- Email: [email protected]
The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae
THE EFFECT OF SIRE SELECTION ON THE RESPONSE OF LAMBS TO VACCINATION WITH IRRADIATED TRICHOSTRONG YLUS COL UBRIFORMIS LARVAE J. K. DINEEN and R. G. WINDON CSIRO, Division of Animal Health, McMaster Laboratory,
Glebe, N.S.W. 2037, Australia
(Received 7 September 1979) Abstract-DINEEN,
J. IL and WINDON, R. G. (1980). The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology 10: 189-196. Rams selected for responsiveness and unresponsiveness to vaccination with irradiated T. colubriformis larvae at an early age were mated to unselected random bred ewes. Progeny were vaccinated with 20,000 irradiated larvae at 8 and 12 weeks of age, given anthelmintic treatment at 16 weeks and challenged with 20.0~ normal larvae at 17 weeks. The results, based on wether worm counts and ewe faecal egg counts, showed significant differences between responder and non-responder progeny. There was a significant correlation between worm counts and faecal egg counts of half-sibs from the same sire group. The occurrence of globule leucocytes was inversely related to worm burdens of wether progeny, however, no clear relationship was found with eosinophils. In virra lymphocyte stimulation using T. colubriformis L3 antigen, concanavalin A and lipopolysaccharide showed that statistically defined responder progeny, pooled from both responder and non-responder sire groups, gave higher responses than non-responder Iambs after vaccination. The results confirm that geneti~aIly-determined factors are invoIved in the response of lambs to vaccination at an early age, and indicate that rapid genetic progress may be achieved in the type of mating usually carried out under field conditions.
INDEX KEY WORDS: T. colubriformis, lambs, vaccination, irradiated larvae, responder progeny, non-responder progeny, in vitro lymphocyte stimulation, globule leucocyter, eosinophils. INTRODUCTION
WINDON, Dineen & Kelly (1980) have described an experiment in which lambs were tested for responsiveness to vaccination with irradiated Trichostrongylus colubrifirmis larvae at an early age. Individual animals were classified as either responders or non-responders to vaccination, and they have formed the parent generation in a breeding programme designed to establish responsive and non-responsive lines of sheep by appropriate assortative matings. The present communication describes the responsiveness of progeny obtained by mating selected rams with random bred unselected flock ewes. There were two main objectives in carrying out such a mating: 1. As it follows conventional practice in which the male, in particular, is selected for desired characteristics, it may indicate whether the breeding of flocks for resistance to internal parasites would be feasible under field conditions. 2. This mating wouId rapidly increase the number of animals available for future genetic studies. MATERIALS AND MBTHODS Sheep. Eighteen-month old Merino rams were mated with 2-3 year old Merino ewes. The rams were selected from those available after testing for responsiveness to
vaccination at an early age (Windon et al., 1980.f Table 1 shows the original treatment group and arithmetic mean egg count ranked over all vaccinated groups for each of the 10 rams selected for mating. Ewes were unselected for responsiveness and were obtained from a general breeding flock. At birth, ewes and lambs were housed in pens to avoid adventitious nematode infection. Male lambs were castrated at 4 weeks of age and all lambs were weaned at 11 weeks of age. Parasitological techniques. Infective larvae were obtained from sheep carrying pure T. colubriformis infections. Methods for irradiating larvae and dosing sheep have been described by Gregg, Dineen and Griffiths (1976). Larvae for vaccination were exposed to 50 krad of yradiation from a 300 Ci cobalt 60 source at 220 radsjmin. Faecal egg counts were performed using the modified McMaster technique (Whitlock, 1948) and the results were expressed as the mean eggs/g (epg) for the experimental period. The number of worms present in each sheep was estimated from two 20 ml samples of the contents and washings of the small intestine suspended in 2 I water. In vitro ceil st~rn~~at~~~.Peripheral blood was obtained by juguIar venipuncture and defibrinated using glass beads. The preparation of leucocytes has been described by Adams & Cripps (1977). Twenty ml of defibrinated blood were subjected to hypotonic lysis for removal of erythrocytes and the leucocytes were then washed twice and suspended in culture medium consisting of RPM1 1640 supplemented with 10% v/v sterile worm-free 189
190
I.J.P. VOL. 10. 1980
J. K. DINEENand R. G. WINDON TABLE ~--SELECTION OF PARENT RAMS FOR F~ MATINGS
Tag Responders D869 D919 D874 D979 A9 Non-responders A5 Al2 D909 D902 D982
Arithmetic mean faecal epg during primary challenge f
Treatment group [No. larvae-months]* 6-[l&0001+3] l-HO.OOO-1+2+31
1 2
124 240 302 324 388
l-~lo;ooo-l+2+3j
3-_[30,000- 21 3-_[30,000- 21 7-[15,000-2+3] 5-]15,0001+ 21 4-[30,00031 6--[15,000-1+3] 5-[15,000-1+21
Rank
4
5 35 36 38 40 41
1956 1982 2006 2291 4262
