0673509A1
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Prior art keywords
animal
animals
response
pigs
antibody
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EP94901714A
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German (de)
French (fr)
Inventor
Brian Wayne Kennedy
Bruce Nicholson Wilkie
Bonnie Allorene Mallard
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University of Guelph
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University of Guelph
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism

Definitions

  • This invention relates to a methodology for developing a superior line of domesticated animals, particularly animals which yield food value and more particularly a methodology which provides an animal line of increase productivity and generally healthier through to maturation.
  • Tumour necrosis factor ⁇ stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature (London) 322: 547- 549.
  • Resistance to infection and disease is the outcome of complex interactions between virulence-related attributes of the pathogen and a spectrum of innate and immunological mechanisms which have a quantitative basis and are under polygenic control (Lie, 0. 1990) .
  • Population variation in resistance to specific diseases could be exploited in. isease control if heritability of the resistance-related attributes was sufficiently high to permit selective breeding (Gavora, J.S., 1990).
  • a procedure for ranking immune system responsiveness in an animal to provide an Estimated Breeding Value (EBV) indicator of the animal's level of ability to resist disease and ability to pass such disease resistance to offspring, such EBV indicator being useful in selecting animals to be bred in order to produce offspring which inherit said level of ability to resist disease.
  • EBV Estimated Breeding Value
  • the procedure comprises: i) testing an animal's response to at least two tests one of which is a general measure and the other antigen specific which determine heritable humoral immunity traits; ii) testing the same animal's response to at least two tests one of which is a general measure and the other antigen specific which determine heritable cell-mediated immunity traits; iii) testing the animal's response to the two tests of humoral immunity traits and two tests of cell- mediated immunity traits beginning as soon as possible after the animal has been weaned from its mother and at a time chosen to negate effects of passive immunity; iv) ranking the animal's EBV indicator relative to other ranked animals based on the animal's level of response to the tests.
  • five traits may be selected on which diagnosis is based are:
  • DHT cellular activity - cutanaceous delay type hypersensitivity
  • BCG Bacillus Calmette Guerin
  • PPD purified protein derivative
  • diagnosis based on these selected traits provides an estimated breeding value (EBV) which determines the superior line of pigs for providing enhanced productivity and of improved health.
  • EBV estimated breeding value
  • the combined EBV can be calculated for each animal, such as pigs, where each pig is assigned to a high, low or controlled breeding group.
  • the methodology may be applied to various livestock to yield a high quality strain of livestock which is disease resistant. It has been found that the traits evaluated in accordance with the above methodology is preserved in generation to generation where, as needed, further EBVs may be calculated in the third and fourth generations to further direct the continued production of a high quality animal or a low quality animal.
  • Figure 1 is a bar chart showing the EBVs for a first generation of Yorkshire pigs
  • Figure 2 is the combined EBV for first generation Yorkshire pigs.
  • Figure 3 is a rate of gain in pigs selected for high-low immune responses.
  • Figure 4 Mean change in weight of M. hyorhinis challenged and control (Placebo) pigs of High (solid bars) and Low (hatched bars) immune response lines of pigs. Weight gain in control pigs is significantly (p ⁇ 0.01) greater in High than in Low line animals. Weight loss is equivalent for animals of each line after infection.
  • Figure 5 Mean clinical scores of arthritis in
  • ESR Erythrocyte sedimentation rate
  • FIG 6a Figure 6a and plasma fibrinogen concentration (Figure 6b) in M. hyorhinis infected pigs of High (solid bars) and Low (hatched bars) immune response lines.
  • ESR is significantly (p ⁇ 0.01) higher in Low than in High line pigs on D3 and elevated in both lines on days 7, 10 and 14.
  • Fibrinogen is increased in pigs of both lines from D3 to D14 (p ⁇ O.001) and on D10 the concentration in the Low line exceeds that of the High line (p ⁇ 0.05).
  • FIG. 7 Serum antibody (passive haemagglutination) titer (log 2 ) in M. hyorhinis infected pigs of High (solid bars) and Low (hatched bars) immune response lines. Titer is significantly (p ⁇ O.001) increased on D3 in High line pigs only and in pigs of both lines at subsequent times. High line pigs have significantly (p ⁇ 0.05) higher titers than Low line pigs on days 3, 7, 10 and 14.
  • FIG. 8 Postmortem lesion scores in M. hyorhinis infected pigs of High (solid bars) and Low (hatched bars) immune response lines. Lines differ in scores of peritonitis (p ⁇ O.008), pleuritis (p ⁇ o.001) and arthritis p ⁇ 0.002) .
  • Figure 9 Antibody responses of High-Low line pigs after vaccination with commercial bacterium against Actinobacillus pleuropneumonia .
  • the ranking procedure of this invention has been applied to pigs; however, it will become apparent that the basis of the ranking procedure may be applied to other types of livestock.
  • the methodology ha ⁇ been applied to the development of a high line of pigs which exhibit increased production, better health where the desirable traits are passed on from one generation to the next.
  • Such increase in productivity and health can result in, for example, one extra pig per litter, moving the pigs to market weight in less time, for example by as much as ten days without reduction in quality of the meat and that such animals respond in a superior way to various vaccination treatments.
  • the procedure, according to this invention, which provides an Estimated Breeding Value is very useful for ranking amongst breeding animals their immune system responsiveness.
  • the Estimated Breeding Value is an indicator of the animal's responsiveness in resisting disease. The greater the animal's ability, that is level of ability to resist disease, then the higher the EBV. Furthermore the EBV indicator is based on the ability of the animal to pass such disease resistance to offspring. Hence the higher the ability of the animal to pass on this disease resistance correspondingly the higher the EBV for that animal.
  • animals may be selected for breeding in order to produce offspring which inherit the higher or lower level of ability to resist disease.
  • the ranking procedure is important in respect of development of strains of animals that are disease resistant, it is also an aspect of the invention, however, to develop at the same time a line of animals which has a very low resistance to disease. Such low animals can be very useful in drug screening programs and other tests to determine efficacies of new drugs, vaccines and the like.
  • the ranking procedure is based on testing the animal's response to several, perhaps related or unrelated traits, which may or may not relate directly to determining immune system responsiveness. These traits are selected from heritable humoral immunity traits and heritable cell-mediated immunity traits and perhaps one or more other traits which are inheritable, such as stress resistance.
  • the heritable humoral immunity traits may be tested by at least two tests. One of the tests is usually a general measure of the immune response and the other test is usually an antigen-specific immune response. Similarly with the heritable cell-mediated immunity trait, this can be tested by at least two tests which are correspondingly directed to either the general or antigen-specific types of tests. These tests are conducted as soon as possible on the animal after it has been weaned form its mother and at a time chosen to negate any effects of passive immunity; thereby ensuring the least amount of interference in respect of getting true values for the ⁇ e test results.
  • the humoral immunity tests more specifically may be selected from tests, such as:
  • the specific test may be an antibody response to an antigen which is not expected to be part of the antigens to which the animal and his parents have previously been exposed.
  • these tests may be: a) peripheral blood lymphocyte blastogenic response to a mitogen not common to the animal and its parents, or b) a cutaneous delayed type hypersensitivity (DTH) to an antigen purified protein derivative of a bacterium previou ⁇ ly used to induce DTH.
  • DTH cutaneous delayed type hypersensitivity
  • Specific examples for the antigen may be hen eggwhite lysozyme. More particularly in respect of the peripheral blood blastogenic response test, the antigen may be concanavalin A.
  • the antigen used to induce DTH may be Bacillus Calmette-Guerin (BCG) or purified protein derived in a human strain of Mycobacterium tuberculosis grown on a protein free synthetic medium.
  • BCG Bacillus Calmette-Guerin
  • the indicator divides all ranked animals into these indicated group ⁇ of high, low or control.
  • the ranking therefore provide ⁇ the ba ⁇ i ⁇ for breeding together only te ⁇ t animals from the same group; that i ⁇ only animal ⁇ ranked high are bred with other animal ⁇ ranked high and corre ⁇ pondingly with re ⁇ pect to the ranked low or control animal ⁇ .
  • the ranking procedure of this invention By virtue of the ranking procedure of this invention, several advantages flow therefrom. particularly in respect of this application to the breeding of pigs, cattle, chickens and fish.
  • the ranking procedure when used in selecting breeding animals from the high group provide offspring which achieve market weight consistently faster than offspring bred from control groups or bred from low groups.
  • the offspring also have a higher percentage of live piglets per litter, such as when applied to pigs, also a lower number of litters with less than three piglets, a lower percentage of deformed piglet ⁇ per litter and a higher production index.
  • the animal ⁇ ranked in the high and low group ⁇ differ in disease manifestations induced by infection.
  • the animals ranked in the high and low groups differ in re ⁇ pon ⁇ e to vaccination ⁇ uch that the animals ranked in the high groups respond earlier, produce more antibody against antigens and have a higher percentage of animals that respond to vaccination.
  • the animals ranked in the high and low group ⁇ differ in re ⁇ pon ⁇ e to immunization, ⁇ uch that animal ⁇ ranked in the high group produce more antibody to antigen ⁇ in addition to tho ⁇ e u ⁇ ed to derive the original EBV ⁇ .
  • Thi ⁇ aspect of the invention will be de ⁇ cribed in more detail in re ⁇ pect of the following Table ⁇ .
  • the animal ⁇ ranked in accordance with the procedure ⁇ of thi ⁇ invention into the high and low group ⁇ differ in re ⁇ pon ⁇ e to immunization, ⁇ uch that animal ⁇ ranked in the high group produce antibody of higher binding strengths (avidity) for the antigen administered. This indicates overall a superior immune respon ⁇ e by the animals in the high group.
  • approximately 120 first generation piglets (Gl) were evaluated in accordance with the ranking procedure.
  • heritability estimates were 0.25, 0.23, 0.08, 0.08 and zero for secondary antibody response to HEWL, blastogenic respon ⁇ e to Con A, cutaneou ⁇ DTH to BCG/PPD, ⁇ erum IgG, and monocyte function, re ⁇ pectively.
  • Lea ⁇ t squares means reflected the ⁇ e estimates in that there were significant (p ⁇ 0.05) differences between High and Low line G, pigs in antibody, blastogenic, and DTH responses. However, there were no significant line differences in serum IgG or uptake and killing of S . typhimurium . Response to selection was determined both by differences in lea ⁇ t square ⁇ means and differences in average EBV between the High and Low line ⁇ .
  • the initial ⁇ creening involved the evaluation of 65 female and 33 male piglets, beginning at approximately 60 days of age.
  • One male and 2 female piglets were sampled from each of 34 litters by 15 sires.
  • Each piglet was evaluated for total serum immunoglobulin G (IgG) and M (I&M) , serum antibody respon ⁇ e to Hen Egg White Ly ⁇ ozyme (HEWL) , a ⁇ ynthetic peptide (TGAL) , and sheep erythrocytes (SRBC) .
  • IgG serum immunoglobulin G
  • I&M serum antibody respon ⁇ e to Hen Egg White Ly ⁇ ozyme
  • TGAL a ⁇ ynthetic peptide
  • SRBC sheep erythrocytes
  • DTH delayed type hyper ⁇ en ⁇ itivity
  • PPD purified protein derivative
  • BCG Bacillus Calmette Guerin
  • DNCB dinitrochlorobenzene
  • CHso Serum hemolytic complement activity
  • PBM peripheral blood monocytes
  • CMI cell mediated immunity
  • pig ⁇ received 0.1 ml (id) of 250 test units of PPD (Connaught, Willowdale, Ontario) and topically 0.2 ml of 5% w/v DNCB in 4:1 acetone and olive oil.
  • Negative control site ⁇ received 0.1 ml of PBS or 0.2 ml of 4:1 acetone and olive oil respectively. Twenty-four hours later cutaneou ⁇ responses were measured by double skin fold thickness.
  • HEWL ICN Biochemicals, Montreal, Quebec
  • TGAL ICN Immunologicals, Lisle, Illinois
  • Plate ⁇ were wa ⁇ hed (EL 403 microplate autowa ⁇ her, Bio-tek In ⁇ truments, Guelph, Ontario) 3 times with 250 ul/well of wash buffer (0.05% Tween-20 in PBS, pH 7.4) then blocked with 200 ul/well of 3% Tween-20 and incubated at room temperature (rt) for 1 hour. Plates were again washed 3 time ⁇ with the wa ⁇ h buffer and ⁇ amples added.
  • a control was provided which included the above reagent ⁇ without the addition of the samples.
  • Antibody responses to SRBCs were determined by a haemagglutination as ⁇ ay described previously (Mallard et al. 1989a) and serum IgG and Igm concentrations by single radial immunodiffusion (Mallard et al. 1989b) .
  • Lymphocyte blastogenesis was evaluated on day 21 of the immunization schedule by measuring lymphocyte proliferative response to the T-cell mitogen Con A and the antigen PPD.
  • Blood was collected from each pig and Ficoll-Hypaque separated peripheral blood lymphocytes (PBLS) were suspended in culture medium (RPMI 1640 plus 20% FCS) to a concentration of 5 x 106 cells/ml.
  • PBLS peripheral blood lymphocytes
  • Delayed Type Hyper ⁇ en ⁇ itivity (DTH) to PPD and DNCB were determined 24 and 48 hour ⁇ after challenge by calculating the increase in double skin fold thickne ⁇ s using the method described previously (Mallard et al. 1989a) .
  • CH J Q Total serum hemolytic complement activity was determined using day 0 (pre-immunization) and day 30 (post-immunization) samples. Blood was collected, held on ice and sera were harvested and frozen (-700°C) until time of analyses. Sera were then analyzed according to the method described previously (Mallard et al. 1989c) . To as ⁇ e ⁇ the ability of peripheral blood monocyte ⁇ to take up and kill S.
  • tvph;»mt ⁇ m r mononuclear cell ⁇ were ⁇ eparated u ⁇ ing a Ficol-Hypaque den ⁇ ity gradient (Specific gravity 1.077, 335 millio ⁇ mole ⁇ ) and resuspended to 5 x 106 cells /ml in culture medium.
  • Ten ml aliquots of suspended cells were dispensed into tis ⁇ ue culture flasks (Nunc, Gibco, Grand Island, NY) and incubated (18 hrs, 37°C) .
  • Medium and nonadherent cell ⁇ were decanted and adherent cells removed by adding 40°C PBS (Bendixen, 1981) .
  • Adherent leukocytes were resuspended at 1 X 106 cells/ml in culture medium and uptake and killing of bacteria determined by a method de ⁇ cribed previou ⁇ ly (Lacey et al. 1989) which is based on the reduction of 3 (4, 5-dimethylthiazoyl-2-yl) 2, 5 diphenyltetrazolium bromide (MTT, Pharmacia, Dorval, Quebec) to a purple formazan by bacterial dehydrogenase ⁇ .
  • Y iju + 9; + Sj + 1 ⁇ + ⁇ yu
  • Y ijkl is a normal rank score on an immune respon ⁇ e measure on the ijkl* pig
  • ⁇ is the population mean
  • g* is the fixed effect of the i* sex of pig (male vs female)
  • i ⁇ the random effect of the ijkl* pig - (0, I ⁇ 2 e ) .
  • the objective was to include one specific and one general indicator of both antibody and cellular immunity and one indicator of innate resistance.
  • the five traits were serum IgG, antibody response to HEWL, blastogenic response to Con A, cutaneous DTH to BCG/PPD and uptake and killing of S . typhimurium .
