Veterinary Parasitology, 37 (1990) 61-77 61 Elsevier Science Publishers B.V., Amsterdam

Is there a relationship between haemoglobin genotype and the innate resistance to

experimental Haemonchus contortus infection in Merino lambs?

T. K a s s a i I , L. F6s i i s 2, W . M . L . H e n d r i k x 3, Cs . Tak~its ~, 12. F o k ~, P. R e d P ,

E. Tak~ics ~, P h . R . N i l s s o n 3, M . A . W . v a n L e e u w e n 3, J. J a n s e n 3, W . E . B e r n a d i n a 3

a n d K . F r a n k e n a 4

J Department of Parasitology and Zoology, University of Veterinary Science, Landler J. u. 2. 1078 Budapest (Hungary)

2Research Institute of Animal Husbandry, 2053 Herceghalom (Hungary) 3Department of Helminthology and Entomology, and Department of Immunology,

Veterinary Faculty, State University of Utrecht, Yale&an 1, 3508 TD Utrecht (The Netherlands) 4Department of Animal Husbandry, Agricultural University Wageningen, Marijkeweg 40,

6700 AH Wageningen (The Netherlands)

(Accepted for publication 22 February 1990)

,ABSTRACT

Kassai, T., F6sfis, L., Hendrikx, W.M.L., Tak~its, Cs,, Fok, 1~., Redl, P., Tak~ics, E., Nilsson, Ph. R., van Leeuwen, M.A.W., Jansen, J., Bernadina, W.E. and Frankena, K., 1990. Is there a relationship between haemoglobin genotype and the innate resistance to experimental Haemonchus contortus infection in Merino lambs? Vet. Parasitol., 37: 61-77.

Responses to a single or repeated infection with 7000 infective larvae of Haemonchus contortus were studied in an experiment using a total of 106 3-month-old lambs with AA, AB or BB haemoglo- bin (Hb) genotypes. Results were assessed by faecal egg counts, adult worm counts, haematocrit val- ues, haemoglobin concentrations, total serum protein and serum antibody IgG~ and IgA ELISA titres. None of these parameters showed a strong relationship to the Hb type.

The prevalence of low responder ( > 500 worms) and of high responder ( < 50 worms) animals in groups AA, AB and BB Hb types was 3.8 and 34.6, 20.6 and 35.2, 28.1 and 43.7%, respectively, sug- gesting that the responsiveness to nematode infection is under the control of gene (s) not closely linked with those determining the Hb genotype. Worm counts of a primary infection are more subject to variation than those of a secondary infection. There is a strong relationship between adult worm counts and faecal egg counts taken close to the time of slaughter. In living animals low and high responder discrimination can be based on individual faecal egg counts around 50 days after a second- ary infection. Haematocrit values proved to be of little value in the low and high responder selection. In this regard neither Hb concentration nor total serum protein values are of practical significance. In 3-month-old lambs primary infection induced partial immunity which could prevent the establish- ment of a part of the secondary infection, irrespective of the presence or absence of the primary worm population. The development of immunity was not associated with an increase of serum lgG~ and IgA antibody levels. Specific antibody production was not influenced by Hb types. Mean antibody

0304-4017/90/$03.50 © 1990 - - Elsevier Science Publishers B.V.

62 T. KASSA1 ET AL.

levels of low responder lambs showed no difference from those of high responders. Thus, serum IgG~ and IgA levels are of no predictive value in identifying lambs which are genetically resistant to tlae- monchus infection.

I N T R O D U C T I O N

It has been well established that host genetics is a key factor in regulating the distribution of parasites in an outbred host population (Wakelin, 1978, 1985; Le Jambre, 1978; Wassom et al., 1986 ). The heritable nature of the host responsiveness raises the possibility of selective breeding of animals which are less susceptible to infection with parasites, or which develop a strong im- mune response to infection, thus limiting worm establishment and /o r sur- vival (Gregory, 1937; Whitlock and Madsen, 1958; Albers and Gray, 1986; Piper and Barger, 1988; Barger, 1989). The crucial prerequisite of the prog- ress towards practical implications of that approach is a readily identifiable genetic marker of the host which is linked to the gene (s) regulating the re- sponsiveness of the host (Outteridge et al., 1985; Courtney, 1986 ).