* Details of treatments and groups from which parent rams were selected are given in Windon et al. (1980). Group number of irradiated T. colubriformis larvae and the age/s of the animals when these were given are included in the present table. t Primary challenge with normal T.colubr(formis larvae was commenced when the animals were 1 month old (for details-see Windon et al., 1980). wether serum, 2 mM L-glutamine, 100 i.u. penicillin G and 100 ug streptomycin per ml. Cells were counted in a haemocytometer under phase contrast. Cultures were made in 10 x 75 mm flint glass tubes and contained 2 x lo6 lymphocytes in 1 ml culture medium. The responsiveness of lymphocytes from each sheep was assessed using triplicate unstimulated control cultures and cultures stimulated with 20 ug (protein) T. colubriformis third stage larval antigen (TcL,) prepared as described by Adams & Cripps (1977) 4 ug concanavalin A (Calbiochem, San Diego, California, U.S.A.) (con A) and 16 ug lipopolysaccharide W (Difco Laboratories, Detroit, Michigan, U.S.A.) (LPS). Cultures were incubated at 37°C in a humidified atmosphere of 5% carbon dioxide in air for 72 h when 0.25 uCi of 3H-thymidine (Thymidine [6-3H], specific activity 5.0 Ci/mMol, NET 355, New England Nuclear, Boston, Mass., U.S.A.) was added to each culture. After a further 18 h incubation cells were collected onto glass fibre discs using a Skatron cell harvester. After drying, the discs were transferred to counting vials containing 5 ml of scintillation fluid (12.21 g PPO, 0.3 g POPOP/2.25 1 toluene) and incorporation of 3H-thymidine was determined. The lymphocyte response was expressed as the stimulation index (SI), where mean counts/min stimulated cultures SI = mean counts/min unstimulated cultures. for each treatment, i.e. TcL,, LPS and Con A. Histological examinations. Duodenal sections were taken 30 cm below the pylorus, fixed in 10 % corrosive formalin and stained with chromotrope (Lendrum, 1944) and haematoxylin. The sections were scanned systematically and a subjective assessment of globule leucocyte and eosinophil numbers was made using a scale of O-3 (i.e., no cells observed to numerous cells present). Mating. Mating was carried outin twogroups with the 5 responder rams joined to 200 ewes and the 5 nonresponder rams joined to 200 ewes. Each ram was fitted with a harness with a different coloured crayon. After
each ram had distinctively marked 20 ewes, these ewes and the ram were placed in a small paddock (approximately 1 ha) for a period of 3 weeks and returns to service were recorded. At the completion of the mating the rams were removed and the ewes combined as a flock until lambing. Testing responsiveness of lambs. The method of determining the responsiveness of lambs involved vaccination with 20,000 irradiated T. colubriformis larvae at 8 and 12 weeks of age, anthelmintic treatment (320 mg levamisole) (Nilverm, ICI Australia Ltd.) at 16 weeks and challenge with 20,000 normal T. colubriformis larvae at 17 weeks. A control group of unvaccinated lambs containing 9 wether lambs from responder sires and 10 wether lambs from non-responder sires were also challenged with 20,000 larvae at 17 weeks. Faecal samples for egg counts were collected from the ewe progeny at weekly intervals and infections were terminated (320 mg levamisole) at 34 weeks when egg counts from both responder and non-responder groups had reached low levels. Faecal egg counts were performed on unvaccinated wethers at 3 and 4 weeks after challenge and both vaccinated and unvaccinated wethers were killed for worm counts and histological examination at 4 weeks after challenge (21 weeks of age). Blood was collected at intervals, as shown in Fig. 2, from 16 vaccinated ewe progeny of responder sires and 16 of nonresponder sires for determination of in vitro cellular responses.
RESULTS Sire influence on the response to vaccination and challenge of half-siblings Fl generation wethers. Worm counts of the wether
progeny are summarised in Table 2. Analysis of variance confirmed that means of progeny of both responder and non-responder sires (3813 and 6387,
I.J.P. VOL.
TABLE
Sire selection and response to vaccination with T. colubriformis
10. 1980
191
Z-TOTAL WORM COUNTS OF VACCINATED WETHER PROGENY, UNVACCINATED CONTROL WETHER PROGENY FAECALEGG COUNTS OF VACCINATED EWE PROGENYFROM RESPONDER AND NON-RESPONDER SIRES
Sires
Vaccinated wether progeny Arithmetic mean No. of lambs worm count
Rank
Tag Responders D869 D919 D874 D979 A9 Mean Non-responder A5 A12 D909 D902 D982 Mean
1 2 3 4 5
7 6 6 6 6 31
35 36 38 40 41
Responders Non-responders Mean
Vaccinated ewe progeny Arithmetic mean No. of lambs faecal epg
6043 3967 5033 1567 2083 3813
6 6 7 5 7 31 Unvaccinated 9 10 19
4941 5192 7257 8900 5986 6387 wether controls 12294 10985 11605
respectively) were significantly less (P -C 0.001) than unvaccinated controls (11605). In addition the effect of selection, i.e. responder vs. non-responder sires, approached significance at the 5% level (P = 0.063 on 8 df). There was no significant correlation between sire arithmetic mean faecal epg, (Table 1) and mean worm counts of the corresponding siblings (Table 2) within responder and nonresponder sire groups. FI generation ewes. Geometric mean faecal epg after challenge of ewe lambs from responderand
AND
8 3 6 7 5 29
125 131 260 26 117 128
9 5 4 5 9 32
515 1076 1158 1277 211 717
non-responder-sire groups and a group of age- and breed-matched unvaccinated control wether lambs are shown in Fig. 1. Mean epg after challenge of ewe half-siblings of individual responder and nonresponder sires are given in Table 2. Fig. 1 shows that the faecal egg counts of both vaccinated groups were significantly less than unvaccinated controls and that egg counts of progeny of responder sires were lower than egg counts of progeny of nonresponder sires. Analysis of variance showed that the effect of selection (responder vs non-responder sires)
Female progeny from : Responder sires(c-o) Non-responder sires (H) Unvaccinated controls(l-n)
I
3
4
5
6
7
6 9 10 il Weeks after challenge
12
13
14
I5
FIG. 1. Geometric mean faecal egg counts of ewe progeny from responder and non-responder controls.