  • a total EBV score on the five traits was combined in an index, as well these five traits can be considered independently.
  • the top, intermediate and bottom ranking 7 young boars were chosen as foundation breeding stock (G Reid) to be sires of High (H) , Control (C) and Low (L) line pigs respectively. Although 7 boars were chosen for each line, only 5 were actually used for breeding with 2 held in reserve in case of reproductive problem ⁇ . similarly, the top, intermediate and bottom ranking gilts were ⁇ elected and mated to H, C and L line boar ⁇ re ⁇ pectively. There were 23, 21 and 19 H, C and L gilt ⁇ . From each litter from the ⁇ e matings, 2 females and 1 male first generation (G,) piglets were randomly sampled and evaluated according to the same immunization schedule a ⁇ the parent ⁇ .
  • a ⁇ is the additive genetic (breeding) value of the ijklm* pig (0, A ⁇ 2 and e ⁇ is the random environmental effect on the ijklm* pig (0,l ⁇ 2 e ).
  • Production data from pigs was also analyzed for high and low immune respon ⁇ e with re ⁇ pect to ⁇ everal generations.
  • back fat was measured.
  • Table 11 the high line of pigs achieved market weight in approximately 10 days less than the control line with les ⁇ back fat to indicate a healthier, more productive line of pig ⁇ with higher quality meat.
  • Thi ⁇ Table presents the rate of weight gain on the basis of days required to reach 90K and backfat thickness in millimeters as determined using an ultrasonic probe of Guelph Yorkshire pigs selected for high and low immune responsivenes ⁇ . The generation of ⁇ election is given down the left margin as Gl, G2 or G3.
  • Arithmetic means and the standard deviations of innate and immune respon ⁇ e trait ⁇ measured in boars and gilts selected as parents to produce High and Low breeding lines are presented in Table 1. These provide baseline data on the traits measured.
  • Serum IgG tended to be negatively correlated with antibody responses to both HEWL and TGAL (Table 2) , and therefore inclusion in the selection index ⁇ hould prevent IgG concentrations from significantly declining.
  • the heritability of porcine serum IgG concentration was estimated at 0.15 in G instinct (Table 2).
  • Cutaneous DTH to PPD and DNCB tended to be negatively but not significantly associated. In the parental generation these traits had heritability estimate ⁇ of 0.27 and 0.17 respectively (Table 2).
  • the DTH respon ⁇ e to PPD was included in the index as one indicator of CMI because it had the higher heritability, and the re ⁇ pon ⁇ e to DNCB wa ⁇ positively correlated with serum IgG which wa ⁇ already marked for inclu ⁇ ion in the index.
  • the lymphocyte proliferative respon ⁇ e ⁇ to PPD and Con A tended to be positively correlated, as were the proliferative and DTH responses to PPD (Table 3) .
  • the heritability estimates of the blastogenic response ⁇ to PPD and Con A in G 0 were 0.15 and 0.37 re ⁇ pectively. For the ⁇ e reasons blastogenic response to the mitogen Con A was chosen as the other indicator of cellular responsivenes ⁇ .
  • Monocyte function mea ⁇ ured a ⁇ anti-bacterial capacity, wa ⁇ positively correlated with hemolytic complement activity (day 0) , but negatively correlated with serum IgM (Table 3) .
  • the heritability of this trait was 0.18 in G 0 and because of the importance of the monocyte in both antibody and cellular immunity it was included as a parameter in the selection index.
  • EBV ⁇ calculated on the five trait ⁇ for each pig within a line, and additively combined EBV, are given in Figure ⁇ 1 and 2.
  • Mean total EBVs for the High, Control and Low lines were 0.66 ⁇ .36, -0.04 ⁇ .19, and -0.55 ⁇ .31 respectively.
  • the respon ⁇ e to selection wa ⁇ determined u ⁇ ing both the lea ⁇ t squares and animal model.
  • the differences between H and L lines are reported in terms of original and standardized units (Table 7) .
  • the results showed that after one generation of ⁇ election the H and L line ⁇ were ⁇ eparated by 1.205 as measured by EBVS, and 1.528 a ⁇ mea ⁇ ured by lea ⁇ t squares which i ⁇ a little more than half a standard deviation of the index (Table 7) .
  • the largest line differences occurred in antibody respon ⁇ e to HEWL and bla ⁇ togenic response to Con A.
  • the multi-trait selection based on predictors of immune and innate resistance demonstrate ⁇ that immunity in mammals i ⁇ regulated not by one, but a complex network of factor ⁇ .
  • EBV ⁇ for each trait were calculated u ⁇ ing an animal model that makes use of all known relationships among animals, and pigs were ranked based on combined EBVs and as ⁇ igned to High, Low or Control breeding groups. Approximately 40 first generation piglets from each line were then similarly evaluated.
  • H and L line ⁇ were ⁇ eparated by 1.205 unit ⁇ as measured by EBVs and 1.508 as measured by least squares EBVs (Table 7).
  • Heritability estimate ⁇ were 0.25, 0.23, 0.08, 0.08 and zero for antibody response to HEWL, lymphocyte ⁇ timulation by Con A, DTH response to BCG/PPD, serum IgG, and monocyte function respectively (Table 5) .
  • pigs and other types of livestock can be separated into high and low breeding lines using aggregate EBV ⁇ .
  • the methodology of thi ⁇ invention provide ⁇ a reproducible technique in determining the high line of pig ⁇ or other livestocks for breeding purposes to increase the productivity and improve general health.
  • this relation ⁇ hip i ⁇ indicated where the high line of pig ⁇ achieves a 90 kilogram weight in first, second and third generations consi ⁇ tently fa ⁇ ter than the control and low group ⁇ .
  • Antibody avidity can also be investigated in the high and low response groups of animal ⁇ as ranked by this invention.
  • Antibody avidity is a measure of the attraction ⁇ of an antibody for an antigen; i.e., the quality of the antibody. It i ⁇ possible to measure this value and has been done for a ranked group of pigs as determined by the procedure of Embodiment #1.
  • Avidity indice ⁇ of antibody to hen eggwhite ly ⁇ ozyme were mea ⁇ ured by chaotropic ion (SCN) elution enzyme-linked immuno ⁇ orbent a ⁇ say (ELISA) in pigs grouped as high control of low for various immune and innate resi ⁇ tance-related trait ⁇ .
  • the avidity index wa ⁇ the molar concentration of SCN " required to reduce by 50% the ELISA optical density value for a given serum. The index was independent of the amount of antibody.
  • Mean avidity indice ⁇ for ⁇ era collected on days 14 and 30 were 0.61 ⁇ 0.43 and 1.22 ⁇ 0.546, with maximum indices of 2.64 and 2.86 respectively.
  • Avidity index wa ⁇ po ⁇ itively correlated with antibody to HEWL on days 14 and 30, but not to preimmunization ⁇ erum IgG concentration or to other mea ⁇ ured trait ⁇ .
  • Animal ⁇ genetically selected as per Embodiment #1 to express high or low immune respon ⁇ e or innate resistance- related trait ⁇ are expected to differ in re ⁇ pon ⁇ e to infection and in development of di ⁇ ease.
  • Pigs were te ⁇ ted a ⁇ ⁇ elected for high (H) or low (L) expre ⁇ ion of ⁇ erum IgG, ⁇ erum antibody to hen egg white ly ⁇ ozyme (HEWL) , peripheral blood lymphocyte blastogene ⁇ i ⁇ after in vitro stimulation with concanavalin A and cutaneous delayed- type hypersensitivity to PPD after sen ⁇ itization with BCG.
  • H and L pig ⁇ of generation G4 which differed ⁇ ignificantly in traits used in selection, were infected with Mycoplasma hyorhinis , in accordance with the following Embodiment #2, by intraperitoneal injection and compared u ⁇ ing a ⁇ plit litter de ⁇ ign on the ba ⁇ i ⁇ of antibody respon ⁇ e, nonspecific indicators of infection and disea ⁇ e ⁇ ign ⁇ , both antemortem and postmortem.
  • the study period began on day -1 and continued for 14 days after infection (day 1) .
  • H line pigs produced M.hyor inis-specific serum antibodies earlier (day 3) (p ⁇ O.OOl) and to higher titer (p ⁇ 0.05).
  • Uninfected H line pigs gained more weight between days -1 and 14 (p ⁇ O.OOl) but within the infected group weight loss was equivalent for pigs of both line ⁇ .
  • H had more blood lymphocytes than L in the absence of challenge (p ⁇ 0.05). Both groups had reduced blood lymphocyte numbers (p ⁇ 0.05) by day 7 with H reverting to prechallenge values by day 10 before L (p ⁇ 0.005).
  • pigs have been genetically selected for high and low response using an index that combined estimated breeding values for ⁇ erum IgG concentration, antibody re ⁇ pon ⁇ e to hen egg white lysozyme, in vitro blastogenesis of peripheral blood lymphocytes stimulated with the mitogen con A and delayed type hypersen ⁇ itivity induced by intradermal injection of tuberculin PPD after ⁇ ensitization with BCG.
  • these pigs differed ⁇ ignificantly for the trait ⁇ incorporated in the ⁇ election index and for certain other trait ⁇ ⁇ uch a ⁇ antibody re ⁇ pon ⁇ e to unrelated antigen ⁇ , antibody avidity and weight gain all of which favor the high re ⁇ pon ⁇ e line.
  • pigs of the high and low response lines were experimentally infected with Mycoplasma hyorhinis (M. hyorhinis) and their respon ⁇ e to infection wa ⁇ a ⁇ ses ⁇ ed a ⁇ de ⁇ cribed in the following Embodiment #2.
  • the challenge strain of M. hyorhinis (497- 14) was originally isolated from joints of a naturally infected pig.
  • the mycopla ⁇ ma ⁇ were cultured in modified Hayflick's broth (Erno, H. et al. 1973), washed by centrifugation, re ⁇ uspended in PBS and stored at -70°c.
  • Pigs in the challenged group received a ⁇ ingle i.p. injection of 2X10 9 M. hyorhinis in 2 ml PBS.
  • the non- challenged pig ⁇ received PBS only.
  • the model used for analyzing the respon ⁇ e during the course of the challenge was: ⁇ *i m -u + DAYj + TREATMENT + LINE k + SET, + (TREATMENTxLINE) jk + (DAYxTREATMENTxLINE) ijk + LITTER(LINExSET) ⁇ + ANIMAL(LITTER LINExSET TREATMENT ) jaam + ERROR. jkt a where
  • Yj jkt ai an observed value for a trait measuring the response to challenge;
  • u population mean for the trait
  • DAY, a fixed effect due to day of observation
  • TREATMENT j a fixed effect due to treatment regime
  • ANIMAL LITTER LINExSET TREATMENT
  • jktam a random effect due to pig nested within litter, line, experimental set and treatment regime
  • ERROR ⁇ - a a random residual error term.
  • the animal mean square was u ⁇ ed a ⁇ the denominator to test the effects of treatment, treatment x line interaction and litter on the various traits.
  • the litter mean square was used as the denominator to test the effects of breeding line and set.
  • the re ⁇ idual mean square wa ⁇ u ⁇ ed a ⁇ the denominator to test the remaining effect ⁇ .
  • Non-challenged pig ⁇ of line H gained ⁇ ignificantly (p ⁇ O.01) more weight than those of line L from day -1 to day 14 ( Figure 4) .
  • Weight loss in challenged pigs wa ⁇ approximately 1 kg and did not differ by line ( Figure 4) .
  • two pig ⁇ of line H and two of line L were euthanized on day 10 due to severe disease.
  • the principal clinical sign of disea ⁇ e wa ⁇ arthriti ⁇ which wa ⁇ ob ⁇ erved earlier and with greater ⁇ everity in H than in L line pig ⁇ .
  • the ESR. increased significantly (p ⁇ O.OOl) from day - 1 and 0 to day 3 in pigs of the L line and to day 7 in H line pig ⁇ ( Figure 6a) .
  • the more rapid rate of increase in ESR in pigs of the low line resulted in a significant (p ⁇ O.Ol) difference between the lines on day 3.
  • the ESR remained elevated in pigs of both lines to the end of the ⁇ tudy.
  • the number of circulating lymphocytes decreased (p ⁇ 0.05) from day 3 to day 7 in pig ⁇ of each line. In those of the H line, lymphocytes reverted earlier to pre- challenge numbers than in L line pigs and there were significantly (p ⁇ 0.05) more blood lymphocytes in H than in L line pigs on day 10. In the non-challenged group there were at all times more (p ⁇ 0.05) blood lymphocytes in H than in L line pigs.
  • H line pig ⁇ produced mycopla ⁇ ma- ⁇ pecific antibody earlier and to higher titers than did the relatively su ⁇ ceptible L line pig ⁇ .
  • In ⁇ ofar a ⁇ the H line developed more severe arthritis than pig ⁇ of the L line in re ⁇ pon ⁇ e to infection with a single pathogen; however, this development may be overcome and may not be a significant result.
  • the two lines differ in a number of traits including tho ⁇ e incorporated in the selection index and correlated traits (Mallard, B.A. et al. 1992), such as antibody production following immunization with other antigens, lytic complement activity and antibody avidity (Appleyard, G.B. et al. 1992), which may have influenced both resi ⁇ tance and development of disease, including arthritis.
  • Antibody avidity is of interest since high avidity antibody has been shown to be mo ⁇ t efficacious in mediating protection in virus infections (Mulchany G. et al. 1992 and Salmi, A.A. 1991) and antibody-dependent disea ⁇ e such as allergic encephaliti ⁇ (Devey, M.E. et al.
  • Pathogene ⁇ is of mycoplas a-associated diseases such as arthriti ⁇ and uveiti ⁇ may involve formation of inflammation-inducing antibody-antigen complexe ⁇ (Thirkill, C.E. et al. 1992).
  • the relative ability of the H and L line pig ⁇ used here to produce toxic immune complexes is not known but favors the H line. Since cutaneous delayed-type hypersensitivity to PPD of tuberculin was significantly higher in the H line pigs they may have a generally higher ability to produce inflammation based upon antigen-specific cell-mediated immune respon ⁇ e which could have re ⁇ ulted in the more ⁇ evere arthriti ⁇ ob ⁇ erved in the H line animal ⁇ .
  • TGAL and sheep erythrocytes a small synthetic peptide known as TGAL and sheep erythrocytes (SRBC)
  • SRBC sheep erythrocytes
  • DNCB topical antigen dinitrochlorobenzene
  • CH50 ⁇ erum haemolytic complement activity
  • Figure 9 depicts least square mean values (i.e, means corrected for unequal sample size, litter effects, and sire effects) of antibody response ⁇ of Guelph High Low line pig ⁇ before (day 0) and after (day ⁇ 14 and 21) vaccination with a commercial bacterium again ⁇ t Actinobacillus pleuropneumonia .
  • Actinobacillus pleuropneumonia i ⁇ a bacteria which causes acute and chronic pneumonia in pigs and presently costs the Canadian pork producers about $4 million annually.
  • Antibody responses to this vaccine were measured using an Enzyme Immunoas ⁇ ay (ELISA) and units of respon ⁇ e are given on the y-axi ⁇ a ⁇ optical den ⁇ ity (OD) of the te ⁇ t sera at a predetermined optimal dilution of 1 ⁇ 800.
  • the different letters above the bars of the graph indicate that the antibody respon ⁇ e ⁇ are ⁇ ignificantly different as determined using a stati ⁇ tical t-te ⁇ t and are reported at a 95% confidence level; i.e, p ⁇ 0.05.