In the last two decades much work has been done to investigate whether haemoglobin (Hb) typing can be used as a tool for identifying responsiveness of sheep to helminth infections. Results obtained by Evans et al. ( 1963 ), Jilek and Bradley (1969), Allonby and Urquhart (1976), Preston and Allonby (1979), Altaif and Dargie ( 1976, 1978a,b) were interpreted to suggest that within breeds, sheep of Hb type AA are more resistant to infection with Hae- monchus contortus than those of the AB or BB types, and less susceptible to the pathologic consequences of worm infection. Other workers however, have found no detectable relationship between Hb type and resistance to H. con- tortus infection (Radhakrishnan et al., 1972; Yazwinski et al., 1980; Riffkin and Yong, 1984; Albers and Gray, 1986).

This paper reports the first part of a more complex study of the genetic determination of the high or low responder character of sheep. An attempt was made further to clarify the relationship between Hb types and the suscep- tibility to establishment and pathogenic effect of a primary and secondary infection with H. contortus. An important aspect of the present study was the practical discrimination of low and high responder characters in living ani- mals. It was also at tempted to relate the worm specific IgG1 and IgA produc- tion to the responsiveness of the host.

MATERIAL AND METHODS

Experimental design

A total of 106 Hungarian Merino lambs of both sexes were reared in con- finement preventing infection with parasites other than coccidia and Stron-

HAEMOGLOBIN GENOTYPE AND H. CONTORTUS INFECTION IN SHEEP 63

TABLE 1

Design o f expe r imen t s on the re la t ionship be tween haemoglob in genotype and innate resistance to Haemonchus contortus infect ion in Mer ino l ambs

Day T r e a t m e n t subgroupsa

1 2 3 4

0 Infect ion Infect ion Infect ion - 7000 L 3 7000 L3 7000 L 3

30 - - T r e a t m e n t 2 37 - Infect ion Infect ion

7000 L 3 7000 L 3 85-88 Necropsy Necropsy Necropsy

T rea tmen t 2 Infect ion 7000 L3 Necropsy

~ Formed in each group o f haemoglob in types: AA n = 29, AB n = 2Oxyclozanide 10 mg k g - ~ and levamisole 7.5 mg k g - ~. e.p.g, and blood exam i na t i ons o f Days 0, 23, 57 and 85.

38, BB n = 3 9 .

gyloides papillosus. When 3 months old, the lambs were allocated to three groups according to Hb type (AA: 29, AB: 38, BB: 39). Each group of lambs of a defined Hb type was divided into four treatment subgroups of 6-10 ani- mals each. Subgroups 1-4 were infected and treated as outlined in Table 1.

Results were assessed by faecal worm egg counts (e.p.g.), examinations of samples of jugular blood for haematocrit values (PCV), Hb concentrations, total serum protein (TSP), antibody titres at Days 0, 23, 57 and 85, and by post mortem individual total worm counts made at Days 85-88 postinfec- tion. During the study, seven lambs died for reasons unrelated to the experi- ment, and another seven lambs lost their identification number. Minor vari- ations in numbers of animals tested as seen in different tables reflect non- availability of individual samples.

Parasitological techniques

A thiabendazole-resistant strain of H. contortus isolated in The Nether- lands, and maintained in the laboratory by passage in worm-free Texel donor sheep was used for the experimental infections. Larvae were stored at 4 ° C. For infections at Days 0 and 37, L3 from the same batch were used. Thus the age of L3 used in the first and second infections varied from 2 to 14 weeks and from 7 to 19 weeks, respectively. Faecal worm egg counts (e.p.g.) were performed using the classical McMaster technique. Post-mortem worm counts were made from the total abomasal content, which was obtained by careful washing with saline of the mucosa of the opened abomasum. The total con- tent was counted for adult worms only (AWC) under dissecting microscope.