16
17
sires and unvaccinated
192
J. K.
and R. G. WINDON
DINEEN
I.J.P. VOL. 10. 1980
TABLE ~--NUMBERS AND MEANS OF RESPONDER AND NON-RESPONDER PROGENY FROMRESPONDER AND NON-RESPONDER SIRES Progeny classification* Responder Non-responder No. Mean No. Mean
Sires Ewe progeny counts Responder Non-responder Overall Wether progeny worm counts Responder Non-responder Overall
27 22 49
70”T 230b 120
27 20 47
2960’ 3960’ 3385
2 10 12
2239” 3322” 2569
4 11 15
9580” 973C’ 9687
* See text. t Means hearing the same superscripts within columns for each sex are not significantly different at the 5 % level by 1.s.d. Ewe egg count data transformed log,, (x + 1) for analysis.
18
-
TcL3 20 yg/culture
lSy
progeny
Non-responder
progeny
T
LPS
Culture no. 0
Responder
-
16 c
16 pg/cuhm
123 6
4 S
10
5 12
14
6 16
7
8
9
20
24
28
10 34
36
i\ge(weeks)
FIG. 2. Mean stimulation indices of lymphocytes from ewe lambs classified as responders and non-responders with TcL, antigen, LPS and con A.
cultured
I.J.P. VOL. 10.
1980
on ewe lamb performance
Sire selection and response to vaccination with 2’. colubriformis was significant
(P = 0.021
on 8 df). However there was no significant correlation between the egg counts of sires and their siblings within responder and non-responder groups.
No. of animals withineach range 2.4 2.2
18
r
T T
of progeny
into responders
and non-
As in a previous study (Windon et al, 1980), progeny were themselves classified as either ‘resnonders’ or ‘non-resvonders’. Resvonders were defined by the formula _ x < x - t o.l 2/ (9/n) + s2 where x is the lamb’s individual performance, X and s2 are the sample mean and variance of unvaccinated controls using egg counts for female progeny and worm counts for wether progeny. The classification of progeny from responder and non-responder sires are shown in Table 3. Table 3 shows that ewes and wethers had similar numbers of responders and non-responders, however the mean differences between progeny within and between sire types are more marked in ewe lambs. In both ewes and wethers, vaccination was most effective in responder progeny from responder sires. Correlation of the response of ewe and wether half-siblings
A significant correlation coefficient (r = 0.681, 8 df, P = 0.03) was calculated for mean faecal egg counts of ewes and mean worm counts of halfsibling-related wethers (see Table 2). In vitro stimulation of peripheral lymphocytes with T. colubriformis Ls antigen, con A and LPS Of the 32 ewe progeny which were bled for cell cultures, those having responder sires were all classified as responder progeny, whereas of those having non-responder sires, 11 were responders and 5 were non-responders. Figure 2 shows mean stimulation indices of lymphocytes from these responder and non-responder progeny cultured with TcL3 antigen, LPS and con A. After vaccination the mean stimulation indices of responder progeny were consistently higher than those of non-responders for both antigen and the mitogens. The relationship between globule leucocytes, eosinophils and worm burdens
The relative abundance of globule leucocytes and eosinophils in tissue sections of small intestine of wether lambs with different worm burdens after challenge are illustrated in Fig. 3. Although the results are only semi-quantitative, being based upon a subjective scoring system, it is obvious that the number of globule leucocytes present in sections is inversely related to the number of worms recovered. This inverse relationship is not clearly defined for eosinophils and worm burdens.
6
I
1'6
Classification responders
193
: 1.6 * ?!1.4 H 1.2 z 1.0 C 1.6 % = 1.6
2001_. 4wl
Worm
FIG.
3.
eosinophil
6000
6000
10000
count ranges
Relationship of globule (0)
leucocyte (a) and score with worm counts from wether progeny.
Whereas eosinophils were confined to lamina propria, globule leucocytes were prominently distributed in epithelium (Fig. 4), particularly of the villi, and intruded into the lamina propria when present in large numbers. Occasionally, morphologically intact globule leucocytes were found in the lumen but this might be due to an artefact of fixation or handling of the tissue. Globule leucocytes were not present in tissues containing worm sections (Fig. 5). DISCUSSION The primary aim was to investigate the influence of parental genetic constitution on the responsiveness of siblings to vaccination and challenge with T. colubriformis. A mating procedure was used which is relevant to normal field practice, i.e. mating of selected sires across an unselected ewe flock. Faecal egg counts of ewe lambs (Fig. 1) and worm counts of wether lambs (Table 2) show that progeny of responder sires are more responsive than progeny of non-responder sires. The influence of the genetic constitution of sire on progeny performance is further emphasised by the finding that the performances of ewe and wether half-siblings were highly positively correlated (r = 0.68). However there was no significant correlation between sibling performance and sire-rank within responder and non-responder sires. Two factors may have contributed to the failure to obtain this correlation: 1. Although all sires were given, as lambs, the same challenge infection, they were selected from treatment groups subjected to different vaccination schedules (see Table 1). Apparently this factor has not affected the broad responder/nonresponder classification, but it may have affected the validity of sire rank within these classifications.
J. K. DINEEN and R. G. WINDON
194
FIG. 4.
Intraepithelial
FIG. 5. Worm
section
(WS)
globule leucocytes
(GL)
I.J.P. VOL.
10. 1980
in the small intestine of a resistant lamb phase contrast.
in small intestine of a susceptible contrast.
lamb. Note absence of globule leucocytes
phase
I.J.P.