  • the nonresponder status reported at the right side of the graph indicates the percentage of pig ⁇ from each line (High, Low and Control pigs of Generation 4) which did not respond in any measurable way to this te ⁇ t vaccine.
  • the breeding selection i ⁇ predictably applicable to other animal ⁇ ⁇ uch a ⁇ cattle, sheep, chickens, fish, horses and other valuable livestock because all of these animals have similar respon ⁇ e to the trait ⁇ u ⁇ ed in developing EDV ⁇ for ranking the animals for further breeding.
  • Animals so developed can reduce husbandry costs through reduced requirements for health-related inputs such as antibiotics and vaccines while enhancing product wholesomene ⁇ by reduced u ⁇ e of extraneous materials .
  • Such superior animals al ⁇ o have an apparent role in pathogenetic studies and in vaccine development and efficacy trials in which the L line animal ⁇ may simulate problematic low responder individual ⁇ in outbred populations.
  • the selection procedure results in an animal model on which drug screening and the like may be conducted.
  • Monocyte function is based on the ability of peripheral blood monocytes to take up and kill S. typhimurium at 30 and 90 minutes respectively.
  • Least squares means and their standard errors in lines (G 0 & G,) of Yorkshire Pigs Selected for high (H), low (L) and control (C) immune and innate resistance.
  • Monocyte function is based on the ability of PBMs to take up and kill S. typhimurium at 30 and 90 minutes respectively. b Probability of significant differences between High and Low lines within a generation.
  • Monocyte function is based on the ability of PBMs to take up and kill S. typhimurium at 30 and 90 minutes respectively.
  • Standardized measures are based on standard deviations of one for ease of comparison between immune response parameters.
  • ITI rri * High differs from Low, p ⁇ 0.05

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Abstract

A procedure for ranking immune system responsiveness in an animal to provide an estimated breeding value (EBV) indicator of the animal's level of ability to resist disease and ability to pass such disease resistance to offspring is provided. The EBV indicator is useful in selecting animals to be bred in order to produce offspring which inherit the level of ability to resist disease. The procedure comprises: 1) testing an animal's response to at least two tests, one of which is a general measure and the other antigen specific which determines heritable humoral immunity traits; 2) testing the same animal's response to at least two tests one of which is a general measure and the other antigen specific which determine heritable cell-mediated immunity traits; 3) testing the animal's response to the two tests of humoral immunity traits and the two tests of cell-mediated immunity traits beginning as soon as possible after the animal has been weaned from its mother and at a time chosen to negate effects of passive immunity; and 4) ranking the animal's EBV indicator relative to other ranked animals based on the animal's level of response to the tests. This procedure is useful in selecting or grouping animals into high and low groups as well as control groups. The procedure is particularly useful in developing superior strains of pigs, cattle, fish, chickens and other like livestock.

Description

METHODOLOGY FOR DEVELOPING λ 8UPERIOR LINE OF DOMESTICATED ANIMALS
FIELD OF THE INVENTION
This invention relates to a methodology for developing a superior line of domesticated animals, particularly animals which yield food value and more particularly a methodology which provides an animal line of increase productivity and generally healthier through to maturation. BACKGROUND OF THE INVENTION
Considerable efforts have been expended in attempting to develop, by way of genetic selection, animal strains which are of a more robust variety and generally yield higher quality meats for food purposes. The following is a list of several references in which investigations have been made in this regard, as well as corresponding methods and diagnosis used in determining various genetic traits.
Appleyard,G. ,B.N.Wil ie,B.W.Kennedy, and B.A.Mallard. 1992. Antibody avidity in Yorkshire pigs of high and low immune response groups. Vet . Immunol , and Immunopathol . 31: 229-240 Archer,R.K., and L.B.Jeffcott. 1977. Comparative Clinical Hae atlogy. Blackwell, Oxford. Bendixen, P.H. 1981. Reversible detachment of blood-derived bovine macrophages by replacement of culture medium with phosphate buffered saline solution. Am J Vet Res 42:687-688.
Berche, P.A. 1985. Resistance to listeriosis in two lines of mice genetically selected for high and low antibody production. Immunology 56:707-716. Biozzi, G., Mouton, D., Sant'Anna, O.A. , Passos, N.C., Gennari, M. , Reis, M.H. , Ferreira, V,C.A., Neumann, A.M., Bouthillier, Y. , lbanez, O.M., Stiffel, C, and Siqueira, M. 1979. Genetics of immunoresporisiveness to natural antigens in the mouse. Curr Top Microbiol Immunol 85:31-98 . Biozzi, G. , Mouton, D. , Stiffel, C. , and Bouthillier, Y. 1984. A major role of the macrophage in quantitative genetic regulation and immunoresponsiveness and antiinfectious immunity. Adv Immunol 36:189-233.
Biozzi G., D.Mouton, C.Stiffel, and Y.Bouthiller. 1984. A major role of the macrophage in quantitative genetic regulation of immunoresponsiveness and antiinfectious immunity. Advances in Immunology 36: 189-234.
Biozzi, G., Mouton, D., Siqueira, M. , and Stiffel, C. 1985. Effect of genetic modification of immune responsiveness on anti-infection and anti-tumor resistance. In Genetic Control of Host Resistance to Infection and Malignancy, pp 3-18, Alan R. Liss, Inc.
Blackwell, J. 1989. The macrophage resistance gene Lsh/Ity/Bcg. Twenty-seventh forum in Immunology 140:767-828. Burton, J.L., Kennedy, B.W. , Burnside, E.B., Wilkie, B.N. , and Burton, J.H. 1989. Variation in serum concentrations of immunoglobulins G, A, and M in Canadian Holstein-Friesian calves. J Dairy Sc! 72:135-149. Buschmann, H. , Krausslich, H. , Herrmann, H. , Meyer, J. , and Kleinschmidt, A. 1985. Quantitative immunological parameters in pigs - experiences with the evaluation of an immunocompetence profile. Tierzuchtg . Zuchtgsbiol 102:189199. Cho H.J. , H.L.Ruhnke, and E.V.Langford. 1976. The indirect hemagglutination test for detection of antibodies in cattle naturally infected with mycoplasmas. Can . J. Comp. Med. 40: 20-29. Corbeil, L.B., Watt, B. , Corbeil, R.R. , Betzen, T.G., Brownson, R.K., and Morril, J.L. 1984.
Immunoglobulin concentrations in serum and nasal secretions of calves at the onset of pneumonia. Am J Ver Res 45(4) :773-778.
Covelli, V., Mouton, D., Di Majo, V., Bouthillier, Y., Bangrazi, C, Mevel, J. , Rebessi, S., Doria, G. , and Biozzi, G. 1989. J Immunology 142:12241234.
Fumoux, F., Traore-Leroux, T., Queval, R. , Pinder, M. , and Roelants, G.E. 1985 High and low responsiveness of bovine lymphocytes to Trypanosome Jbrucei in vitro: lack of correlation with resistance to trypanosomiasis. Immunology 54:195-203.
Covelli V., D.Mouton , V.di Majo, Y.Bouthillier, C.Bangrazi, J.-C.Mevel, S.Rebessi, G.Doria, and G.Biozzi. 1989. Inheritance of immune responsiveness, life span and disease resistance in interline crosses of mice selected for high or low multispecific antibody production. J. Immunol . 142: 1224-1234.
Devey,M.E., P.J.Major, K.M.Bleasdale-Barr, G.P.Holland, M.C.Dal Canto and P.Y.Paterson. 1990. Experimental allergic encephalomyelitis (EAE) in mice selectively bred to produce high affinity (HA) or low affinity (LA) antibody responses. Immunology 69: 519-524 Edfors-Lilja,I. , M.Bergstrom, U.Gustafsson, U.Magnusson, and C.Fossum. 1991. Genetic variation in Con A-induced production of interleukin 2 by porcine peripheral blood mononuclear cells. Vet . Immunol . Immunopathol . 27: 351-363. Edfors-Lilja,I. , E.Wattrang, U.Magnusson and C.Fossum. 1993. Genetic variation in parameters reflecting immune competence of swine. Vet . Immun . and Immunopathol . In press. Erno,H., and L.Stripkovitz. 1973. Bovine mycoplasmas: cultural and biochemical studies. II Acta Vet . Scand. 14: 450-463.
Gavora, J.Si 1990. Genetic disease resistance: mechanisms and strategies for improvement. Proceedings of the 4th World Congress on Genetics Applied to Livestock Production, 427-436. Gavora, J.S. and Spencer, L. 1983. Breeding for immune responsiveness and disease resistance. Animal Blood Groups and Biochemical Genetics 14:159- 180.
Gill,H.S., G.D.Gray, D.L.Watson, and A.J.Husband. 1993. Isotype-specific antibody responses to Haemonchus contortus in genetically resistant sheep. Parasite Immunology 15: 61-67.
Groves,T.C., B.N.Wilkie, B.W.Kennedy and B.A.Mallard. 1993. The effect of selection of swine for high and low immune-responsiveness on monocyte superoxide anion production and class II MHC antigen expression. In press. Vet . Immunol , and Immunopathol .
Harmen, B.G., Templeton, J.W. , Crawford, R.P., Heck, F.C., Williams, J.D., and Adams, L.G. 1985. Macrophage function and immune response of naturally resistant and susceptible cattle to Brucella abortus . In Genetic Control of Host Resistance to
Infection and Malignancy, pp 345-354, Alan R. Liss,
Inc.
Helwig,T.T. , and K.A.Council. 1979. SAS users guide. SAS Institute, Raleigh,N.C.
Hopkins,S.J. and A.Meager. 1988. Cytokines in synovial fluid: II The presence of tumour necrosis factor and interferon. Clin . exp. Immunol . 73: 8-92. Ibanez, O.M. , Reis, M.H. , Gennari, M. , Ferreira, V.C.A., Sant'Anna, O.A., Siqueira, M. , and Biozzi, G. 1980. Selective breeding of high and low antibody responder lines of guinea pigs. Immunogenetics 10:283-293. Kennedy, B.W. 1990. Use of mixed model in analyses of designed experiments. In: Advances in statistical methods for genetic improvement of livestock. D. Gionola and K. Hammond (eds.).
Springer-Verlag , pp.77-97.
Kennedy, B.W. and Sorensen, D.A. 1988. Properties of mixed model methods for prediction of genetic merit. In: Proc. 2nd Int. Conf. Quant. Genet. B.S.
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Mallard, B.A. 1989. Genetic and other effects on bacterial phagocytosis and killing by cultural peripheral blood monocytes of SLA-defined miniature pigs. Animal Genetics 20:371-382.
LaGrange, P.H. , and Hurtrel, B. 1985. Lister ia monocytogenes infection in Biozzi mouse lines with High or low responses to phytohemagglutinin.
Cellular Immunology 96:210-222.
Lai,W.C, M.Bennett, Y.-S.Lu, and S.P.Pakes. 1991.
Vaccination of Lewis rats with temperature-sensitive mutants of Mycoplasma pulmonis . Adoptive transfer of immunity by spleen cells but not sera. Infect.
Immun. 59: 346-350.
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Applied to Livestock Production, 421-425. Lillehoj , H. S. , Ruf f , M. D. , Bacon, L. D. ,
Lamont, S. J. , and Jeffers, T. K. 1989.
Genetic control of immunity to Eimeria tenella . Vet
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Dairy Sci 66:862-866.
Mallard, B.A. , Wilkie, B.N. , and Kennedy, B.W.
1989a. Genetic and other effects on antibody and cell mediated immune response in SLA-defined miniature pigs. Animal Genetics 20:167-178. Mallard, B.A., Wilkie, B.N. , and Kennedy, B.W. 1989b. The influence of the swine major histocompatibility genes on variation in serum immunoglobulin concentration. Vet Immunol and Isopath 21:139-151.
Mallard, B.A. , Wilkie, B.N. , and Kennedy, B.W. 1989c. Influence of major histocompatibility genes on serum hemolytic complement activity in miniature swine. Am J Vet Res 50(3) :359-363. Mallard, B.A. , Wilkie, B.N., and Kennedy, B.W. 1989d. Genetic selection for improved immune response in Yorkshire pigs. Immunobiology (Supplement) Vol 4:104-105. Mallard, B.A. , Wilkie, B.N., and Kennedy, B.W. 1990. Variation in number of circulating leukocytes in lines of pigs selected for high and low immune and innate resistance mechanisms. (Abstract) Seventy- first Conference of Research Workers in Animal Diseases, Chicago, Nov. 5-6, pp 22. Mallard,B.A. , B.N.Wilkie, S.Rosendal, and S.Keay. 1991. Induction of tumour necrosis factor alpha in SLA-defined mini pigs. Abstract. Proceedings of the 72nd Conference of Research Workers in Animal Disease, Chicago, November 11-12. Mallard B.A., B.N.Wilkie, B.W.Kennedy, and
M.Quinton. 1992. Use of estimated breeding values in a selection index to breed Yorkshire pigs for high and low immune and innate resistance factors. Anim. Biotech . 3(2): 257-280. Mazengara, K.E., Kennedy, B.W. , Burnside, E.B. , Wilkie, B.N., and Burton, J.H. 1985. Genetic parameters of bovine serum immunoglobulins. J Dairy Sci 68:2309-2314. Meyer, K. 1988. DFRF-ML - a set of programs to estimate variance components under an individual animal model, J . Dairy Sci . 71 (Suppl.) 2:33-34. Morris, R.S. 1988. The effects of disease on productivity and profitability of livestock: How should it be assessed? Proc New Zealand Soc of An Prodn 48:117-125.
Morse E.E., S.Panek, and R.Menga. 1970-71. Automated fibrinogen determination. Am .J. Clin .Pathol . 55: 671- 676.
Mouton, D., Heumann, A., Bouthillier, Y., Mevel, J., and Biozzi, G. 1979. Interaction of H-2 and Non H-2 linked genes in the regulation of antibody response to a threshold dose of sheep erythrocytes. Immunogenetics 8:475486.
Mouton, D., Stiffel, C. , and Biozzi, G. 1985. Genetic factors of immunity against infection. Ann Inst Pasteur 1 Immunol 136D:131-141. Mulchany G. , E.Reid, R.D.Di archi, and T.R.Noel.
1992. Maturation of functional antibody affinity in animals immunized with synthetic foot-and-mouth disease virus. Res . in Vet . Sci . 52: 133-140 Nicholas, F.W. 1987. Veterinary Genetics, Oxford Science Publications .
Niewland, M.G.B., Kreuknief, M.B. , Hepkema, B.G., Pinard, M.H., and Van der Zijpp, A.J. 1989. Breeding for high and low antibody production in chickens: Effects on disease resistance, MHC- haplotypes and production traits. JmmunoJbiology, Supplement 4:106.
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Saklatvala,J. 1986. Tumour necrosis factor α stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature (London) 322: 547- 549.
Salmi,A.A. 1991. Antibody affinity and protection in virus infections. Current Opinion in Immunology 3: 503-506 Sant'Anna, O.A. , Ferreira, V.C.A., Reis, M.H.,
Gennari, M. , Ibanez, O.M. , Esteves, M.B., Mouton, D., and Biozzi, G. 1982. Genetic parameters of the polygenic regulation of antibody responsiveness to flagellar and somatic antigens of salmonellae. J . Immunogenetics 9 :191-205.