64 T. KASSAI ET AL.

Haematology

Haemoglobin types were determined by starch gel electrophoresis using the procedure described by Gahne et al. (1960). Haematocrit values (packed- cell volumes, PCV) were measured by a microhaematocrit technique (Son- nenwirth and Jarrett, 1980). Haemoglobin concentrations were determined by the cyanmethaemoglobin method (Sunderman et al., 1953 ). TSP was de- termined by Biuret's method (Sonnenwirth and Jarrett, 1980).

Serological technique

Serum antibodies were measured by the enzyme linked immunosorbent as- say (ELISA). Somatic antigen was prepared from infective larvae of H. con- tortus. Concentrated larvae were disrupted in a dismembrator at 4°C. The antigen was extracted in phosphate buffered saline (PBS) overnight, centri- fuged for 1 hour (45 100 g), and the supernatant dialyzed against distilled water for 18 h, passed through a 45 ~tm millipore filter, liophilized and stored at 4 ° C. L 3 crude antigen extract was dissolved in PBS and the protein concen- tration determined by the Lowry method (Lowry et al., 1951 ). Wells of mi- crotiterplates (Inotech) were incubated overnight at room temperature with 100/~l of a crude extract of L3 of H. contortus, protein concentration being 5 and 2/tg m l - 1, respectively, for IgA and lgG1, testing in a morpholino-ethan- sulfonic acid buffer 0.025 M, pH 6.0 (Merck), with 0.1% N/3-dimethylami- nopropyl/N-ethyl-carbodiimidhydrochlorid 0.1% (Merck), followed by an incubation for 1 h at 37°C with 150 #1 1% bovine serum albumin (BSA) (Sigma), in PBS, pH 7.3. Serum dilutions were prepared in 1% BSA in PBS. Lamb serum dilutions included 1 / 10, 1/20, 1/40 and 1/80 for IgA and 1/80, 1 / 160, 1 / 320 and 1/640 for IgGl testing. Sera were incubated for 2 h at 37 ° C. Rabbit anti-sheep IgA and IgG~ antisera, 100 #1 per well, dilution 1 / 600, were incubated for 1 h at 37°C, followed by incubation with 1/1500 conjugate (ICN ImmunoBiologicals) goat anti rabbit Ig (heavy/light chains)-peroxi- dase, 100/zl per well for 1 h at 37 ° C. Substrate solution was freshly prepared by dissolving 60 mg 5-amino-2-hydroxybenzoic acid (Sigma) in 100 ml H20, and the acidity was adjusted to pH 6.0 with NaOH. To 90 ml of this solution 9 ml H20 and 1 ml 1% H202 were added. Finally, 100/A of the latter substrate solution was added to each well and the microtiterplates were left for 1 h at room temperature. Between each step the plates were washed twice for 15 s each time with tapwater. The reaction was stopped by adding 50 pl 0.1 N NaOH. Microtiterplates were read at 405 nm (Titertek MCC Multiscan).

The extinction value of each of the four serum dilutions (animal) date was In transformed and the mean of these In values was calculated and retrans- formed, resulting in the In-extinction value, which was used for further statis- tics. During preliminary study for optimalization of the ELISA technique,

HAEMOGLOBIN GENOTYPE AND 14. C O N T O R T U S I N F E C T I O N IN SHEEP 65

evidence was obtained that both sheep IgG1 and IgA could be used to discrim- inate between the antigens of H. contortus and those of other common para- sites of sheep.

Statistical analysis

Parasitological data were analysed by analysis of variance. The e.p.g, values and worm count data were transformed by log (X+ 1 ) to normalize data. Multiple comparisons were made by the calculation of Bravis' correlation coefficient, and by regression analysis. Differences were considered signifi- cant when P < 0.05.

Statistical treatment of ELISA data was carried out by the use of the statis- tical software BMPD (Biomedical Programs, University of California). Be- fore analysis, data were checked for normality by means of skewness and kur- tosis values. Homogeneity of variances was checked by Levene's test. The Brown-Forsythe test was used to draw conclusions about the significance of factors in the model when Levene's test was significant (P< 0.05 ). For anal- ysis of variance Fisher's F-test was used to demonstrate significance. When F was significant, differences between pairs of means were inspected by the test of Bonferroni (multiple range test). The significance of the differences be- tween mean extinctions of low and high responder lambs was calculated by the t-test.