VOL. 10. 1980
Sire selection and response to vaccination with T. coi~~rifor~ri~
2. It is likely that mating with unselected ewes contributed additional genetic variance which could affect mean progeny rank within responder and non-responder sires. Accepting that faecal egg counts and worm counts are closely related in young Iambs infected with T. colubriformis (Gregg, Dineen, Rothwell & Kelly, 1978) the relationship between egg counts of ewe progeny and worm counts of wether progeny of responder and non-responder sires (128 : 717 and 3813 : 6387 respectively, see Table 2) suggests that selection was expressed more vigorously in ewe than wether lambs. This apparent interaction with sex is considered in greater detail in a subsequent study. The occurrence of circulating antigen-reactive cells and lymphocytes responding to the T- and B-cell mitogens con A and LPS respectively, was monitored by the in vitro uptake of 3H-thymidine. Although cell cultures prepared from blood of statistically defined responders gave higher SI’s than preparations from non-responders, this only occurred after vaccination was commenced (Fig. 2). This may indicate that the genetic influence has affected a specific component of immunological responsiveness, that is, the recognition of antigen which precedes the sequence of non-specific events causing worm expulsion (see Dineen, Gregg, Windon, Donald & Kelly, 1977). Either quantitative or qualitative aspects of antigen recognition may be deficient in non-responder animals. It was disappointing that there was no significant difference between responder and non-responder cultures set up before vaccination (cultures 1-3 inclusive). If such a difference had occurred it would have indicated that selection, based on response to vaccination and challenge with T. colubriformis, was resulting in a general improvement of immunological performance. In addition a strong correlation between cell SI’s before vaccination and resistance to challenge, would provide a valuable marker for selection. However, the present results indicate that in vitro culture of lymphocytes with T- and B-cell mitogens is unlikely to provide such a marker. Previously (Windon et al, 1980) and again in the present study, we were not able to show a relationship between haemoglobin-type and the responsiveness of vaccinated lambs which might have been expected from the work of Evans, Blunt & Southcott (1963), Allonby & Urquhart (1976) and Altaif & Dargie (1978). On the other hand, the previously described relationship between intestinal globule leucocytes and resistance (O’Sullivan & Donald, 1973; Gregg et al, 1978) was again evident (Fig. 3). Although there was a tendency for the number of eosinophils to be reduced in intestinal lamina propria of wether lambs harbouring substantial worm burdens after challenge, the association of eosinophils with resistance to challenge is not convincing.
195
The results confirm that substantial progress towards increased responsiveness of lambs to vaccination and challenge with the nematode can be achieved in one generation of selection+even though the relevant genetic component was contributed by sire alone. Currently we are assessing the significance of the genetic component of resistance in iambs which are the progeny of both selected dams and sires.
Acknowledgements-me
wish to thank Mrs. Elizabeth Townsend, Messrs Athol Luker and Stephen Ferguson for their excellent technical assistance and Mr. Cyril Samways of the School of Chemical Engineering, University of New South Wales, for irradiation of T. colubriformis infective larvae. We are also indebted to Mr. Robert Do&on for assistance with statistical analyses. This work was supported by a grant from the
Rural Credits Development Fund (Reserve Bank of Australia).
ADAMSD. B. & CRIPPS A. W. 1977. Cellular changes in the intestinal lymph of sheep infected with the enteric nematode, Trichostrongylus colubriformis. The Australian Journal of Experimental Biology and Medical Science 5s: 509-522.
A~LONBYE. W. & URQUHARTG. M. 1976. A possible relationship between haemonchosis and haemoglob~n polymorphism in Merino sheep in Kenya. Research in Veterinary Science. 20: 212-214.
ALTAIFK. 1. & DARGIEJ. D. 1978. Genetic resistance to helminths. Comparison of the development of Ostertagia circumcincta infections in Scottish Blackface sheep of different haemoglobin type. Research in Veterinary Science 24: 391-393.
EVANSJ. V., BLUNT M. H. & SOU~HCOTTW. H. 1963. The effects of infection with Haemonchus contortus on the sodium and potassium concentrations in the erythrocytes and plasma, in sheep of different haemoglobin types. Australian Journal of Agricultural Research 14: 549-558.
DINEENJ. K., GREGG P., WINDONR. G., DONALDA. D. & KELLY J. D. 1977. The role of immunologically specific and non-specific components of resistance in cross-protection to intestinal nematodes. international Journal for Parasitology 7: 211-215. GREGG P., DINEEN J. K. & GRIFFITHSD. A. 1976. The effect of y-radiation on the development of infective larvae of Trichostrongylus colubriformis in guinea pigs and sheep. Veterinary Parasitology 2: 363-375. GREGG P., DINEEN3. K., ROTHWELLT. L. W. & KELLY J. D. 1978. The effect of age on the response of sheep with irradiated Trichostrongylus to vaccination colubriformis larvae. Veterinary Parasitology 4: 35-48. LENDRUMA. C. 1944. The staining of eosinouhil oolvmorphs and enterochromaffin cells in h&tological sections. Journal of Pathology and Bacteriology. 56: 441.
196
J. K. DINEENand R. G. WINDON
O’SULUVANB. M. & DONALDA. D. 1973. Responses to infection with Haemonchus contortus and Trichostrongylus colubriformis in ewes of different reproductive status. International Journal for Parasitology 3: 521-530.
WHITLOCK H. V. 1948. Some modifications of the McMaster helminth egg-counting technique apparatus.
Journal of the Council for Research 21: 177-180.