Schurr, E. , Morgan, K. , Skamene, E. , and Gros, P. 1989. The search for a human homologue of the mouse Beg host resistance locus. Twenty - seventh fozrum in Immunology 140:767-828. Sher,T., S.Rottem and R.Gallily. 1990. Mycoplasma capricolum membranes induce tumour necrosis factor α by a mechanism different from that of lipopolysaccharide. Cancer Immunol . Immunother. 31: 86-92. Simonsen, M. 1988. ' The MHC of the chicken, genomic structure, gene products, and resistance to oncogenic DNA and RNA viruses. In B.N. Wilkie and P.E. Shewen (eds): Veterinary Immunology, pp 243- 253, Elsevier. Skamene, E. , Kongshavn, P.L., and Landi, M. 1980. Genetic control of natural resistance to infection and malignancy. London, New York. Academic Press. Sorensen, D.A. and Kennedy, B.W. 1983. The use of the relationship matrix to account for genetic drift variance in the analyses of genetic experiments. Theor. Appl . Genet . 66:217-220. Sorensen, D.A. and Kennedy, B.W. 1986. Analyses of selection experiments using mixed model methodology. J. Anim . Sci . 63:245-258.
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Van der Zijpp, A.P. , Scott, T.R. , Glick, B. , and Kreukniet, M.B. 1988. Interference with the humoral immune response in diverse genetic lines of chickens. Vet Immunol and Immunopath 20:53-60. Vangen, O. 1979. Studies on a two trait selection experiment in pigs. Acta Agr Scand 29:305-319. Vespa, L. , and Zwilling, B.S. 1989. Expression of I-A by macrophages from Bcgr and Beg* mice. J Immunology 143:214-220. Wakelin, D., and Donachie, A.M. 1983. Genetic control of eosinophilia. Mouse strain variation in response to antigens of parasitic origin. Clinical and Experimental Immunology 51:239-246. Warner, CM., Meeker, D.L., and Rothschild, M.F. 1987. Genetic control of immune responsiveness: A review of its use as a tool for selection for disease resistance. J Animal Sci 64:394-406. Wassom, D.L., and Kelly, A.B. 1990. The role of the major histocompatibility complex in resistance to parasite infections. Critical Reviews in Immunology 10 :31-52 .
Windon, R.G. , and Dineen, J.K. 1981. The effect of selection of both sire and dam on the response of F, generation lambs to vaccination with irradiated Trichostrongylus colubriformis larvae . Int J Parasit 11 :11-18 . Windon,R.G. 1993. Selection of sheep for immunological responsiveness to the intestinal nematode Trichostrongylus colubriformis . In Workshop report: Selection experiments to alter disease resistance traits in domestic animals. In press. Vet . Immunol . and Immunopathol .
Wright, P. 1987. Enzyme Immunoassay : Observations on aspects of quality control. Vet Immunol and Immunopath 17:441-452. For ease of reference to these references in the remainder of the specification, they will be identified by the first author and the date of the reference.
A variety of techniques have been investigated to provide for genetic selection of better strains of animals. In making such selection, particular attention has been paid to disease resistance or susceptibility thereto. Normally with domestic livestock, selection has been based primarily on production rather than disease traits. There is a continued push, however, to reduce health costs in developing livestock and to minimize chemical residues in the food chain. Such outside pressure has renewed interest in the selection of beneficial strains of animals for food production. From a health standpoint, selection experiments have been based on a single trait selection and often antagonistic relationships surface between specific and non-specific indicators of such resistance, for example, antibody and macrophage function (Biozzi et al 1979; Harmon et al 1985) . Resistance to infection and disease is the outcome of complex interactions between virulence-related attributes of the pathogen and a spectrum of innate and immunological mechanisms which have a quantitative basis and are under polygenic control (Lie, 0. 1990) . Population variation in resistance to specific diseases could be exploited in. isease control if heritability of the resistance-related attributes was sufficiently high to permit selective breeding (Gavora, J.S., 1990). There is however an apparent risk in altering population resistance to selected infectious diseases since the underlying resistance mechanisms may represent only some of those needed to enhance overall population resistance to common pathogens. Cattle remaining healthy when naturally infected with the intracellular pathogen Brucella abortus , resistance to which is correlated with effective mononuclear phagocytic function, may be relatively susceptible to other infectious diseases (Templeton, J.W. et al. 1990) .
Genetic and phenotypic correlations of this nature are not unusual in mammals and have been described in a number of species. For instance, in a study compiling immune profiles on a variety of German pig breeds a negative association was reported between per cent peripheral blood monocytes and lymphocytes (Buschmann et al. 1985) . A similar correlation was seen in the Guelph pigs (r = -0.35, p < 0.002) (Mallard et al. 1990). Positive associations between lymphocyte blastogenesis using PHA, Con A, LPS and PWM have also been reported in pigs (Buschmann et al. 1985) . No association however was apparent between antibody response to tetanus toxoid or ovalbumin and blastogenic response to PHA, Con A or PWM, but a positive correlation existed between antibody response and lymphocyte stimulation using LPS (Buschmann et al. 1985). In lambs selected for responsiveness to T. colubriformis there was again an indication of an inverse relationship between B and T-cell mitogen response. In this case the B-cell response (LPS stimulation) was greater in non-responder lambs, whereas the T-cell response (Con A stimulation) was greater in the responder progeny (Windon and Dineen, 1981) . Conversely, in cattle selected for trypanoresistance or trypanosensitivity no correlation was noted between the level of resistance and lymphocyte stimulation by trypanoso es (Fumoux et al. 1985) . In the Biozzi High and Low line mice selected for antibody response to SRBC (Selection I) , and in cattle determined as resistance or susceptibility to B . abortus , an antagonistic relationship existed between antibody responsiveness and various indicators of macrophage activity (Biozzi et al. 1979, Harmen et al. 1985).
However, in chickens selected over 8 generations for high and low antibody response to SRBCS, innate macrophage activity appeared to be unaffected (van der Zijpp et al. 1988) . It becomes obvious that selection for one trait almost always produces alterations in other parameters, but these associations are often species and breed dependent. At present it is therefore difficult to generalize as to the nature and direction of these relationships. In accordance with this invention, a methodology has been developed for multi-trait selection for several indicators of disease resistance which have proven to yield a superior line of livestock. This methodology has particular application with pigs, but as well cattle, chickens and other valuable livestock.
In accordance with an aspect of this invention, a procedure is provided for ranking immune system responsiveness in an animal to provide an Estimated Breeding Value (EBV) indicator of the animal's level of ability to resist disease and ability to pass such disease resistance to offspring, such EBV indicator being useful in selecting animals to be bred in order to produce offspring which inherit said level of ability to resist disease. The procedure comprises: i) testing an animal's response to at least two tests one of which is a general measure and the other antigen specific which determine heritable humoral immunity traits; ii) testing the same animal's response to at least two tests one of which is a general measure and the other antigen specific which determine heritable cell-mediated immunity traits; iii) testing the animal's response to the two tests of humoral immunity traits and two tests of cell- mediated immunity traits beginning as soon as possible after the animal has been weaned from its mother and at a time chosen to negate effects of passive immunity; iv) ranking the animal's EBV indicator relative to other ranked animals based on the animal's level of response to the tests. According to another aspect of the invention, five traits may be selected on which diagnosis is based are:
1. a measure of antibody - serum IgG;
2. antibody response to hen eggwhite lysozyme (HEWL) ; 3. cellular activity - blastogenetic response to concanavalinA (ConA) ;
4. cellular activity - cutanaceous delay type hypersensitivity (DHT) to Bacillus Calmette Guerin (BCG)/purified protein derivative (PPD) ; and
5. indicator of ainnate monocyte function - uptake in killing of S. typhimurium .
In accordance with this invention, diagnosis based on these selected traits provides an estimated breeding value (EBV) which determines the superior line of pigs for providing enhanced productivity and of improved health.
In accordance with the more specific aspect of the invention based on th«M selected traits, the combined EBV can be calculated for each animal, such as pigs, where each pig is assigned to a high, low or controlled breeding group.
The methodology, according to this invention, may be applied to various livestock to yield a high quality strain of livestock which is disease resistant. It has been found that the traits evaluated in accordance with the above methodology is preserved in generation to generation where, as needed, further EBVs may be calculated in the third and fourth generations to further direct the continued production of a high quality animal or a low quality animal. Various aspects of the invention are described with respect to the drawings wherein:
Figure 1 is a bar chart showing the EBVs for a first generation of Yorkshire pigs;
Figure 2 is the combined EBV for first generation Yorkshire pigs; and
Figure 3 is a rate of gain in pigs selected for high-low immune responses.
Figure 4 Mean change in weight of M. hyorhinis challenged and control (Placebo) pigs of High (solid bars) and Low (hatched bars) immune response lines of pigs. Weight gain in control pigs is significantly (p < 0.01) greater in High than in Low line animals. Weight loss is equivalent for animals of each line after infection. Figure 5 Mean clinical scores of arthritis in
M. hyorhinis infected pigs of High (solid bars) and Low (hatched bars) immune response lines. Difference between lines is significant (D7 p≤O.001, D10 p<0.05, D14 p<0.05) . Figure 6 Erythrocyte sedimentation rate (ESR,
Figure 6a) and plasma fibrinogen concentration (Figure 6b) in M. hyorhinis infected pigs of High (solid bars) and Low (hatched bars) immune response lines. ESR is significantly (p<0.01) higher in Low than in High line pigs on D3 and elevated in both lines on days 7, 10 and 14. Fibrinogen is increased in pigs of both lines from D3 to D14 (p≤O.001) and on D10 the concentration in the Low line exceeds that of the High line (p<0.05).
Figure 7 Serum antibody (passive haemagglutination) titer (log2) in M. hyorhinis infected pigs of High (solid bars) and Low (hatched bars) immune response lines. Titer is significantly (p≤O.001) increased on D3 in High line pigs only and in pigs of both lines at subsequent times. High line pigs have significantly (p<0.05) higher titers than Low line pigs on days 3, 7, 10 and 14.
Figure 8 Postmortem lesion scores in M. hyorhinis infected pigs of High (solid bars) and Low (hatched bars) immune response lines. Lines differ in scores of peritonitis (p≤O.008), pleuritis (p≤o.001) and arthritis p<0.002) .
Figure 9 Antibody responses of High-Low line pigs after vaccination with commercial bacterium against Actinobacillus pleuropneumonia .
In order to demonstrate various aspects of the invention, the ranking procedure of this invention has been applied to pigs; however, it will become apparent that the basis of the ranking procedure may be applied to other types of livestock. The methodology haε been applied to the development of a high line of pigs which exhibit increased production, better health where the desirable traits are passed on from one generation to the next. Such increase in productivity and health can result in, for example, one extra pig per litter, moving the pigs to market weight in less time, for example by as much as ten days without reduction in quality of the meat and that such animals respond in a superior way to various vaccination treatments.
The procedure, according to this invention, which provides an Estimated Breeding Value is very useful for ranking amongst breeding animals their immune system responsiveness. The Estimated Breeding Value is an indicator of the animal's responsiveness in resisting disease. The greater the animal's ability, that is level of ability to resist disease, then the higher the EBV. Furthermore the EBV indicator is based on the ability of the animal to pass such disease resistance to offspring. Hence the higher the ability of the animal to pass on this disease resistance correspondingly the higher the EBV for that animal. By way of ranking the immune system responsiveness of the animal, animals may be selected for breeding in order to produce offspring which inherit the higher or lower level of ability to resist disease. Although the ranking procedure is important in respect of development of strains of animals that are disease resistant, it is also an aspect of the invention, however, to develop at the same time a line of animals which has a very low resistance to disease. Such low animals can be very useful in drug screening programs and other tests to determine efficacies of new drugs, vaccines and the like.
The ranking procedure is based on testing the animal's response to several, perhaps related or unrelated traits, which may or may not relate directly to determining immune system responsiveness. These traits are selected from heritable humoral immunity traits and heritable cell-mediated immunity traits and perhaps one or more other traits which are inheritable, such as stress resistance. The heritable humoral immunity traits may be tested by at least two tests. One of the tests is usually a general measure of the immune response and the other test is usually an antigen-specific immune response. Similarly with the heritable cell-mediated immunity trait, this can be tested by at least two tests which are correspondingly directed to either the general or antigen-specific types of tests. These tests are conducted as soon as possible on the animal after it has been weaned form its mother and at a time chosen to negate any effects of passive immunity; thereby ensuring the least amount of interference in respect of getting true values for theεe test results.
The humoral immunity tests more specifically may be selected from tests, such as:
1. serum concentration of immunoglobulin G (IgG) . This is directed to the general measure of the animal's response in respect of immunity, traits. 2. The specific test may be an antibody response to an antigen which is not expected to be part of the antigens to which the animal and his parents have previously been exposed. For example, these tests may be: a) peripheral blood lymphocyte blastogenic response to a mitogen not common to the animal and its parents, or b) a cutaneous delayed type hypersensitivity (DTH) to an antigen purified protein derivative of a bacterium previouεly used to induce DTH. Specific examples for the antigen may be hen eggwhite lysozyme. More particularly in respect of the peripheral blood blastogenic response test, the antigen may be concanavalin A. In respect of the antigen used to induce DTH, that may be Bacillus Calmette-Guerin (BCG) or purified protein derived in a human strain of Mycobacterium tuberculosis grown on a protein free synthetic medium. By carrying out these tests, the animals may be grouped into high, low or control. By virtue of this ranking procedure, the indicator divides all ranked animals into these indicated groupε of high, low or control. The ranking therefore provideε the baεiε for breeding together only teεt animals from the same group; that iε only animalε ranked high are bred with other animalε ranked high and correεpondingly with reεpect to the ranked low or control animalε. Cross-breeding of a high animal with a low animal would not produce the deεired result, becauεe of the resultant degeneracy in the offspring inεofar aε developing an animal line with the increasing ability to resiεt disease and other advantages and features of this invention.