RESULTS

Worm counts

Individual adult worm counts varied between 0 and 2771. Using the anal- ysis of variance of the logarithmically transformed mean worm counts of the AA, AB and BB groups, irrespective of the experimental variables of the subgroups, the group mean worm counts of the different Hb types were found not to show statistically significant variations (AA: 188.4, AB: 281.7, BB: 439.4) (Table 2 ). An assessment of the distribution of animals with low or high worm burdens in groups of lambs of different Hb types revealed that individual worm counts showed extremely high variations within each group of l ib type (AA: 7-1700, AB: 0-1673, BB: 0-2771 ).

Since the worm count is the most direct measurement of the response of host to infection with helminths, lambs harbouring more than 500 adult worms were classified as low responders (LR), and those harbouring less than 50 worms as high responders (HR) . The prevalence of low and high responder individuals in groups of AA, AB and BB Hb types was 3.8, 20.6, 28.1, and 34.6, 35.3, 43.7%, respectively (Table 2).

Considering the obvious lack of close association of responsiveness to any

66 T. KASSAI ET AL.

TABLE 2

Adul t worm counts and the d is t r ibut ion of low and high responder an imals in groups o f different haemoglob in types

Haemog- n Adult worm coun ts j Low responder 2 High responder 3 lobin an imals an imals type

G r o u p m e a n +- SD X ' +-SD 4 n (%) n (%)

AA 26 188.4+,332.5 1.9181+,0.5715 1 3.8 9 34.6 AB 34 281.7_+422.4 1.8617+_0.8900 7 20.6 12 35.2 BB 32 439.4+_659.0 1.7574+_ 1.1344 9 28.1 14 43.7

Total 92 17 18.5 35 38.0

*Differences o f t r ans fo rmed m e a n adul t worm coun ts are not significant. 2Low responder = worm counts > 500. 3High responder = worm counts < 50. 4 X ' = l o g ( X + 1).

TABLE 3

Adul t worm counts and the d is t r ibut ion of low and high responder an imals in different t r ea tment subgroups

T r e a t m e n t n Adult worm coun ts Low responder High responder subgroups an imals an imal s

Group m e a n _+ SD X '+_SD I n (%) n (%)

1 21 75.6+,109.3 1.3171a°2+_0.8015 0 0 13 67.9 2 22 271.1+,410.5 1.7469 b +-0.9592 5 22.7 10 45.5 3 27 384.7+,691.9 1.8351 ad +_0.9006 5 18.5 II 40.7 4 22 498.9+__489.5 2.4494b~d_+ 0.6007 7 31.9 1 4.5

Total 92 17 18.5 35 38.0

~X'=log (X+ 1). 2a, d = P < 0.05, b = P < 0 . 0 1 , c = P < 0 . 0 0 1 , LSD5%=0.4888, LSD~o/o=0.6459, LSDo.I , ,=0.8351.

of the Hb types, results were assessed also according to treatment subgroups, irrespective of their Hb type classification.

Comparing the transformed mean adult worm counts by the analysis of variance, significantly more worms were found in Subgroup 4 than in any other subgroup, and the least number of worms occurred in Subgroup 1 (Ta- ble 3 ). Relatively few worms were found in Subgroups 2 and 3, both given double infection. Note that less worms were present in Subgroup 2, where two infections were superimposed, than in Subgroup 3, where worms of the pri-

HAEMOGLOBIN GENOTYPE AND H. CONTORTUS INFECTION IN SHEEP 67

mary population were removed prior to the challenge infection, although the difference was below the level o f statistical significance (Table 3 ).

Faecal worm egg counts

Logarithmically transformed mean e.p.g, values of sheep of different Hb types did not show any statistically significant differences, either at Days 20- 23, or at Days 54-57 (Table 4).

When comparing mean e.p.g, values of treatment subgroups at Days 23, 57 and 85 of the experiment, the 85-day values of Subgroup 1 was significantly lower, and of Subgroup 4 was significantly higher than means of the other three subgroups (Table 5 ).