I.J.P. VOL. 10. 1980 Scientific
and Industrial
WINDONR. G., DINEEN J. K. & KELLY J. D. 1980. The segregation of lambs into ‘responders’ and ‘nonresponders’: response to vaccination with irradiated Trichostrongylus colubriformis larvae before weaning. International Journal for Parasitology 10: 65-73.
Glebe, N.S.W. 2037, Australia
(Received 7 September 1979) Abstract-DINEEN,
J. IL and WINDON, R. G. (1980). The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology 10: 189-196. Rams selected for responsiveness and unresponsiveness to vaccination with irradiated T. colubriformis larvae at an early age were mated to unselected random bred ewes. Progeny were vaccinated with 20,000 irradiated larvae at 8 and 12 weeks of age, given anthelmintic treatment at 16 weeks and challenged with 20.0~ normal larvae at 17 weeks. The results, based on wether worm counts and ewe faecal egg counts, showed significant differences between responder and non-responder progeny. There was a significant correlation between worm counts and faecal egg counts of half-sibs from the same sire group. The occurrence of globule leucocytes was inversely related to worm burdens of wether progeny, however, no clear relationship was found with eosinophils. In virra lymphocyte stimulation using T. colubriformis L3 antigen, concanavalin A and lipopolysaccharide showed that statistically defined responder progeny, pooled from both responder and non-responder sire groups, gave higher responses than non-responder Iambs after vaccination. The results confirm that geneti~aIly-determined factors are invoIved in the response of lambs to vaccination at an early age, and indicate that rapid genetic progress may be achieved in the type of mating usually carried out under field conditions.
INDEX KEY WORDS: T. colubriformis, lambs, vaccination, irradiated larvae, responder progeny, non-responder progeny, in vitro lymphocyte stimulation, globule leucocyter, eosinophils. INTRODUCTION
WINDON, Dineen & Kelly (1980) have described an experiment in which lambs were tested for responsiveness to vaccination with irradiated Trichostrongylus colubrifirmis larvae at an early age. Individual animals were classified as either responders or non-responders to vaccination, and they have formed the parent generation in a breeding programme designed to establish responsive and non-responsive lines of sheep by appropriate assortative matings. The present communication describes the responsiveness of progeny obtained by mating selected rams with random bred unselected flock ewes. There were two main objectives in carrying out such a mating: 1. As it follows conventional practice in which the male, in particular, is selected for desired characteristics, it may indicate whether the breeding of flocks for resistance to internal parasites would be feasible under field conditions. 2. This mating wouId rapidly increase the number of animals available for future genetic studies. MATERIALS AND MBTHODS Sheep. Eighteen-month old Merino rams were mated with 2-3 year old Merino ewes. The rams were selected from those available after testing for responsiveness to
vaccination at an early age (Windon et al., 1980.f Table 1 shows the original treatment group and arithmetic mean egg count ranked over all vaccinated groups for each of the 10 rams selected for mating. Ewes were unselected for responsiveness and were obtained from a general breeding flock. At birth, ewes and lambs were housed in pens to avoid adventitious nematode infection. Male lambs were castrated at 4 weeks of age and all lambs were weaned at 11 weeks of age. Parasitological techniques. Infective larvae were obtained from sheep carrying pure T. colubriformis infections. Methods for irradiating larvae and dosing sheep have been described by Gregg, Dineen and Griffiths (1976). Larvae for vaccination were exposed to 50 krad of yradiation from a 300 Ci cobalt 60 source at 220 radsjmin. Faecal egg counts were performed using the modified McMaster technique (Whitlock, 1948) and the results were expressed as the mean eggs/g (epg) for the experimental period. The number of worms present in each sheep was estimated from two 20 ml samples of the contents and washings of the small intestine suspended in 2 I water. In vitro ceil st~rn~~at~~~.Peripheral blood was obtained by juguIar venipuncture and defibrinated using glass beads. The preparation of leucocytes has been described by Adams & Cripps (1977). Twenty ml of defibrinated blood were subjected to hypotonic lysis for removal of erythrocytes and the leucocytes were then washed twice and suspended in culture medium consisting of RPM1 1640 supplemented with 10% v/v sterile worm-free 189
190
I.J.P. VOL. 10. 1980
J. K. DINEENand R. G. WINDON TABLE ~--SELECTION OF PARENT RAMS FOR F~ MATINGS
Tag Responders D869 D919 D874 D979 A9 Non-responders A5 Al2 D909 D902 D982
Arithmetic mean faecal epg during primary challenge f
Treatment group [No. larvae-months]* 6-[l&0001+3] l-HO.OOO-1+2+31
1 2
124 240 302 324 388
l-~lo;ooo-l+2+3j
3-_[30,000- 21 3-_[30,000- 21 7-[15,000-2+3] 5-]15,0001+ 21 4-[30,00031 6--[15,000-1+3] 5-[15,000-1+21
Rank
4
5 35 36 38 40 41
1956 1982 2006 2291 4262