By virtue of the ranking procedure of this invention, several advantages flow therefrom. particularly in respect of this application to the breeding of pigs, cattle, chickens and fish. The ranking procedure when used in selecting breeding animals from the high group provide offspring which achieve market weight consistently faster than offspring bred from control groups or bred from low groups. The offspring also have a higher percentage of live piglets per litter, such as when applied to pigs, also a lower number of litters with less than three piglets, a lower percentage of deformed pigletε per litter and a higher production index. Furthermore, the animalε ranked in the high and low groupε differ in disease manifestations induced by infection. The animals ranked in the high and low groups differ in reεponεe to vaccination εuch that the animals ranked in the high groups respond earlier, produce more antibody against antigens and have a higher percentage of animals that respond to vaccination. The animals ranked in the high and low groupε differ in reεponεe to immunization, εuch that animalε ranked in the high group produce more antibody to antigenε in addition to thoεe uεed to derive the original EBVε. Thiε aspect of the invention will be deεcribed in more detail in reεpect of the following Tableε. Furthermore the animalε ranked in accordance with the procedureε of thiε invention into the high and low groupε differ in reεponεe to immunization, εuch that animalε ranked in the high group produce antibody of higher binding strengths (avidity) for the antigen administered. This indicates overall a superior immune responεe by the animals in the high group. As an example of an embodiment of the invention, approximately 120 first generation piglets (Gl) were evaluated in accordance with the ranking procedure. Based on G0 plus G,, heritability estimates were 0.25, 0.23, 0.08, 0.08 and zero for secondary antibody response to HEWL, blastogenic responεe to Con A, cutaneouε DTH to BCG/PPD, εerum IgG, and monocyte function, reεpectively. Leaεt squares means reflected theεe estimates in that there were significant (p < 0.05) differences between High and Low line G, pigs in antibody, blastogenic, and DTH responses. However, there were no significant line differences in serum IgG or uptake and killing of S . typhimurium . Response to selection was determined both by differences in leaεt squareε means and differences in average EBV between the High and Low lineε. After one generation of selection lines were separated by 1.508 (least squareε) and 1.205 (EBV) index pointε, or slightly more than half a standard phenotypic deviation. The following materials and methods and asεay procedureε for the variouε traits are provided to illustrate various embodiments of the invention in carrying out the methodology thereof. MATERIALS AND METHODS - EMBODIMENT #1
Experimental Animals and Traits Measured
To determine the effectiveness of using a composite selection index for breeding high and low immune response lines, a random bred population of Yorkshire pigs housed in an Specific Pathogen Free (SPF) research facility
(Arkell Station, University of Guelph) was evaluated for various indicators of antibody, cell-mediated and innate resiεtance-related traitε. The initial εcreening involved the evaluation of 65 female and 33 male piglets, beginning at approximately 60 days of age. One male and 2 female piglets were sampled from each of 34 litters by 15 sires. Each piglet was evaluated for total serum immunoglobulin G (IgG) and M (I&M) , serum antibody responεe to Hen Egg White Lyεozyme (HEWL) , a εynthetic peptide (TGAL) , and sheep erythrocytes (SRBC) . Cellular immune response was assessed by measuring delayed type hyperεenεitivity (DTH) to a purified protein derivative (PPD) of Bacillus Calmette Guerin (BCG) , aε well aε by contact εenεitivity to dinitrochlorobenzene (DNCB) . In addition lymphocyte proliferative responses to concanavalin A (Con A) and PPD were assessed. Serum hemolytic complement activity (CHso) , and the ability of peripheral blood monocytes (PBM) to take up and kill Salmonella typhimurium were evaluated as indicators of innate resistance.
Immunisation and Sampling Schedule
A standard protocol of immunization and sampling began on day 0 when piglets were about 60 days of age, since at this point interfering maternal antibodieε would be minimal, particularly to inert antigens not previously encountered to negate thereby effects of passive immunity. Piglets were weaned at an average of 21 days of age. To induce antibody production, HEWL (ICN Biochemicals, Montreal, Quebec) and TGAL (ICN Immunologicals, Liεle, 111.) were separately disεolved in phoεphate buffered εaline (PBS, 0.10 M, pH 7.4) εuch that 10 ug waε contained in 1.0 and 0.10 ml reεpectively. Solutions were emulsified with an equal volume of Freund's complete adjuvant (FCA, Difco, Detroit, MI.) and injected intramuεcularly (im) to different siteε delivering 10 ug of antigen to each pig. One ml of 40% v/v washed SRBCs in Alεever'ε solution was injected intraperitoneally (ip) . Immunizations were repeated on day 14 uεing HEWL with FCA and TGAL without FCA and 0.5 ml of the SRBC suspension. Two 10 ml tubes of blood were collected from each pig on day 0 in order to evaluate pre-immunization serum titerε, serum IgG and IgM concentrations, and hemolytic complement activity (one tube kept on ice) . Additional sera were collected on days 9, 14, 21, and 30 for titration of primary and secondary antibody responseε and evaluation of CH^ at the end of the immunization protocol (day 30) .
On day 9, for induction of cell mediated immunity (CMI) , the pigs received 0.5 mg of BCG (Connaught, Willowdale, Ontario) suspended in 0.5 ml of PBS injected intradermally (id). Each pig also received 0.1 ml of 5% w/v DNCB (Sigma, St. Louis, Missouri) in 9.5% ethanol mixed with an equal volume of aqueous 90% dimethyl sulfoxide (DMSO, Fisher Scientific, Ottawa, Ontario) . This mixture waε applied to a designated skin surface area on the outside of the thigh and evaporated to drynesε. On day 21 pigε received 0.1 ml (id) of 250 test units of PPD (Connaught, Willowdale, Ontario) and topically 0.2 ml of 5% w/v DNCB in 4:1 acetone and olive oil. Negative control siteε received 0.1 ml of PBS or 0.2 ml of 4:1 acetone and olive oil respectively. Twenty-four hours later cutaneouε responses were measured by double skin fold thickness.
On day 21, 35 mis of whole blood waε collected in 50 ml conical tubes containing 5.0 mis of εodium heparin (Sigma, St. Louis, Misεouri, 100 unitε/ml in PBS) , and peripheral blood lymphocyteε (PBL) iεolated by Ficoll- Hypaque εeparation (specific gravity 1.077, 260 milliosmoleε) in order to evaluate blastogenic response to Con A and PPD. On day 14 whole blood was collected in a similar fashion and adherent cells obtained for assessment of uptake and killing of S . typhimurium .
Antibody Assays
An ELISA assay was performed to quantify primary and secondary antibody responses to HEWL and TGAL on days 0, 9, 14, 21, and 30 of the immunization schedule. Flat bottom polystyrene 96 well microplates (Dynatech,
Alexandria, Virginia) were coated with 100 ul/well of 1 x 10"* moles/liter HEWL (ICN Biochemicals, Montreal, Quebec) or 1 x 10"8 moles/liter TGAL (ICN Immunologicals, Lisle, Illinois) and incubated 2 dayε at 4°C for HEWL or overnight for TGAL. Plateε were waεhed (EL 403 microplate autowaεher, Bio-tek Inεtruments, Guelph, Ontario) 3 times with 250 ul/well of wash buffer (0.05% Tween-20 in PBS, pH 7.4) then blocked with 200 ul/well of 3% Tween-20 and incubated at room temperature (rt) for 1 hour. Plates were again washed 3 timeε with the waεh buffer and εamples added. In addition, a control was provided which included the above reagentε without the addition of the samples.
A 1:5 and a 1:125 dilution of each of the aforementioned test sample waε prepared using 0.05% Tween-20 as diluent and 100 ul diεpenεed into each of 4 replicate wells using a quadrant system (Wright 1987) . Replicates of pooled positive and negative pig sera were also included on each plate. Plates were incubated (2 hours, rt) and washed 3 times with wash buffer. A rabbit anti-swine IgG (H + L) alkaline phosphatase conjugate
(Sigma, St. Louiε, Missouri) was diluted 1:1500 for HEWL and 1:1000 for TGAL in 0.05% Tween-20 and 100 ul dispenεed into each well. Plateε were incubated (2 hours, rt) and waεhed 3 times. The substrate (p- nitrophenophoεphateε, Kirkegaard and Perry, Gaitherεburg, Maryland) was dissolved in diethanolamine (10% in dH20) to a concentration of 1 mg/ml and 50 ul added to each well. Plateε were incubated at 37°C until the abεorbance reading (EL 311 Automatic ELISA Scanner, Biotek Instruments) of the standard positive control is 1.0 at 405 run. This reading was generally achieved in 20 to 30 minutes. The mean, standard deviation and coefficient of variation were calculated for the 4 replicates of each dilution and the mean sum of the 2 dilutions recorded as the antibody titer.
Antibody responses to SRBCs were determined by a haemagglutination asεay described previously (Mallard et al. 1989a) and serum IgG and Igm concentrations by single radial immunodiffusion (Mallard et al. 1989b) .
Cellular Assays
Lymphocyte blastogenesis was evaluated on day 21 of the immunization schedule by measuring lymphocyte proliferative response to the T-cell mitogen Con A and the antigen PPD. Blood was collected from each pig and Ficoll-Hypaque separated peripheral blood lymphocytes (PBLS) were suspended in culture medium (RPMI 1640 plus 20% FCS) to a concentration of 5 x 106 cells/ml. For each pig tested 100 ul of this cell suspension was dispensed into an entire row of a 96 well tissue culture plate (Nunclon, Gibco, Grand Island, NY) . Wells in columns 1 through 4 then received an additional 100 ul of culture medium providing unstimulated controlε (4 replicateε/pig) , whereaε wellε in columnε 5 through 12 (8 replicateε/pig) received 50 ul of culture medium pluε 50 ul of either Con A (10 ug/ml in RPMI) or PPD (400 ug/ml in RPMI, M. bovis strain AN5 kindly provided by Dr. B. Stemshorn, Agriculture Canada) . Plateε were incubated (18 hours, 370βC) and then "pulsed" with 20 ul/well tritiated thymidine (25 pCi/ml in RPMI. Plates were covered with foil, incubated for a further 24 hours (37°C) and then frozen (-20βC) until a convenient time for harvesting. To harvest cellε, plates were thawed (30 minutes, 37°C) , and cells deposited onto glasε fiber filter discs (PHD Cell Harvester, Cambridge Technology Inc., Watertown, Ma). Filter discs were placed into scintillation vialε with 5.0 mlε of scintillation fluid and counts per minute (cpm) obtained (Packard, Liquid Scintillation System, Downers Grove, IL) . The change in cpm waε calculated aε the mean of the εtimulated wells (with mitogen or antigen) minus the mean of the unstimulated control wells.
Delayed Type Hyperεenεitivity (DTH) to PPD and DNCB were determined 24 and 48 hourε after challenge by calculating the increase in double skin fold thickneεs using the method described previously (Mallard et al. 1989a) .
Nonspecific Resistance Assays
Total serum hemolytic complement (CHJQ) activity was determined using day 0 (pre-immunization) and day 30 (post-immunization) samples. Blood was collected, held on ice and sera were harvested and frozen (-700°C) until time of analyses. Sera were then analyzed according to the method described previously (Mallard et al. 1989c) . To asεeεε the ability of peripheral blood monocyteε to take up and kill S. tvph;»mt^^mr mononuclear cellε were εeparated uεing a Ficol-Hypaque denεity gradient (Specific gravity 1.077, 335 millioεmoleε) and resuspended to 5 x 106 cells /ml in culture medium. Ten ml aliquots of suspended cells were dispensed into tisεue culture flasks (Nunc, Gibco, Grand Island, NY) and incubated (18 hrs, 37°C) . Medium and nonadherent cellε were decanted and adherent cells removed by adding 40°C PBS (Bendixen, 1981) . Adherent leukocytes were resuspended at 1 X 106 cells/ml in culture medium and uptake and killing of bacteria determined by a method deεcribed previouεly (Lacey et al. 1989) which is based on the reduction of 3 (4, 5-dimethylthiazoyl-2-yl) 2, 5 diphenyltetrazolium bromide (MTT, Pharmacia, Dorval, Quebec) to a purple formazan by bacterial dehydrogenaseε.
Statistical Analyses and Selection of Animals The initial population (G0) consisted of 65 females and 33 males from 34 litters sired by 15 boars. Because distributional properties of the traits were not known and the data set at G0 waε relatively small (n = 98) , observations on all traits were standardized using rank normal scores. Estimates of heritabilities of the standardized records were then obtained by restricted maximum likelihood according to the following model:
Yiju = + 9; + Sj + 1^+ βyu where Yijkl is a normal rank score on an immune responεe measure on the ijkl* pig, μ is the population mean, g* is the fixed effect of the i* sex of pig (male vs female) , Sj iε the random effect of the j* εire (0, Iσ2,) , 1^ iε the random effect of the jk* litter - (0, Iσ2j) and e^, iε the random effect of the ijkl* pig - (0, Iσ2 e) . The litter term containε the additive genetic contribution of the dam pluε environmental and dominance genetic effects common to littermates. Solutions from the restricted maximum likelihood analyses were used to compute an estimated breeding value of each tested pig for each trait as follows (Kennedy and Sorensen, 1988) : EBV^ - s, + l^ + h2 w (y^, - g. - Sj - 1*) where k = σ2, / o2 λ and h2 w = 2σ2, / 2σ2, + σ
Baεed on initial estimates of heritabilities and correlations between EBVs of the traits, five traits were chosen as εelection criteria to be included in a composite index. The objective was to include one specific and one general indicator of both antibody and cellular immunity and one indicator of innate resistance. The five traits were serum IgG, antibody response to HEWL, blastogenic response to Con A, cutaneous DTH to BCG/PPD and uptake and killing of S . typhimurium . A total EBV score on the five traits was combined in an index, as well these five traits can be considered independently. The top, intermediate and bottom ranking 7 young boars, according to combined EBV score, were chosen as foundation breeding stock (G„) to be sires of High (H) , Control (C) and Low (L) line pigs respectively. Although 7 boars were chosen for each line, only 5 were actually used for breeding with 2 held in reserve in case of reproductive problemε. similarly, the top, intermediate and bottom ranking gilts were εelected and mated to H, C and L line boarε reεpectively. There were 23, 21 and 19 H, C and L giltε. From each litter from theεe matings, 2 females and 1 male first generation (G,) piglets were randomly sampled and evaluated according to the same immunization schedule aε the parentε.
Response to selection was calculated from the data of both generations by leaεt squares and according to an animal model (Kennedy, 1990) .
The least squareε analyεiε was according to the following model: Yijum = + g, + j + c, + aljklm + eijUm where y^, μ, and g* are as defined previously, tj is the effect of the j* generation, lk is the effect of the k* line, and tl^ is the line by generation interaction, and e^u the random error. All effects, except the error, were fixed.
Responεe to εelection waε measured on the difference between least squares means for the H and L lines at generation 1. Standard errors of the least squareε estimates do not account for drift variance and an adjustment for drift was made using the procedure of Sorensen and Kennedy (1983) .
Heritabilitieε of the five traitε and breeding values of the pigs of all lines and both generations were estimated under an individual animal model using the derivative free REML programs of Meyer (1988) according to the following model: y*_m = μ + g; + tj + Cj + a^ + e^ where y^, μ, g; and tj are as previously defined, c, is the common environmental effect due to littermates (0,
Iσ2 c) , a^ is the additive genetic (breeding) value of the ijklm* pig (0, Aσ2 and e^ is the random environmental effect on the ijklm* pig (0,lσ2 e).
The average genetic value of pigs of the jk* generation and line waε eεtimated aε ∑ilm a^ / Njk where Nj,- iε the number of pigε. Reεponεe to εelection was estimated as differences in average estimated genetic value between the H and L lines in generation 1. Standard errors of response were according to Sorensen and Kennedy (1986).
Both the leaεt squares and animal model analyseε were done for the five εelection traitε on the data on the original scale of measurement because significant departures from normality were not found. After the analyseε, the traitε were converted to a εtandardized scale (μ = 0, σ = 1) and combined EBVs for the five traitε were again computed and 5 boars and 21 gilts from each of the three lines were selected as breeding stock to produce the next generation.
Data haε been gathered from two generation of pigε G0 and G, to develop a production index. The reεultε of these investigationε are summarized in Table 9, where it is demonstrated that the high line of pigs reach 90 kilograms at 144 days versus the low line of pigs which reach the necessary weight in 154 days, such pigs having a greater amount of fat than the high line of pigs. This result in a production index of 156 versuε 141 for the low line.
Several generationε of pigε have been analyzed for reproduction data selected for the high and low immune responεe traits. As demonstrated in Table 10, there is a significant increase in the number of live piglets per litter, usually in excesε of 1, with less poorly performing sows and lesε deformed and mummified pigε. Thiε Table describes the average (mean +\-standard deviation) number of live pigletε born per litter, the number of sows (%) farrowing 3 or lesε pigletε per litter, and the number of deformed and mummified piglets per litter (%) obtained from Guelph Yorkshire sows selectively bred for high and low immune responsiveness. This data was obtained from computerized record sheets kept on all pigs housed at the University of Guelph Arkel Research Center and significant differences between high and low lines are determined based on statistical t-tests and reported at either a 90% (*) or 95% (**) confidence level. Generation of selection is given down th left margin as GO to G3.