The correlation between group mean worm counts at Day 85-88 and mean

TABLE 4

Transformed e.p.g, values in groups of different haemoglobin types on Days 23 and 57 of the primary and on Days 20 and 54 of the secondary infection

Haemog- Transformed EPG values lobin type On Days 20-23 On Days 54-57

n X' -+SD l n Y~' -+SD 1

AA 26 3.3325 -+ 0.7957 24 3.0899 -+ 0.9671 AB 34 3.1882-+ 1.0761 34 3.1866-+ 1.0123 BB 32 3.5185 -+ 0.3967 31 3.2754-+ 0.8081

IX' =log ( X + 1 ). Differences are not significant.

TABLE 5

Transformed e.p.g, values in treatment subgroups on Days 23, 57 and 85

Treatment Transformed e.p.g, values subgroups

On Day 23 On Day 57

n X' -+SD 1 n X' -+SD

On Day 85

n X' -+SD

1 21 3.4454--+0.4853 21 3.3710--+0.6671 21 2 22 3.6416-+0.3279 22 3.3713_+0.9327 22 3 27 3.3746--+0.7328 25 2.7318--+ 1.2459 26 4 22 Not done 22 2.8541 -+ 1.2512 22

1.3756abc2_+ 1.1919 2.1227 ad -+1.4525 2.6240 be _+1.1370 3.3673 ode _+0.4633

~X'=log ( X + I ) 2a, e = P < 0.05, b, c, d = P < 0.001, LSDs~=0.6670, LSD~o~,=0.8816, LSDo,~o,~= 1.1397.

68 T. KASSAI ET AL.

e.p.g, values at Days 23, 57 and 85 was also investigated by calculating the Bravis' correlation coefficient. Close correlation was found between adult worm counts and e.p.g, at Day 85 of the experiment, i.e. some 50 days after the secondary infection. As the time interval between post-mortem worm counts and faecal examination increased, the correlation became progres- sively less certain (Table 6 ). High correlation of individual adult worm counts on the respective e.p.g, values taken at Day 85 was also shown by using the regression analysis (r= 0.760, Fig. 1 ).

As the data of Subgroups 2 and 3 are likely to provide the most reliable basis for discrimination of LR and HR lambs, in these subgroups the levels of correlation between worm counts and 85-day e.p.g.s were checked sepa- rately by regression analysis. High levels of correlation were found, both for all animals belonging to these subgroups ( n = 4 8 , r=0 .769) , and for those designated as low and high responders ( n = 31, r=0 .782) . In Subgroup 4, which was given a single infection only, the level of correlation was much lower (n=22, r=0.511 ).

TABLE6

The correlation between 85-88-day adult w o r m counts and e.p.g.s observed in different treatment subgroups on Days 23, 57 and 85

Treatment subgroups

Bravis' correlation coefficient

On Day 23 On Day 57 On Day 85

1 21 0.09 0.21 0.56** 2 20 0.37 0.63*** 0.90*** 3 27 0.25 0.23 0.66*** 4 22 - -0 .02 0.53*

* = P < 0 . 0 5 , * * = P < 0 . 0 1 , ***=P<0.001.

log EPG85

5

3

2

1

L n i 1 2 3

tog odutt worm counts

n ~ O.L,02 b = 1. )01 r = 0 . 7 6 0

Fig. 1. C o r r e l a t i o n o f i n d i v i d u a l adul t w o r m c o u n t s o n the faecal w o r m egg c o u n t s (e.p.g.) at D a y 85 ( n = 9 2 ) .

HAEMOGLOBIN GENOTYPE AND H. CONTORTUS INFECTION IN SHEEP

H a e m a t o l o g i c a l p a r a m e t e r s

69

Changes of haematological parameters such as PCV, Hb concentration, and TSP measured at Days 0, 23, 57 and 85 of the experiment were related to groups of different Hb types as well as to different treatment subgroups. Group mean values reflected little if any characteristic differences attributable either to the Hb genotypes or to the variations in experimental treatments. Appar- ently all values tended to decline during a 3-week period after infection, there-

PCV %

37

35

33

31

mmol/I 7.5

7

6,5

6!

g/100 ml 6,3-

6,1-

5,9-

5,7-

55

5,3.