* Details of treatments and groups from which parent rams were selected are given in Windon et al. (1980). Group number of irradiated T. colubriformis larvae and the age/s of the animals when these were given are included in the present table. t Primary challenge with normal T.colubr(formis larvae was commenced when the animals were 1 month old (for details-see Windon et al., 1980). wether serum, 2 mM L-glutamine, 100 i.u. penicillin G and 100 ug streptomycin per ml. Cells were counted in a haemocytometer under phase contrast. Cultures were made in 10 x 75 mm flint glass tubes and contained 2 x lo6 lymphocytes in 1 ml culture medium. The responsiveness of lymphocytes from each sheep was assessed using triplicate unstimulated control cultures and cultures stimulated with 20 ug (protein) T. colubriformis third stage larval antigen (TcL,) prepared as described by Adams & Cripps (1977) 4 ug concanavalin A (Calbiochem, San Diego, California, U.S.A.) (con A) and 16 ug lipopolysaccharide W (Difco Laboratories, Detroit, Michigan, U.S.A.) (LPS). Cultures were incubated at 37°C in a humidified atmosphere of 5% carbon dioxide in air for 72 h when 0.25 uCi of 3H-thymidine (Thymidine [6-3H], specific activity 5.0 Ci/mMol, NET 355, New England Nuclear, Boston, Mass., U.S.A.) was added to each culture. After a further 18 h incubation cells were collected onto glass fibre discs using a Skatron cell harvester. After drying, the discs were transferred to counting vials containing 5 ml of scintillation fluid (12.21 g PPO, 0.3 g POPOP/2.25 1 toluene) and incorporation of 3H-thymidine was determined. The lymphocyte response was expressed as the stimulation index (SI), where mean counts/min stimulated cultures SI = mean counts/min unstimulated cultures. for each treatment, i.e. TcL,, LPS and Con A. Histological examinations. Duodenal sections were taken 30 cm below the pylorus, fixed in 10 % corrosive formalin and stained with chromotrope (Lendrum, 1944) and haematoxylin. The sections were scanned systematically and a subjective assessment of globule leucocyte and eosinophil numbers was made using a scale of O-3 (i.e., no cells observed to numerous cells present). Mating. Mating was carried outin twogroups with the 5 responder rams joined to 200 ewes and the 5 nonresponder rams joined to 200 ewes. Each ram was fitted with a harness with a different coloured crayon. After
each ram had distinctively marked 20 ewes, these ewes and the ram were placed in a small paddock (approximately 1 ha) for a period of 3 weeks and returns to service were recorded. At the completion of the mating the rams were removed and the ewes combined as a flock until lambing. Testing responsiveness of lambs. The method of determining the responsiveness of lambs involved vaccination with 20,000 irradiated T. colubriformis larvae at 8 and 12 weeks of age, anthelmintic treatment (320 mg levamisole) (Nilverm, ICI Australia Ltd.) at 16 weeks and challenge with 20,000 normal T. colubriformis larvae at 17 weeks. A control group of unvaccinated lambs containing 9 wether lambs from responder sires and 10 wether lambs from non-responder sires were also challenged with 20,000 larvae at 17 weeks. Faecal samples for egg counts were collected from the ewe progeny at weekly intervals and infections were terminated (320 mg levamisole) at 34 weeks when egg counts from both responder and non-responder groups had reached low levels. Faecal egg counts were performed on unvaccinated wethers at 3 and 4 weeks after challenge and both vaccinated and unvaccinated wethers were killed for worm counts and histological examination at 4 weeks after challenge (21 weeks of age). Blood was collected at intervals, as shown in Fig. 2, from 16 vaccinated ewe progeny of responder sires and 16 of nonresponder sires for determination of in vitro cellular responses.
RESULTS Sire influence on the response to vaccination and challenge of half-siblings Fl generation wethers. Worm counts of the wether
progeny are summarised in Table 2. Analysis of variance confirmed that means of progeny of both responder and non-responder sires (3813 and 6387,
I.J.P. VOL.
TABLE
Sire selection and response to vaccination with T. colubriformis
10. 1980
191
Z-TOTAL WORM COUNTS OF VACCINATED WETHER PROGENY, UNVACCINATED CONTROL WETHER PROGENY FAECALEGG COUNTS OF VACCINATED EWE PROGENYFROM RESPONDER AND NON-RESPONDER SIRES
Sires
Vaccinated wether progeny Arithmetic mean No. of lambs worm count
Rank
Tag Responders D869 D919 D874 D979 A9 Mean Non-responder A5 A12 D909 D902 D982 Mean
1 2 3 4 5
7 6 6 6 6 31
35 36 38 40 41
Responders Non-responders Mean
Vaccinated ewe progeny Arithmetic mean No. of lambs faecal epg
6043 3967 5033 1567 2083 3813
6 6 7 5 7 31 Unvaccinated 9 10 19
4941 5192 7257 8900 5986 6387 wether controls 12294 10985 11605
respectively) were significantly less (P -C 0.001) than unvaccinated controls (11605). In addition the effect of selection, i.e. responder vs. non-responder sires, approached significance at the 5% level (P = 0.063 on 8 df). There was no significant correlation between sire arithmetic mean faecal epg, (Table 1) and mean worm counts of the corresponding siblings (Table 2) within responder and nonresponder sire groups. FI generation ewes. Geometric mean faecal epg after challenge of ewe lambs from responderand
AND
8 3 6 7 5 29
125 131 260 26 117 128
9 5 4 5 9 32
515 1076 1158 1277 211 717
non-responder-sire groups and a group of age- and breed-matched unvaccinated control wether lambs are shown in Fig. 1. Mean epg after challenge of ewe half-siblings of individual responder and nonresponder sires are given in Table 2. Fig. 1 shows that the faecal egg counts of both vaccinated groups were significantly less than unvaccinated controls and that egg counts of progeny of responder sires were lower than egg counts of progeny of nonresponder sires. Analysis of variance showed that the effect of selection (responder vs non-responder sires)
Female progeny from : Responder sires(c-o) Non-responder sires (H) Unvaccinated controls(l-n)
I
3
4
5
6
7
6 9 10 il Weeks after challenge
12
13
14
I5
FIG. 1. Geometric mean faecal egg counts of ewe progeny from responder and non-responder controls.