Production data from pigs was also analyzed for high and low immune responεe with reεpect to εeveral generations. In achieving shipping weight of 90 kilograms in carrying out the production, back fat was measured. As summarized in Table 11, the high line of pigs achieved market weight in approximately 10 days less than the control line with lesε back fat to indicate a healthier, more productive line of pigε with higher quality meat. Thiε Table presents the rate of weight gain on the basis of days required to reach 90K and backfat thickness in millimeters as determined using an ultrasonic probe of Guelph Yorkshire pigs selected for high and low immune responsivenesε. The generation of εelection is given down the left margin as Gl, G2 or G3. The difference in rate of gain between the high and low line pigs (i.e., about 10 days) was reported as significantly different using a statiεtical t-teεt at a 95% confidence level; i.e., p < 0.05 and iε depicted uεing an ** εymbol.
RESULTS FOR EMBODIMENT #1
Parental Generation (Go) : Arithmetic Means
Arithmetic means and the standard deviations of innate and immune responεe traitε measured in boars and gilts selected as parents to produce High and Low breeding lines are presented in Table 1. These provide baseline data on the traits measured.
Analysis of variance
Resultε from the leaεt squares analysis of data from G0 are summarized in Table 2. Results of these analyses indicated that the sire significantly contributed to the variation observed in secondary antibody response to HEWL and TGAL, serum IgG, DTH to PPD, and hemolytic complement activity (Day 0 and 30) . The litter significantly influenced the secondary antibody response to HEWL, serum IgG and IgM, blaεtogenic reεponεe to PPD and Con A, and complement (day 0) . The εex of the pig influenced the primary antibody reεponεe to HEWL, εerum IgM and complement activity (day 0 and 30) . The analyεis of variance for monocyte function showed that sire did not influence this trait, however age and group were εignificant factorε (Table 2) . Correlation and Heritability Estimates
To determine which of the fourteen parameters measured might be most representative of the pigs overall potential to respond to subsequent infection; correlations, analysiε of variance, and heritabilitieε were taken into account. Based on thiε information, 5 traitε were choεen to be included in the selection index as follows.
Correlations based on EBVs (Table 3) indicated a positive and significant correlation between primary and secondary antibody responses to TGAL and HEWL in both boars and gilts. There was also a slight positive, but nonsignificant correlation between antibody reεponεe to HEWL and TGAL. Due to theεe aεεociationε and becauεe heritability estimates (Table 2) of secondary antibody responεeε were higheεt, the secondary responεe to HEWL waε choεen aε one of the indicatorε of humoral immunity. Since εerum IgG concentrationε have previouεly been reported to be aεεociated with diεeaεe incidence (Mallard et al. 1983, Burton et al. 1989, Corbeil et al. 1984), this was also included as an indicator of humoral immunity. Serum IgG tended to be negatively correlated with antibody responses to both HEWL and TGAL (Table 2) , and therefore inclusion in the selection index εhould prevent IgG concentrations from significantly declining. The heritability of porcine serum IgG concentration was estimated at 0.15 in G„ (Table 2).
Cutaneous DTH to PPD and DNCB tended to be negatively but not significantly associated. In the parental generation these traits had heritability estimateε of 0.27 and 0.17 respectively (Table 2). The DTH responεe to PPD was included in the index as one indicator of CMI because it had the higher heritability, and the reεponεe to DNCB waε positively correlated with serum IgG which waε already marked for incluεion in the index. The lymphocyte proliferative responεeε to PPD and Con A tended to be positively correlated, as were the proliferative and DTH responses to PPD (Table 3) . The heritability estimates of the blastogenic responseε to PPD and Con A in G0 were 0.15 and 0.37 reεpectively. For theεe reasons blastogenic response to the mitogen Con A was chosen as the other indicator of cellular responsivenesε.
In termε of innate immunity, hemolytic complement activity on dayε 0 and 30 were significantly and positively correlated. Preimmunization complement activity was also poεitively correlated with secondary antibody responεe to HEWL and serum IgG (Table 3) . The heritability of complement activity was 0.13 and 0.31 pre (day 0) and post (day 30) immunization respectively. Due to the poεitive correlation with antibody response to HEWL, which was already included in the index, complement activity was not included in the index.
Monocyte function, meaεured aε anti-bacterial capacity, waε positively correlated with hemolytic complement activity (day 0) , but negatively correlated with serum IgM (Table 3) . The heritability of this trait was 0.18 in G0 and because of the importance of the monocyte in both antibody and cellular immunity it was included as a parameter in the selection index.
Estimated Breeding Values of the Parents Selected to Produce High, Low, and Control Line Groups
The five EBVs for each of the selected traits were added to give each pig a total composite Estimated
Breeding Value. Boars and gilts were then ranked on this basis. The five top ranking fertile boars were asεigned to the high group and had a mean total EBV of 0.24 ± 0.12. The intermediate five boars were asεigned to the Control group (x = 0.02 ± 0.03), and the bottom five to the Low group (x = -0.16 ± 0.03) (Table 4). Similarly the high, intermediate, and low one third of the sows were assigned to H (x = 0.21 ± .10), C (x = -0.02 ± .06) and L (x s -0.18 i .07) line breeding groups. Mean combined EBVs indicated significant differenceε (p < 0.001) between the breeding groups (Table 4).
First Generation (0,) : Means, Heritability and Environmental Correlations
Estimates of means, phenotypic standard deviations, heritabilities, and environmental correlations among litter mates of the five selected immune and innate resistance factors were calculated using the combined data from the parents and first generation piglets (Table 5) . The heritability of εecondary antibody reεponεe to HEWL waε estimated at 0.25 and the similarity amongst litter mates due to a common maternal environment (c2) was 0.23 (Table 5). Least squares means (Lsmeanε) for antibody response to HEWL of first generation piglets showed a significant (p < 0.04) difference between H and L lines (Table 6) . The heritability of lymphocyte blastogeneεiε to Con A uεing the combined (G0 + Glj data waε 0.23 and the environmental correlation waε eεtimated at 0.24 (Table 5). Lsmeanε indicated significant (p < 0.03) differenceε between H and L line pigletε for thiε trait (Table 6) . The heritability of the DTH reεponse to BCG/PPD was 0.08 and the environmental correlation was 0.28. Again there were significant (p < 0.007) differences in lεmean responses between H and L line piglets. The heritability estimate of serum IgG concentrations uεing the combined data from both generationε had dropped from 0.15 based on G0 data to 0.08, and the variation amongst littermates waε 0.26 (Table 5) . Lεmeans revealed no differences between H and L lineε in either generation (Table 6) . Similarly there were no differenceε in Lsmeans for monocyte function (Table 6) and the heritability was now estimated to be zero (Table 5) . Analysis of Variance
Results from the least squareε analysis performed using the combined data from the parental and first generation piglets indicated that the generation factor was significant (p ≤ 0.0001) for each of the five traits and that line, and generation by line also significantly contributed to the variation in each of the traits, except monocyte function (Table 7) . There were no significant sex effects on any of these traits.
Estimated Breeding Values and Response to Selection
EBVε calculated on the five traitε for each pig within a line, and additively combined EBV, are given in Figureε 1 and 2. Mean total EBVs for the High, Control and Low lines were 0.66 ± .36, -0.04 ± .19, and -0.55 ± .31 respectively.
The responεe to selection waε determined uεing both the leaεt squares and animal model. The differences between H and L lines are reported in terms of original and standardized units (Table 7) . The results showed that after one generation of εelection the H and L lineε were εeparated by 1.205 as measured by EBVS, and 1.528 aε meaεured by leaεt squares which iε a little more than half a standard deviation of the index (Table 7) . As expected from the heritability estimates the largest line differences occurred in antibody responεe to HEWL and blaεtogenic response to Con A.
Correlated Traits Differences between the H and L lines were also apparent for several non-selected traits (Table 8) . High line pigs produced more antibody to TGAL (p < 0.02) and SRBC (p < 0.10) aε measured on day 21 of the immunization schedule. Low line pigs however, had a substantially greater (p < 0.07) DTH responεe to DNCB (Table 8). Genetic εelection, in accordance with the methodology of thiε invention, enhances inherent diεeaεe resistance and increases livestock productivity and profitability without the hazards and costs associated with the traditional use of medications and vaccines. Recent analysis revealed that preventive treatment of animal disease yielded a much higher return on funds invested (500-1500%) than curative methods (Morris, 1988) . In addition, High/Low and Resiεtant/ Susceptible lines provide a powerful tool to investigate mechanisms of host resistance to a variety of infectious agents. Genetically selected lines of pigs are also useful in gene mapping studies.
The multi-trait selection based on predictors of immune and innate resistance demonstrateε that immunity in mammals iε regulated not by one, but a complex network of factorε.
A random bred population of Yorkshire pigε waε characterized uεing variouε indicators of immune and innate resiεtance (Table 1) aε deεcribed. Based on first estimates of heritability and correlations of eεtimateε of breeding values (Tables 2 and 3) , serum IgG and secondary antibody response to HEWL were selected aε predictorε of antibody reεponεiveneεs, while lymphocyte stimulation by Con A and DTH to BCG/PPD were chosen aε predictorε of cellular reεponεe, and innate reεiεtance waε evaluated in termε of monocyte function. EBVε for each trait were calculated uεing an animal model that makes use of all known relationships among animals, and pigs were ranked based on combined EBVs and asεigned to High, Low or Control breeding groups. Approximately 40 first generation piglets from each line were then similarly evaluated.
After one generation of selection there were significant differenceε between the H and L lineε. In fact the H and L lineε were εeparated by 1.205 unitε as measured by EBVs and 1.508 as measured by least squares EBVs (Table 7). Heritability estimateε were 0.25, 0.23, 0.08, 0.08 and zero for antibody response to HEWL, lymphocyte εtimulation by Con A, DTH response to BCG/PPD, serum IgG, and monocyte function respectively (Table 5) . Least squareε meanε (Table 6) and line differenceε, both in termε of original and standardized meaεures, (Table 7) reflected these heritability estimates in that the largest differences were apparent in the more highly heritable traits, whereas there were no εignificant differenceε between the lineε in εerum IgG concentration on monocyte function. Traitε not included aε original εelection criteria were alεo modified aε a result of selection or drift. Antibody responεeε to SRBCs and TGAL were significantly higher in the H line and lower in the L line (Table 8) . This iε εimilar to the nonspecific effects of selection described in the Biozzi mice (Biozzi et al. 1984) . Consequently the genetic regulation of antibody responεiveness in the H and L line pigε iε not reεtricted to a single antigen, but may operate at least in part aε a more general level εuch aε antigen preεentation, rather than B-cell function alone. Converεely, immune baεed inflammatory reεponεe to DNCB waε εignificantly higher in the L line pigε (Table 8) . Thiε parameter has also been previously reported to be negatively associated with antibody response in SLA defined miniature pigs (Mallard et al. 1989a) .
In accordance with the methodology of this invention, pigs and other types of livestock can be separated into high and low breeding lines using aggregate EBVε. Baεed on the above-identified traitε, the methodology of thiε invention provideε a reproducible technique in determining the high line of pigε or other livestocks for breeding purposes to increase the productivity and improve general health. With reference to Figure 3, this relationεhip iε indicated where the high line of pigε achieves a 90 kilogram weight in first, second and third generations consiεtently faεter than the control and low groupε. Antibody avidity can also be investigated in the high and low response groups of animalε as ranked by this invention. Antibody avidity is a measure of the attractionε of an antibody for an antigen; i.e., the quality of the antibody. It iε possible to measure this value and has been done for a ranked group of pigs as determined by the procedure of Embodiment #1.
Avidity indiceε of antibody to hen eggwhite lyεozyme (HEWL) were meaεured by chaotropic ion (SCN) elution enzyme-linked immunoεorbent aεsay (ELISA) in pigs grouped as high control of low for various immune and innate resiεtance-related traitε. The avidity index waε the molar concentration of SCN" required to reduce by 50% the ELISA optical density value for a given serum. The index was independent of the amount of antibody.
Eight- to ten-week old Yorkshire pigs were immunized with HEWL and serum antibody measured by ELISA as one of five traits used ot asεign them to high, low or control reεponεe groupε. Serum antibody avidity for HEWL waε evaluated on day 14 and day 30 after primary (day 0) and εecondary (day 14) immunization. The effects of responεe group, gender, litter, εerum IgG concentration and anti- HEWL antibody on avidity were determined using a linear model. Antibody avidity indices varied amongst individuals. Mean avidity indiceε for εera collected on days 14 and 30 were 0.61 ± 0.43 and 1.22 ± 0.546, with maximum indices of 2.64 and 2.86 respectively. Avidity of secondary responεe antibody waε εignificantly higher (p < 0.05). Pigs of the high response group had significantly higher secondary antibody avidity than thoεe of the control (p < 0108) and low groupε (p ≤ 0.01). Avidity index waε poεitively correlated with antibody to HEWL on days 14 and 30, but not to preimmunization εerum IgG concentration or to other meaεured traitε.
Animalε genetically selected as per Embodiment #1 to express high or low immune responεe or innate resistance- related traitε are expected to differ in reεponεe to infection and in development of diεease. Pigs were teεted aε εelected for high (H) or low (L) expreεεion of εerum IgG, εerum antibody to hen egg white lyεozyme (HEWL) , peripheral blood lymphocyte blastogeneεiε after in vitro stimulation with concanavalin A and cutaneous delayed- type hypersensitivity to PPD after senεitization with BCG. H and L pigε of generation G4 which differed εignificantly in traits used in selection, were infected with Mycoplasma hyorhinis , in accordance with the following Embodiment #2, by intraperitoneal injection and compared uεing a εplit litter deεign on the baεiε of antibody responεe, nonspecific indicators of infection and diseaεe εignε, both antemortem and postmortem. The study period began on day -1 and continued for 14 days after infection (day 1) . H line pigs produced M.hyor inis-specific serum antibodies earlier (day 3) (p≤O.OOl) and to higher titer (p≤0.05). Uninfected H line pigs gained more weight between days -1 and 14 (p≤O.OOl) but within the infected group weight loss was equivalent for pigs of both lineε. Erythrocyte εedimentation rate waε elevated on day 3 in L and day 7 (p≤O.OOl) in H and remained elevated in H and L. Fibrinogen waε elevated by day 3 (p≤O.OOl) with L>H on day 10 (p≤0.005). H had more blood lymphocytes than L in the absence of challenge (p≤0.05). Both groups had reduced blood lymphocyte numbers (p≤0.05) by day 7 with H reverting to prechallenge values by day 10 before L (p<0.005). Blood neutrophil counts were higher in L than in H (p≤O.OOl) in the absence of challenge and were elevated in both groups (p≤O.01) after challenge without line-related differences. The principal antemortem diseaεe εign was arthritis which had onset at day 7 in H and 10 in L (p≤O.OOl) with H>L on days 7 (p≤O.OOl),10 and 14 (p≤0.005). Postmortem, H displayed lesε severe peritonitis (p≤O.008) and pleuritis (p≤O.OOl) while pericarditis, although lesε in H was not εignificantly different. Arthritis was more severe in H (p<0.002) with more M. hyorhinis in synovial fluid (p<0.005) but not in blood. Synovial fluid antibody did not differ by line. Theεe findings indicate that differences in responεe to infection have been induced by indirect εelection for resistance-related attributes and that animals selected in this way have utility in husbandry aε well as in experimentation directed towards vaccine development or investigation of disease pathogenesiε. As demonstrated in Embodiment #1, pigs have been genetically selected for high and low response using an index that combined estimated breeding values for εerum IgG concentration, antibody reεponεe to hen egg white lysozyme, in vitro blastogenesis of peripheral blood lymphocytes stimulated with the mitogen con A and delayed type hypersenεitivity induced by intradermal injection of tuberculin PPD after εensitization with BCG. By the third generation of selection these pigs differed εignificantly for the traitε incorporated in the εelection index and for certain other traitε εuch aε antibody reεponεe to unrelated antigenε, antibody avidity and weight gain all of which favor the high reεponεe line.