Haematocrit

i i i i J /

2 4 6 8 10 1 2 14 week s

Fig. 2. Effect of H. contortus infection on haematocrit, haemoglobin concentration, and total serum protein in groups of l ib types AA D - D , AB + - - + , BB O- -O , bars representing S.E.M. Larval infect ions (7000 L3) were given at Days 0 and 37.

70 T. KASSAI ET AL

PCV ° [o

37

35

33.

31.

mmo[/I 7,5

7

6,5

6

g/100 6,3

6,1-

5,9

5,7

5,5

5,3

Haematrocit

T

I I 14 weeks

Fig. 3. Effect of H. contortus infection on haematocrit, haemoglobin concentration, and total serum protein in treatment subgroups 1 O-F1, 2 + +, 3 0 - 0 , 4 A - , ~ , bars representing S.E.M. Larval infections (7000 L3) were given at Days 0 and 37.

after to rise (Figs. 2 and 3 ). Regression analysis revealed no correlation be- tween individual worm counts and 85-day haematocrit values (r= 0.028, Fig. 4).

ELISA results

Geometric mean serum IgGl and IgA levels suggested that antibody pro- duction was not influenced by Hb types (Fig. 5a,b). IgG1 levels were higher than IgA levels, however, mean extinctions did not show any tendency to in-

HAEMOGLOBIN G E N O T Y P E A N D H. CONTORTUS INFECTION IN SHEEP 7 1

PCV

.35

. 3

• 25

• o • • •

• . . • . - . . ; . • • • • •

0 1 2 3 4

Log odut t worm c o u n t s

ct = O. 3t+O

b : 7 .106e -&

r = 0 . 0 2 8

Fig. 4. Correlation of individual adult worm counts on the haematocrit values (PCV) at Day 8 5 ( n = 9 2 ) .

700

×

8 5~0

520

460

400

700

% 640

0 580

~ 520

4 6 0

.........

(a)

23 57 experimental days

J 400 85 o

I

23 57 85 experimental days

21o I 21o

~ I14L ~ ~ . . . . . . . . ~ 194 178

146

(b) ] a.

o 23 57 s5 experir~ental days

\ , \ \ \

\ , \

(6) , , 130 0

23 57 85 exper;mental d~ys

Fig. 5. ELISA serum IgG, and IgA geometric mean extinctions. (a) IgG, levels in Hb type groups. (b) IgA levels in Hb type groups AA - + - AB . . . • . . . BB --(3--. (c) IgG~ levels in treatment subgroups. (d) IgA levels in treatment subgroups 1 - + - 2 --A--- 3 ---(3--- 4 "" + " '.

crease. Similarly, the variations in either of treatment Subgroups 1-4, re- sulted in no significant increase in the IgG1 and IgA levels throughout the experiment (Fig. 5c,d). A comparison by Student's t-test o f group mean ex- tinctions of low responder lambs with those of high responders revealed no statistically significant differences (Table 7 ).

72 T. KASSAI ETAL.

TABLE 7

Mean serum IgG~ and IgA extinctions ( × 103) of low and high responder lambs on Days 0, 23, 57 and 85

Anti- body iso- types

Subgroups Mean extinctions ( × 103) on Days

0 23 57 85

n ) ( ± S D n 5~+-SD n 5~_+SD n 7~_+SD

IgGl Lowresponders 15 654.8_+202.8 16 678.2+_191.8 16 609.3_+129.8 16 610.9+-240.5 Highresponders 33 598.7+_217.6 34 610.5_+190.7 34 585.4+169.1 34 547.1+175.8

lgA Lowresponders 16 180.6+_15.8 16 195.2+_18.2 13 180.6_+19.4 16 182.2_+17.8 Highresponders 32 184.9+-35.9 34 204.3_+40.9 30 190.5+-47.3 34 196.3_+82.4

Differences between low and high responder subgroups are not significant.