16
17
sires and unvaccinated
192
J. K.
and R. G. WINDON
DINEEN
I.J.P. VOL. 10. 1980
TABLE ~--NUMBERS AND MEANS OF RESPONDER AND NON-RESPONDER PROGENY FROMRESPONDER AND NON-RESPONDER SIRES Progeny classification* Responder Non-responder No. Mean No. Mean
Sires Ewe progeny counts Responder Non-responder Overall Wether progeny worm counts Responder Non-responder Overall
27 22 49
70”T 230b 120
27 20 47
2960’ 3960’ 3385
2 10 12
2239” 3322” 2569
4 11 15
9580” 973C’ 9687
* See text. t Means hearing the same superscripts within columns for each sex are not significantly different at the 5 % level by 1.s.d. Ewe egg count data transformed log,, (x + 1) for analysis.
18
-
TcL3 20 yg/culture
lSy
progeny
Non-responder
progeny
T
LPS
Culture no. 0
Responder
-
16 c
16 pg/cuhm
123 6
4 S
10
5 12
14
6 16
7
8
9
20
24
28
10 34
36
i\ge(weeks)
FIG. 2. Mean stimulation indices of lymphocytes from ewe lambs classified as responders and non-responders with TcL, antigen, LPS and con A.
cultured
I.J.P. VOL. 10.
1980
on ewe lamb performance
Sire selection and response to vaccination with 2’. colubriformis was significant
(P = 0.021
on 8 df). However there was no significant correlation between the egg counts of sires and their siblings within responder and non-responder groups.
No. of animals withineach range 2.4 2.2
18
r
T T
of progeny
into responders
and non-
As in a previous study (Windon et al, 1980), progeny were themselves classified as either ‘resnonders’ or ‘non-resvonders’. Resvonders were defined by the formula _ x < x - t o.l 2/ (9/n) + s2 where x is the lamb’s individual performance, X and s2 are the sample mean and variance of unvaccinated controls using egg counts for female progeny and worm counts for wether progeny. The classification of progeny from responder and non-responder sires are shown in Table 3. Table 3 shows that ewes and wethers had similar numbers of responders and non-responders, however the mean differences between progeny within and between sire types are more marked in ewe lambs. In both ewes and wethers, vaccination was most effective in responder progeny from responder sires. Correlation of the response of ewe and wether half-siblings
A significant correlation coefficient (r = 0.681, 8 df, P = 0.03) was calculated for mean faecal egg counts of ewes and mean worm counts of halfsibling-related wethers (see Table 2). In vitro stimulation of peripheral lymphocytes with T. colubriformis Ls antigen, con A and LPS Of the 32 ewe progeny which were bled for cell cultures, those having responder sires were all classified as responder progeny, whereas of those having non-responder sires, 11 were responders and 5 were non-responders. Figure 2 shows mean stimulation indices of lymphocytes from these responder and non-responder progeny cultured with TcL3 antigen, LPS and con A. After vaccination the mean stimulation indices of responder progeny were consistently higher than those of non-responders for both antigen and the mitogens. The relationship between globule leucocytes, eosinophils and worm burdens
The relative abundance of globule leucocytes and eosinophils in tissue sections of small intestine of wether lambs with different worm burdens after challenge are illustrated in Fig. 3. Although the results are only semi-quantitative, being based upon a subjective scoring system, it is obvious that the number of globule leucocytes present in sections is inversely related to the number of worms recovered. This inverse relationship is not clearly defined for eosinophils and worm burdens.
6
I
1'6
Classification responders
193
: 1.6 * ?!1.4 H 1.2 z 1.0 C 1.6 % = 1.6
2001_. 4wl
Worm
FIG.
3.
eosinophil
6000
6000
10000
count ranges
Relationship of globule (0)
leucocyte (a) and score with worm counts from wether progeny.
Whereas eosinophils were confined to lamina propria, globule leucocytes were prominently distributed in epithelium (Fig. 4), particularly of the villi, and intruded into the lamina propria when present in large numbers. Occasionally, morphologically intact globule leucocytes were found in the lumen but this might be due to an artefact of fixation or handling of the tissue. Globule leucocytes were not present in tissues containing worm sections (Fig. 5). DISCUSSION The primary aim was to investigate the influence of parental genetic constitution on the responsiveness of siblings to vaccination and challenge with T. colubriformis. A mating procedure was used which is relevant to normal field practice, i.e. mating of selected sires across an unselected ewe flock. Faecal egg counts of ewe lambs (Fig. 1) and worm counts of wether lambs (Table 2) show that progeny of responder sires are more responsive than progeny of non-responder sires. The influence of the genetic constitution of sire on progeny performance is further emphasised by the finding that the performances of ewe and wether half-siblings were highly positively correlated (r = 0.68). However there was no significant correlation between sibling performance and sire-rank within responder and non-responder sires. Two factors may have contributed to the failure to obtain this correlation: 1. Although all sires were given, as lambs, the same challenge infection, they were selected from treatment groups subjected to different vaccination schedules (see Table 1). Apparently this factor has not affected the broad responder/nonresponder classification, but it may have affected the validity of sire rank within these classifications.
J. K. DINEEN and R. G. WINDON
194
FIG. 4.
Intraepithelial
FIG. 5. Worm
section
(WS)
globule leucocytes
(GL)
I.J.P. VOL.
10. 1980
in the small intestine of a resistant lamb phase contrast.
in small intestine of a susceptible contrast.
lamb. Note absence of globule leucocytes
phase
I.J.P.