To determine whether or not selection for potentially resiεtance-mediating traitε had altered capacity to resist infection and diseaεe, pigs of the high and low response lines were experimentally infected with Mycoplasma hyorhinis (M. hyorhinis) and their responεe to infection waε aεsesεed aε deεcribed in the following Embodiment #2.
MATERIALS AND METHODS - EMBODIMENT #2
Animals. Forty four weaned Yorkεhire pigε were 40 to 76 dayε old at the start of the experiment. The pigs were from the fourth (G4) generation of one of two breeding lineε, εelected for high (high line, H) and low (low line, L) immune reεponse, respectively (Mallard, B.A. et al. 1992) . In brief, the εelection waε baεed on an index that combined estimated breeding values for εerum concentration of IgG, antibody reεponse to hen egg white lyεozyme (HEWL) , cutaneouε delayed type hyperεenεitivity to PPD and lymphocyte blastogenesis to concanavalin A in vitro . Estimated breeding values were determined using all genetic relationships among individualε in the population (Mallard, B.A. et al. 1992) .
Experimental design. The study was performed in three sets, comprising 8, 6 and 8 pairs of litter-mates, respectively. In each set, half of the pairs were from line H and the others from line L. Within each pair, one pig was challenged with M. hyorhinis while the other received phosphate buffered saline (PBS) as a placebo. The challenged and non-challenged groupε of pigε were housed separately in adjacent rooms of an isolation unit. Jugular blood was collected into appropriate additives in evacuated tubes on the dayε immediately preceding and following challenge or placebo treatment and on dayε 3, 7, 10 and 14 thereafter (designated day -1, 0, 3, 7, 10 and 14) . The pigs were euthanized on day 14 for postmortem examination.
Challenge. The challenge strain of M. hyorhinis (497- 14) was originally isolated from joints of a naturally infected pig. The mycoplaεmaε were cultured in modified Hayflick's broth (Erno, H. et al. 1973), washed by centrifugation, reεuspended in PBS and stored at -70°c. Pigs in the challenged group received a εingle i.p. injection of 2X109 M. hyorhinis in 2 ml PBS. The non- challenged pigε received PBS only.
Antemortem observations. Arthritis was εcored aε described in Table 12. Pigs given score 3 were euthanized and aεεigned a score of 3 for each remaining day of the experimental period. Scoring was performed without reference to treatment by the same perεon throughout the εtudy.
Microbiology. At necropεy, 10 μl of blood collected in tubes with EDTA or 10 μl of εynovial fluid were uεed to inoculate modified Hayflick medium agar in Petri plateε for incubation at 37 C for 8 dayε. Colony countε were recorded aε follows: no colonies = 0, 1-10 = 1, 11- 100 = 2, > 100 = 3. The maximum score for mycoplasma growth from 4 joints of l pig was thus 12 (4 X 3) .
Clinical pathology. White blood cells were counted electronically (Coulter S +4, Coulter Electronics of Canada, Burlington, Ont. Canada) and differential cell counts were made on Wright's stained blood smears. The erythrocyte sedimentation rate (ESR) was determined by the Westergren method (Archer, R.K. et al. 1977) . Plaεma fibrinogen concentration was measured by coagulation analyzer (Fibrometer, Becton Dickinson, Canada Inc. , Miεsisauga, Ont., Canada) according to Morse e al. (Morεe, E.E. et al. 1970-71).
Antibody titres to Mycoplasma hyorhinis . Indirect haemagglutination (Cho, H.J. et al. 1976) , waε uεed to titrate antibody to M. hyorhinis in εerum and εynovia. For εtatiεtical analyεis, titers were converted to log2 : titre < 1:2 = 0, titre 1:2 = 1, titre 1:4 = 2, titre 1:8 = 3, etc.
Postmortem observations. Pigs were euthanized by intravenous injection of pentobarbital sodium. Synovial fluids were obtained aseptically from the tarsal and carpal joints prior to macroεcopic examination. The abdominal and thoracic cavitieε were examined for εignε of serositiε. All obεervationε and scoring were done by a single person without knowledge of breeding line. Scores were aεεigned to reflect relative εeverity of reεponse in each pig aε deεcribed in Table 13.
Statistical analysis. Breeding line and other effectε on response to mycoplasma-challenge were analyzed by least squareε uεing the SAS general linear model (GLM) procedure (Helwig, T.T. et al. 1979) . Normality of distribution of the data was assessed using the univariate procedure (Helwig, T.T. et al. 1979) and for moεt traitε loge-transformations were required to obtain normal distributionε for εtatiεtical analyεiε. For graphic presentation these data were converted to anti- loge. For the data describing the course of the reεponεe to challenge, the S.E.M. at variouε days were compared and it waε found possible to use pooled S.E.M.
The model used for analyzing the responεe during the course of the challenge was: ϊ*im -u + DAYj + TREATMENT + LINEk + SET, + (TREATMENTxLINE)jk + (DAYxTREATMENTxLINE)ijk + LITTER(LINExSET)^ + ANIMAL(LITTER LINExSET TREATMENT )jaam + ERROR.jkta where
Yjjktai = an observed value for a trait measuring the response to challenge; u = population mean for the trait
DAY, = a fixed effect due to day of observation; TREATMENTj = a fixed effect due to treatment regime
(challenge/placebo) ; LINEk = a fixed effect due to breeding line; SET, = a fixed effect due to experimental set; LITTER (LINExSET)kta
= a random effect due to litter nested within line and experimental set; ANIMAL (LITTER LINExSET TREATMENT)jktam = a random effect due to pig nested within litter, line, experimental set and treatment regime; ERROR^-a = a random residual error term. The animal mean square was uεed aε the denominator to test the effects of treatment, treatment x line interaction and litter on the various traits. The litter mean square was used as the denominator to test the effects of breeding line and set. The reεidual mean square waε uεed aε the denominator to test the remaining effectε.
The model used for analyzing the data obtained at necropsy waε:
Y—U + LINE; + SETj + ERRORjj, where all termε are aε previouεly defined and y iε the value of a trait recorded at necropεy.
RESULTS
Antemortem observations. The effects of M. hyorhinis challenge by line are presented in Table 14. Unlesε specifically indicated there were no differences between pigε of the H and L lineε in the non-challenged group.
Non-challenged pigε of line H gained εignificantly (p≤O.01) more weight than those of line L from day -1 to day 14 (Figure 4) . Weight loss in challenged pigs waε approximately 1 kg and did not differ by line (Figure 4) . In the challenged group, two pigε of line H and two of line L were euthanized on day 10 due to severe disease. The principal clinical sign of diseaεe waε arthritiε which waε obεerved earlier and with greater εeverity in H than in L line pigε. Arthritiε waε firεt obεerved on dayε 7 and 10 reεpectively in pigε of the H and L reεponεe lineε reεulting in a highly significant (p≤O.OOl) difference in clinical scores on day 7 (Figure 5). Scores alεo differed (p≤0.05) on dayε 10 and 14, the laεt day of antemortem observation.
The ESR. increased significantly (p≤O.OOl) from day - 1 and 0 to day 3 in pigs of the L line and to day 7 in H line pigε (Figure 6a) . The more rapid rate of increase in ESR in pigs of the low line resulted in a significant (p≤O.Ol) difference between the lines on day 3. The ESR remained elevated in pigs of both lines to the end of the εtudy.
Blood fibrinogen concentration increased (p≤O.OOl) from day -1 and 0 to day 3 in pigs of each line (Figure 6b) and remained elevated. On day 10 however, the fibrinogen concentration was significantly (p≤0.05) higher in L than in H line pigs.
The number of circulating lymphocytes decreased (p≤0.05) from day 3 to day 7 in pigε of each line. In those of the H line, lymphocytes reverted earlier to pre- challenge numbers than in L line pigs and there were significantly (p≤0.05) more blood lymphocytes in H than in L line pigs on day 10. In the non-challenged group there were at all times more (p≤0.05) blood lymphocytes in H than in L line pigs.
The numbers of circulating segmented neutrophils (PMN) increased significantly (p≤O.10) from day -1 and 0 to day 3 and remained elevated in pigs of both lineε. On dayε -1 and 0, PMNε were higher (p<0.05) in the L than in the H line pigε but did not differ by line after challenge. Serum antibody titreε to M. hyorhinis increased
(p≤O.OOl) from day -1 and 0 to day 3 in H line pigs and to day 7 in pigs of the L line (Figure 7) . Titres thuε increaεed more rapidly in the H line pigε and were significantly (p≤0.05) higher than in pigε of the L line on dayε 7, 10 and 14.
Postmortem observations. There were no postmortem signs of diseaεe in pigε of the non-challenged group. In pigε infected with M. hyorhinis , signs of both peritonitis and pleuritis were εignificantly (p≤O.008 and p≤O.OOl, reεpectively) more εevere in pigs of the L than of the H line (Figure 8). The lines did not differ significantly in scores for pericarditis (Figure 8) . Arthritis waε significantly (p<0.002) more severe in H than in L line pigs (Figure 8) .
There was no difference between the lines in the numbers of M. hyorhinis cultured from blood collected at necropsy but significantly (p≤0.005) more M . hyorhinis grew in cultured synovial fluid of H than of L line pigε.
There waε no difference between the lineε in synovial antibody titres to M. hyorhinis .
DISCUSSION
Yorkshire pigs selectively bred to differ in a number of traitε that may relate to their ability to reεiεt infectious diseases (Appleyard, G. et al. 1992 Mallard, B.A. et al. 1992 and Mallard, B.A. et al. 1991) have been shown here to differ in a line-related manner in their responεe to challenge with a single pathogen, Mycoplasma hyorhinis . Infection was associated with antemortem signs of arthritiε, pleuritiε, pericarditis and peritonitis evident poεtmortem aε expected for infection with thiε organism (Robert, E.D. et al. 1963a and Roberts, E.D. et al 1963b) . Correlates of reεponεe εuggeεt that pigε of the H line are more able than thoεe of the L line to resist production of systemic diseaεe signs εuch as peritonitis, pleuritis and pericarditiε but that the L line pigε are leεs prone to develop arthritis. Protection againεt infection and disease due to mycoplaεmaε is usually asεociated with production of antibody although in the case of M. pulmonis infection of rats, but not mice, adoptive transfer of resistance could only be achieved using immune εpleen cellε (Lai, W.C. et al. 1991) . In the preεent εtudy, H line pigε produced mycoplaεma-εpecific antibody earlier and to higher titers than did the relatively suεceptible L line pigε. The H lineε of pigε, aε developed from the foregoing Embodiment #1, realized general improvement in disease resiεtance indirectly by εelecting for a range of εpecific and innate attributeε reflecting both humoral and cell-mediated resistance-mediating mechanismε. Inεofar aε the H line developed more severe arthritis than pigε of the L line in reεponεe to infection with a single pathogen; however, this development may be overcome and may not be a significant result. For example, the two lines differ in a number of traits including thoεe incorporated in the selection index and correlated traits (Mallard, B.A. et al. 1992), such as antibody production following immunization with other antigens, lytic complement activity and antibody avidity (Appleyard, G.B. et al. 1992), which may have influenced both resiεtance and development of disease, including arthritis. Antibody avidity is of interest since high avidity antibody has been shown to be moεt efficacious in mediating protection in virus infections (Mulchany G. et al. 1992 and Salmi, A.A. 1991) and antibody-dependent diseaεe such as allergic encephalitiε (Devey, M.E. et al. 1990) . Pathogeneεis of mycoplas a-associated diseases such as arthritiε and uveitiε may involve formation of inflammation-inducing antibody-antigen complexeε (Thirkill, C.E. et al. 1992). The relative ability of the H and L line pigε used here to produce toxic immune complexes is not known but favors the H line. Since cutaneous delayed-type hypersensitivity to PPD of tuberculin was significantly higher in the H line pigs they may have a generally higher ability to produce inflammation based upon antigen-specific cell-mediated immune responεe which could have reεulted in the more εevere arthritiε obεerved in the H line animalε. Although εerum antibody to the challenge agent was produced earlier and to higher titre in the H than L line animals, εynovial fluid antibody titreε were equivalent while the numbers of M. hyorhinis were greater in the joint fluid of H line pigs. This may suggest that within jointε the lineε do not differ with regard to antibody production and that antibody quantity may not influence the development of arthritis. However, there was no attempt in the present study to determine the relative amounts, isotype or avidity of M. yorhinis-specific antibody present in serum or synovial fluid in response to infection and if the lines differ in these traitε thiε may have influenced the outcome of challenge. Given the fastidiouε nature of mycoplasmas and their dependence upon intimate asεociation with hoεt cellε in order to obtain essential nutrients (Ruuth, E. et al. 1989) it may be that intraarticular growth conditions for mycoplaεma were more favorable in H line pigε.