DISCUSSION

The association between disease resistance or susceptibility and Hb poly- morphism of sheep has been sought for many years. Nevertheless, owing to the conflicting results of numerous studies referred to in the Introduction there remained certain vagueness as to the significance of the Hb genotype in de- termining the within-breed variations of resistance to infections with gas- trointestinal nematodes.

The fact that both high and low adult worm burdens do occur in sheep of any Hb types following a uniform infection (Table 2 ) indicates that Hb types cannot be considered as suitable correlates which could predict resistance or susceptibility to haemonchosis. This may imply that the responsiveness to helminth (H. contortus) infection is under the control of gene (s) not closely linked with those determining the Hb genotype. This conclusion is in line with the observations reported by Radhakrishnan et al. (1972), Le Jambre (1978), Yazwinski et al. (1980), Riffkin and Yong (1984), Courtney et al. (1985) and Albers and Gray (1986).

The identification of LR and HR lambs was based, in the present study, on adult worm counts. It was found, however, that the duration of the parasite population appears to be an important factor in determining the actual size of the primary worm burden. This was shown by the relatively high number of lambs harbouring less than 50 worms at Day 85 (Subgroup 1 ), as well as by the high number of animals harbouring more than 500 worms at Day 48 after infection (Subgroup 4). Because the expression of that character is sub- ject to variation, the size of a primary worm population does not provide a reliable basis for low and high responder selection. The prevalence of LR and HR lambs in various subgroups suggests that distribution between LR and

HAEMOGLOBIN GENOTYPE AND 1-1. C O N T O R T U S INFECTION 1N SHEEP 7 3

HR animals is apparent only in Subgroups 2 and 3 given double infection. Thus adult worm counts at around 50 days after a secondary infection appear to moni tor the responsiveness of the host, allowing LR and HR discrimina- tion (Table 3).

Although it is obvious that the most direct and reliable index of the respon- siveness of sheep to helminth infection is the size of the established worm population, a major drawback of applying this measurement is the necessity of slaughtering the animals, and necropsy renders lambs unfit for breeding. It is, therefore, important to look for an alternative measurement, such as faecal egg count.

It had been known that after a prepatent period of 18-21 days the egg pro- duction of female H. contortus reached a peak after about 50-60 days of in- fection (Whitlock et al., 1972). Roberts and Swan ( 1981 ) demonstrated a strong relationship ( r=0.83, P<0.001 ) between faecal egg counts and the total number of adult H. contortus. Evidence appears to indicate that factors such as the size and sex ratio of the worm population, the time of year etc. are only minor sources of variation (Poteet and Conway, 1966; Le Jambre et al., 1971; Courtney et al., 1985 ). As a measurement of resistance the use of the e.p.g, values at 4 or 6 weeks after a secondary infection was proposed (Albers et al., 1987; Barger and Dash, 1987).

In the present study, no significant differences between mean e.p.g, values of sheep of different Hb types could be seen either at Days 20-23, or at Days 54-57 of infection (Table 4). On the other hand our results confirmed the strong relationship between worm counts and e.p.g.s taken close to the time of slaughter, in our experiment at some 50 days of a secondary infection (Ta- bles 5 and 6, Fig. 1 ). It can therefore be concluded that in living animals LR and HR classification can be based on individual faecal egg counts taken around 50 days after a secondary infection.

A rough estimation of the levels ofe.p.g, corresponding to HR and LR lambs in the present study was up to 200 and above 2000, respectively. Riffkin and Dobson ( 1979 ) reported group mean e.p.g.s at Days 28-35 after primary in- fection with 12 000 L 3 o f n . contortus to be 899_+ 237 and 13 801 + 2875 for resistant and susceptible sheep, respectively. Faecal egg counts of lambs showing high responsiveness to H. contortus larval infections (Gray et al., 1987 ), or following vaccination with irradiated Trichostrongylus colubrifor- mis larvae and artificial or natural challenge (Windon and Dineen, 1984) ranged from 5 to 50% of those of the low responder counterparts. Thus the actual e.p.g, levels characteristic for high and low responder animals should be identified in each individual instance, because the take and egg production partly depend on factors such as breed and sex of host, strain of parasite, variations in infection and nutrition regimens.