VOL. 10. 1980
Sire selection and response to vaccination with T. coi~~rifor~ri~
2. It is likely that mating with unselected ewes contributed additional genetic variance which could affect mean progeny rank within responder and non-responder sires. Accepting that faecal egg counts and worm counts are closely related in young Iambs infected with T. colubriformis (Gregg, Dineen, Rothwell & Kelly, 1978) the relationship between egg counts of ewe progeny and worm counts of wether progeny of responder and non-responder sires (128 : 717 and 3813 : 6387 respectively, see Table 2) suggests that selection was expressed more vigorously in ewe than wether lambs. This apparent interaction with sex is considered in greater detail in a subsequent study. The occurrence of circulating antigen-reactive cells and lymphocytes responding to the T- and B-cell mitogens con A and LPS respectively, was monitored by the in vitro uptake of 3H-thymidine. Although cell cultures prepared from blood of statistically defined responders gave higher SI’s than preparations from non-responders, this only occurred after vaccination was commenced (Fig. 2). This may indicate that the genetic influence has affected a specific component of immunological responsiveness, that is, the recognition of antigen which precedes the sequence of non-specific events causing worm expulsion (see Dineen, Gregg, Windon, Donald & Kelly, 1977). Either quantitative or qualitative aspects of antigen recognition may be deficient in non-responder animals. It was disappointing that there was no significant difference between responder and non-responder cultures set up before vaccination (cultures 1-3 inclusive). If such a difference had occurred it would have indicated that selection, based on response to vaccination and challenge with T. colubriformis, was resulting in a general improvement of immunological performance. In addition a strong correlation between cell SI’s before vaccination and resistance to challenge, would provide a valuable marker for selection. However, the present results indicate that in vitro culture of lymphocytes with T- and B-cell mitogens is unlikely to provide such a marker. Previously (Windon et al, 1980) and again in the present study, we were not able to show a relationship between haemoglobin-type and the responsiveness of vaccinated lambs which might have been expected from the work of Evans, Blunt & Southcott (1963), Allonby & Urquhart (1976) and Altaif & Dargie (1978). On the other hand, the previously described relationship between intestinal globule leucocytes and resistance (O’Sullivan & Donald, 1973; Gregg et al, 1978) was again evident (Fig. 3). Although there was a tendency for the number of eosinophils to be reduced in intestinal lamina propria of wether lambs harbouring substantial worm burdens after challenge, the association of eosinophils with resistance to challenge is not convincing.
195
The results confirm that substantial progress towards increased responsiveness of lambs to vaccination and challenge with the nematode can be achieved in one generation of selection+even though the relevant genetic component was contributed by sire alone. Currently we are assessing the significance of the genetic component of resistance in iambs which are the progeny of both selected dams and sires.
Acknowledgements-me
wish to thank Mrs. Elizabeth Townsend, Messrs Athol Luker and Stephen Ferguson for their excellent technical assistance and Mr. Cyril Samways of the School of Chemical Engineering, University of New South Wales, for irradiation of T. colubriformis infective larvae. We are also indebted to Mr. Robert Do&on for assistance with statistical analyses. This work was supported by a grant from the
Rural Credits Development Fund (Reserve Bank of Australia).
ADAMSD. B. & CRIPPS A. W. 1977. Cellular changes in the intestinal lymph of sheep infected with the enteric nematode, Trichostrongylus colubriformis. The Australian Journal of Experimental Biology and Medical Science 5s: 509-522.
A~LONBYE. W. & URQUHARTG. M. 1976. A possible relationship between haemonchosis and haemoglob~n polymorphism in Merino sheep in Kenya. Research in Veterinary Science. 20: 212-214.
ALTAIFK. 1. & DARGIEJ. D. 1978. Genetic resistance to helminths. Comparison of the development of Ostertagia circumcincta infections in Scottish Blackface sheep of different haemoglobin type. Research in Veterinary Science 24: 391-393.
EVANSJ. V., BLUNT M. H. & SOU~HCOTTW. H. 1963. The effects of infection with Haemonchus contortus on the sodium and potassium concentrations in the erythrocytes and plasma, in sheep of different haemoglobin types. Australian Journal of Agricultural Research 14: 549-558.
DINEENJ. K., GREGG P., WINDONR. G., DONALDA. D. & KELLY J. D. 1977. The role of immunologically specific and non-specific components of resistance in cross-protection to intestinal nematodes. international Journal for Parasitology 7: 211-215. GREGG P., DINEEN J. K. & GRIFFITHSD. A. 1976. The effect of y-radiation on the development of infective larvae of Trichostrongylus colubriformis in guinea pigs and sheep. Veterinary Parasitology 2: 363-375. GREGG P., DINEEN3. K., ROTHWELLT. L. W. & KELLY J. D. 1978. The effect of age on the response of sheep with irradiated Trichostrongylus to vaccination colubriformis larvae. Veterinary Parasitology 4: 35-48. LENDRUMA. C. 1944. The staining of eosinouhil oolvmorphs and enterochromaffin cells in h&tological sections. Journal of Pathology and Bacteriology. 56: 441.
196
J. K. DINEENand R. G. WINDON
O’SULUVANB. M. & DONALDA. D. 1973. Responses to infection with Haemonchus contortus and Trichostrongylus colubriformis in ewes of different reproductive status. International Journal for Parasitology 3: 521-530.
WHITLOCK H. V. 1948. Some modifications of the McMaster helminth egg-counting technique apparatus.
Journal of the Council for Research 21: 177-180.
I.J.P. VOL. 10. 1980 Scientific
and Industrial
WINDONR. G., DINEEN J. K. & KELLY J. D. 1980. The segregation of lambs into ‘responders’ and ‘nonresponders’: response to vaccination with irradiated Trichostrongylus colubriformis larvae before weaning. International Journal for Parasitology 10: 65-73.