In that the Biozzi (Biozzi, G. et al. 1984 and Covelli, V. et al. 1989) εelection of mice on the basis of immune response resulted in lines divergent in macrophage function it waε surpriεing that in the preεent εelection of pigs based upon a multi-trait scheme, including aspectε of both cell-mediated and antibody reεponεe, there waε no line-related difference in macrophage uptake and killing of bacteria, production of oxygen metaboliteε or expression of MHC II gene products (Groves, T.C. et al. 1993). It is therefore unlikely that macrophage-mediated events such as phagocytosiε, killing or cytokine releaεe contributed to the obεerved differenceε between H and L line pigε in reεponεe to challenge infection with M. hyorhinis . However, it cannot be excluded that differences do exist between the lines in ability of macrophages to be activated by mycoplaεmas with subsequent production of monocyte or lymphocyte- derived inflammatory mediators; events which have been implicated in pathogeneεiε of mycoplaεma-associated arthritiε (Hopkinε, S.J. et al. 1988; Saklatvala, J. 1986; Shet, T. et al. 1990 and Thomεon, B.M. et al. 1987) . We and otherε have confirmed that pigs which differ genetically may vary in their ability to produce cytokines (Edford-Lilja, I. et al. 1991; Edford-Lilja, I. et al. 1993 and Mallard, B.A. et al. 1991). Table 15 describes a number of resiεtance-related traitε of the Guelph Yorkεhire pigε (High, Low and Control Lineε) that are not part of the εelection criteria but nonetheless may be relied upon in predicting diseaεe outcome. These traitε include antibody reεponεe to a small synthetic peptide known as TGAL and sheep erythrocytes (SRBC) , skin thickness responεe to a topical antigen dinitrochlorobenzene (DNCB) , and εerum haemolytic complement activity (CH50) . The averages (means +\- standard deviations) are reported for particular days using specific tests which are described on the left margin under each test. Significant differences between the lines are determined based on statistical t-testε and are reported at either a 90% (*) or 95% (**) confidence level. Generation of selection is indicated as Gl or G3. Figure 9 depicts least square mean values (i.e, means corrected for unequal sample size, litter effects, and sire effects) of antibody responseε of Guelph High Low line pigε before (day 0) and after (dayε 14 and 21) vaccination with a commercial bacterium againεt Actinobacillus pleuropneumonia . Actinobacillus pleuropneumonia iε a bacteria which causes acute and chronic pneumonia in pigs and presently costs the Canadian pork producers about $4 million annually. Antibody responses to this vaccine were measured using an Enzyme Immunoasεay (ELISA) and units of responεe are given on the y-axiε aε optical denεity (OD) of the teεt sera at a predetermined optimal dilution of 1\800. The different letters above the bars of the graph indicate that the antibody responεeε are εignificantly different as determined using a statiεtical t-teεt and are reported at a 95% confidence level; i.e, p<0.05. The nonresponder status reported at the right side of the graph indicates the percentage of pigε from each line (High, Low and Control pigs of Generation 4) which did not respond in any measurable way to this teεt vaccine. The experiments reported here confirm that resistance to infectious diseaεe can be altered indirectly by breeding using selection for immune response-related and other traitε. There is a general tendency for resistance enhancement in the high reεponder animals but it is already evident that the H line animals have superior production performance in termε of weight gain (Mallard, B.A. et al. 1992. This may be due to improved resistance to subclinical infection and diseaεe. The relatively low blood neutrophil numberε in the H animals may be an indicator of this while the higher lymphocyte countε of the H animalε may reflect immune εyεtem fitneεε. Leucocyte numberε and neutrophil function have been εhown to be heritable traitε in animalε, pigε (Edford-Lilja, I. et al. 1993).
The breeding selection, according to this invention, iε predictably applicable to other animalε εuch aε cattle, sheep, chickens, fish, horses and other valuable livestock because all of these animals have similar responεe to the traitε uεed in developing EDVε for ranking the animals for further breeding. Animals so developed can reduce husbandry costs through reduced requirements for health-related inputs such as antibiotics and vaccines while enhancing product wholesomeneεε by reduced uεe of extraneous materials . Such superior animals alεo have an apparent role in pathogenetic studies and in vaccine development and efficacy trials in which the L line animalε may simulate problematic low responder individualε in outbred populations. Hence, the selection procedure results in an animal model on which drug screening and the like may be conducted. There are alεo animal welfare implicationε if inherent ability to maintain health iε improved in production systems and the selected lines allow experimentε to be conducted with fewer animalε.
Although preferred embodimentε of the invention are deεcribed herein in detail, it will be underεtood by those skilled in the art that variationε may be made thereto without departing from the εpirit of the invention or the εcope of the appended claimε.
TABLE 1
Arithmetic Means (and Their Standard Deviations) of Immune and Innate Resistance-Related Traits in Yorkshire Boars and Gilts Selected to Produce High and Low Breeding Lines
* Significant differences between boars and gilts: * p < 0.05; ** p < 0.01. b nt = not tested c Monocyte function is based on the ability of peripheral blood monocytes to take up and kill at 30 (TJO) and 90 Tw) minutes respectively
SUBSTITUTE SHΞΞT Table 2
Analysis of Variance and Heritability Estimates of Traits Evaluated in Yorkshire Pigs Selected to Produce High and Low Breeding Lines.
Variance component estimation was by Maximum Likelihood.
The significance of sire was tested using the type 111 mean square for litter
(Sire) as the error term.
Traits chosen as actual selection criteria. ns = not significant (p > 0.10)
SUESTfTUTE SHEET Table 3
Correlation Coefficients Based on Estimated Breeding Values Between
Immune and Innate Resistance-Related Traits Evaluated in Yorkshire
Pigs Selected to Produce High and Low Breeding Lines
Correlated Traits
IgG (day O)/IgM (day 0) HEWL (day 14)/HEWL (day 21) TGAL (day 14)/TGAL (day 21) HEWL (day 21)/TGAL (day 21) IgG (day O)/HEWL (day 21) IgG (day O)/TGAL (day 21) IgM (day O)/HEWL (day 21) IgM (day O)/TGAL (day 21)
PPD (DTH,24 hr)/PPD (48 hr) DNCB (DTH, 24 hr)/DNCB (48 hr) PPD (24 hr)/DNCB (24 hr) PPD (48 hr)/DNCB (48 hr) PPD (24 hr)/DNCB (48 hr) DNCB (24 hr)/IgG (day 0) DNCB (48 hr)/IgG (day 0)
PPD (blastyCon A (blast) PPD (blastyPPD (24 hr) Con A (blast)/HEWL (day 21) Con A (blast)/TGAL (day 21) PPD (blast)/TGAL (day 21) PPD (blastyigG (day 0)
CHso (day OyCH*, (day 30) CHso (day O)/HEWL (day 21) CHJO (day O)/IgG (day 0)
Mφ (T^-T^/IgG (day 0)
* IgG ; serum IgG, HEWL and TGAL antibody response to hen lysozyme and the synthetic peptide TGAL, PPD and DNCB (24 or 48 hr) = cutaneous DTH measured at 24 or 48 hours post challenge, PPD and Con A (blast) - blastogenic response, CH50 = hemolytic complement activity, M' = monocyte ability to take up and kill S. typhimuri m measured at 30 (T30) and 90 (T^) minute respectively. b* p < 0.01. c nt = not tested.
SUBSTITUTE SHEET Table 4 Selection Indexes of Boars Based on Combined Estimated Breeding Values (EBV) of Antibody, Cell Mediated Immunity and Monocyte Function.
Boar
139 148 112 172 190
157 124 193 187 175
106 199 178 184 181
* Combined EBV was calculated as the mean EBV of the five traits chosen for selection. The selection index for gilts was similarly determined.
SUBSTITUT Table 5.
Estimates of means (x), Phenotypic Standard Deviations (SD),
Heritabilities (h2), and Environmental Correlations (C2) of Immune Response
Traits in Selected lines (G0 + G,) of Yorkshire Pigs
Number of Trait Observations x SDA h2(%) C2( )
HEWL 217 1.052 0.56 25.00 23.10
(day 21, ELISA)
IgG 218 1008.2 292.5 07.60 25.70
(day 0, mg/lOOml)
PPD 217 0.957 0.684 8.20 28.30
(24 hr, mm)
Con A 211 197109. 77808 22.50 23.50
(blastogenesis, cpm)
Monocyte fc 81 0.241 0.138 0.00 0.00
(T30-T50, OD)°
1 Standard deviations are within generation, line and sex. 0 Monocyte function is based on the ability of peripheral blood monocytes to take up and kill S. typhimurium at 30 and 90 minutes respectively.
SUBSTITUTE SHEET Table 6.
Least squares means and their standard errors in lines (G0 & G,) of Yorkshire Pigs Selected for high (H), low (L) and control (C) immune and innate resistance.
Number of Least squares means
Trait Generation Observations H Line C Line L Line P> FD
HEWL G0 96 Λ (day 21 , ELISA)
G, 121 l PPD G0 97
(24 hr, mm) G, 120
Con A G„ 97 (blastogenesis, > cpm) G! 114
Monocyte Fc G0 33 ^o-T^ 0D)'
G, 48
' Monocyte function is based on the ability of PBMs to take up and kill S. typhimurium at 30 and 90 minutes respectively. b Probability of significant differences between High and Low lines within a generation.
t 1
to T
Table 7. Differences between High and Low lines of Yorkshire pigs after one generation of selection (by average estimated breeding value and least squares).
HEWL IgG PPD Con A Monocyte
(day 21, ELISA) (day 0,mg/ 100ml) (24hr,mm) (24hrr, cpm) functionO Index
in
^_s_α
0.120 33903 0.00
0.383 30556 -0.018
0.170 23115. 0.043 rπ
Standardized measures(b)
0.175 0.436 0.000 1.205
ΓTI m
0.560 0.393 -0.013 1.528
0.249 0.297 0.313
' Monocyte function is based on the ability of PBMs to take up and kill S. typhimurium at 30 and 90 minutes respectively. b Standardized measures are based on standard deviations of one for ease of comparison between immune response parameters. c Adjusted for drift variance.
Table 8.
Response of the Correlated Traits in Pigs (G,) Selected for High and Low Immune and Innate Resistance Factors.
Traits Line Mean ± (Std. Dev.) Significance
Measured High Low p < F
TGAL (ELISA titer, day 21) 1.16(0.73) 0.84(0.69) p<0.02
SRBC (HA titer, day 21) 354.70(815) 167.40(250) P <0.10 DNCB (24 hr, mm) 20.27(22.24) 28.56(21.81) p<0.07
SUBSTITUTE SHEET
TABLE 9
Production Data from G0 and G, Generation Pigs Selected for High and Low Immune Responsiveness
MEAN (STD. DEV.)
A A TRAIT HIGH LOW CONTROL
Age (days) 144.00 (16.28) 154.37 (24.12)* 147.43 (22.30)
EBV.Age -2.20 (4.42) 0.80 (4.77)* -0.99 (4.60)
Fat (mm) 13.79 (2.38) 14.02 (3.23) 14.29 (2.45)
EBV.Fat -2.70 (0.88) -2.76 (1.26) -2.88 (1.06) m Production 156.78 (20.87) 141.93 (24.32)* 155.76 (23.47) Index
ITI rri * High differs from Low, p < 0.05
Table 10
Reproduction Data From Yorkshire Pigs Selected for High and Low Imune Response
TRAIT MEAN (Std. Dev.) HIGH LINE CONTROL LINE LOW LINE
*High differs from Low line, p 0.10 ** High differs from Low line, p < 0.05
SUBSTiTUTE SHEET Table 11
Reproduction Data From Yorkshire Pigs Selected for High and Low Imune Response
** High differs from Low, p 0.05
SUBSTITUTE SHEET Table 12 Scores Assigned for Antemorten Signs of Arthritis
No clinical signs of arthritis 0
One or more joints slightly swollen 1 Two or more joints moderately swollen with lameness and reluctance to move 2 Two or more joints severely swollen, severe lameness and difficulty in moving 3
SUBSTITUTE SHEET Table 13
Postmortem Observations and Scores of Relative Severity
ore
of joint capsule with moderate increase of normal synovial fluid in one or two joints
Moderately increased turbid synovial fluid and moderate, edematous villous hyperthropy in one or more joints
Highly increased very turbid synovial fluid and very edematous villous hypertrophy in one or more joints
SUBSTITUTE SHEET Table 14 Analysis of Variance for Traits Recorded After Mycoplasma hyorhinis Challenge of Yorkshire pigs.
Dependent variable Source of variation (level of significance)
CO c.v. Set Line Litter Challenge Treatment x Line
W { Clinical signs of arthritis
-I Erythrocyte sedimentation rate Fibrinogen in plasma c H Lymphocytes in blood m Segmented neutrophils in blood Antibody titre in serum m m n.s. = Not significant ■H * Coefficient of determination b Coefficient of variation c Probability > F.
Table 15
Response to Non-Selected Traits in Yorkshire Pigs
Bred for High and Low Immune Responsiveness
HIGH DIFFERS FROM LOW, » p < 0.10 and »*p < 0.05
SUBSTITUTE SHEET

Claims

1. A procedure for ranking immune system responsiveness in an animal to provide an Estimated Breeding Value (EBV) indicator of the animal's level of ability to resist disease and ability to pass such disease resistance to offspring, such EBV indicator being useful in selecting animals to be bred in order to produce offspring which inherit said level of ability to resist disease, said procedure comprising:
i) testing an animal's response to at least two tests one of which is a general measure and the other antigen specific which determine heritable humoral immunity traits;
ii) testing the same animal's response to at least two tests one of which is a general measure and the other antigen specific which determine heritable cell-mediated immunity traits;
iii) testing the animal's response to said two tests of humoral immunity traits and said two tests of cell-mediated immunity traits beginning as soon as possible after the animal has been weaned from its mother and at a time chosen to negate effects of passive immunity;
iv) ranking said animal's EBV indicator relative to other ranked animals based on said animal's level of response to said tests.
2. The ranking procedure of claim 1, in which the humoral immunity tests are:
i) serum concentration of immunoglobulin G (IgG); ii) antibody response to an antigen not expected to be part of the antigens to which the animal and its parents have been previously exposed, and the cell- mediated immunity tests consisting of:
a) peripheral blood lymphocyte blastogenic response to a mitogen: and b) cutaneous delayed type hypersensitivity (DTH) to an antigen purified protein derivative of a bacterium used to induce DTH.
3. The ranking procedure of claim 2 in which the immunoglobulin measured in said humoral immunity test is IgG.
4. The ranking procedure of claim 2 in which the antigen in said antibody response test is hen eggwhite lysozyme.
5. The ranking procedure of claim 2 in which the mitogen in said peripheral blood blastogenic response test is concanavalin A.
6. The ranking procedure of claim 2 in which the agent used to induce DTH is Bacillus Calmette-Guerin and said purified protein derivative in said delayed type
hypersensitivity test is from a human strain of
Mycobacterium tuberculosis grown on a protein-free synthetic medium.
7. The ranking procedure of claim 2 in which the animal's indicator according to the animal's immune responsiveness is designated high, low or control.
8. The ranking procedure of claim 2, in which the animals are selected from the group consisting of pigs, cattle, fish and chickens.
9. The ranking procedure in claim 7 wherein said indicator divides all ranked animals into indicated groups of high, low or control, such ranking providing the basis for breeding together only test animals from the same group.
10. The ranking procedure of claim 9 wherein said animals are pigs ranked in said high group in accordance with this procedure and bred together to yield offspring which achieve market weight consistently faster than pig offspring from bred control groups or bred low groups, said pigs of said high group have a higher percentage of live piglets per litter, a lower percentage of litters with less than three piglets, a lower percentage of deformed piglets per litter and a higher production index.
11. The ranking procedure of claim 9 wherein said animals ranked in said high and low groups differ in disease manifestations induced by infection.
12. The ranking procedure of claim 9 wherein said animals ranked in said high and low groups differ in response to vaccination such that animals ranked in the high group respond earlier, produce more antibody to antigens and have a higher percentage of animals that respond to vaccination.
13. The ranking procedure of claim 9 wherein said animals in said high and low groups differ in response to immunization such that animals ranked in the high group produce more antibody to antigens in addition to those used to derive the EBV.
14. The ranking procedure of claim 9 wherein said animals ranked in said high and low groups differ in response to immunization such that the animals ranked in the high group produce antibody of higher binding
strength for an antigen.
EP94901714A 1992-12-09 1993-12-09 Methodology for developing a superior line of domesticated animals Withdrawn EP0673509A1 (en)

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US6287564B1 (en) 1997-12-24 2001-09-11 Lauraine Wagter-Lesperance Method of identifying high immune response animals
US7258858B2 (en) 1997-12-24 2007-08-21 University Of Guelph Method of identifying high immune response animals
CA2487099A1 (en) * 2001-05-24 2002-11-28 Bruce N. Wilkie Methods for selecting and producing animals having a predicted level of immune response
EP1581043A4 (en) * 2002-06-21 2006-08-09 Pig Improvement Co Uk Ltd Methods for selection for efficient animal growth
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