Haematocrit values were especially considered since it had been suggested by Albers et al. (1987) that in view of the putative high genetic correlation between faecal egg count and haematocrit, a selection for resistance based on

74 T. KASSAI ET AL.

haematocrit values might be an alternative to the faecal egg count assay. Our own results, however, did not suggest that haematocrit values may prove a reliable or useful parameter in the selection of high responder individuals of a flock (Fig. 4). In this regard neither Hb concentration nor total serum pro- tein values are of practical significance (Figs. 2 and 3 ).

The present investigation provides data also on the relationship between Hb types of sheep and parasite-specific serum antibodies. In 3- to 6-month- old Merino lambs no relationship between Hb type and IgG~ or IgA levels was found (Fig. 5a,b).

There has been evidence to suggest that in sheep the high level of resistance to infection in the high responder genotype may have an immunological basis (Altaifand Dargie, 1978b; Adams and Davies, 1982; Wakelin, 1985; Presson et al., 1988; Barger, 1989 ), though it was also suggested that the inherent im- mune responsiveness, and increased immunological competence in older sheep may operate by different mechanisms (Riffkin and Dobson, 1979 ).

The relatively low number of worms in Subgroups 2 and 3, both given dou- ble infection, as compared with mean worm counts of Subgroup 4, given the second infection only, (Table 3), appears to indicate that a certain degree of immuni ty was induced by the primary infection in lambs prior to puberty, which could prevent the establishment of a part of the secondary worm pop- ulation. The acquisition of a partial resistance, however, was not associated with any apparent increase of serum antibody levels (Fig. 5c,d).

In the studies of Manton et al. ( 1962 ) a primary infection with H. contortzts in 2- to 4-month-old lambs conferred no protection against subsequent chal- lenge. Similarly, vaccination of lambs up to 5 months of age with irradiated H. contortus larvae failed to induce immuni ty (Urquhart et al., 1966a,b).

As regards the antibody producing capacity, Duncan et al. (1978) and Charley-Poulain et al. (1984) did not find significant increase in serum IgG levels in young lambs after infection with H. contortus, and Shubber et al. ( 1984 ) found no haemagglutinating antibody response in lambs infected with H. contortus. In older sheep Smith and Christie ( 1978 ) stimulated protection by irradiated H. contortus larvae, serum antibody titers, however, failed to correlate with resistance. By using an ELISA technique Neilson and van de Walle ( 1987 ) found enhanced serum antibody titers in vaccinated or primary infected and subsequently challenged lambs, but these titers were not related to the resistance of the host. The abomasal IgA response was reported to be more closely associated with protection to H. contortus infection (Smith and Christie, 1978; Smith et al., 1984).

In line with these observations were the results of the present study, where after primary or challenge infection with H. contortus of 3- to 4-month-old lambs no enhancement of the circulating IgG~ and IgA production could be demonstrated. Moreover, by comparing the mean ELISA titres of the lambs classified as low or high responders, no significant differences in the levels of

HAEMOGLOBIN GENOTYPE AND H. CONTORTUSINFECTION IN SHEEP 75

IgG~ and IgA antibodies were found (Table 7 ). Though it is possible that high responder lambs are simply able to mount an earlier or faster immune re- sponse, this potential is not reflected in serum antibody titers, therefore, that measurement cannot be used for the improvement of high responder host selection.

The possibility of using faecal egg counts as a selection criterion in breeding programs for innate worm resistance clearly promotes the search for the re- lationship between the presence or absence of a readily identifiable genetic marker and resistance to nematode parasites.

ACKNOWLEDGEMENTS

The work was supported by grant No. 1535 from the Hungarian Research Found (OTKA). Thanks are due to Dezs~5 Reinwald, Veterinary student, for his contribution to the haematological assessment. Emma K6sa, M~irta Maku, Piroska K~itain6 (Hungary) are thanked for excellent technical assistance, and Judith Kor6nyin6 for the preparation of the manuscript.

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