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The seven-day growth of haemonchus contortus compared in worm-free and experienced sheep
J. COMP. PATH. 1967. VOL. 77.
g9
T H E S E V E N - D A Y G R O W T H OF HAEMONCHUS CONTORTUS C O M P A R E D IN W O R M - F R E E AND EXPERIENCED
SHEEP
By
M. G. CHRISTIE and M. R. BRAMBEL5 Parasitology Department, Moredun Research Institute, Edinburgh INTRODUCTION
In an earlier experiment (Christie, Brambell and Charleston, 1964) we showed that a group of lambs dosed first with infective larvae of Haemonchus contortus and then, after 10 days, with an anthelmintic to cut short the parasitic career of the larvae, acquired a measure of resistance to a further challenge with this worm. This was shown by comparing the numbers of adult worms surviving from a challenge dose given 26 days previously in a protected and a control group. The experiment described in this paper followed a similar pattern up to the time of challenge, but the sheep were slaughtered 7 days after challenge, in order to see whether any difference was discernible early in the life of the worms of the challenge dose. This experimental design has the advantage that, while worms from the challenge were 7 days old at slaughter, any persisting from the immunising course were 24 days old and, therefore, easily recognised. METHODS
Experimental animals. The lambs were ¼ Scottish Hill Blackface all born over a period of l0 days. They were taken from their mothers 24 hours after birth and reared by hand on cows' milk under worm free conditions. Weaning was completed by the age of 6 weeks. The lambs were 7½ months old at the time of challenge. Allocation of animals to groups. Two groups, each of 3 males and 5 females, were made up with a table of random numbers from the 20 female and 12 male lambs which were available in the weight range 85 to 95 lb. A further random choice designated one as the protected group. Infective larvae. Both for immunisation and challenge, larvae were used which had been stored for 5 to 11 days at 5°C. after harvesting from the cultures. They were grown and harvested by the method of Christie and Patterson (1963). For administration to the lambs they were taken up on fragments of paper tissue packed into No. 0 gelatin capsules. Immunising treatment. Lambs in the protected group were each given 10 daily doses of 25,000 larvae of H. contortus. On the 11th and 16th day after the first immunising dose of larvae, both protected and control animals were dosed with thiabendazole suspension at the rate of 90 mg./kg, of body weight. Both groups were challenged with 50,000 larvae 9 days after the second dose of thiabendazole. Examination of worm populations. The lambs were shot with a humane killer 7 days after receiving the challenge dose. The abdomen was opened within 5 minutes of death, and ligatures and a label were tied round the omasum and the proximal end of the duodenum. The abomasum was removed and taken to the laboratory where it was opened and washed in 3 changes of 2,000 ml. of 1 per cent. NaHCO~, all the mucosal surfaces being rubbed gently. The worms were left to relax and die overnight at 5°C. The next morning about 4,500 ml. of supernatant liquid was
100
H a e m o n c h u s contortus IN SHEEP
drawn off from the surface and the remaining material was treated with formalin and made up to 2,000 ml. This suspension was thoroughly mixed and an aliquot was taken by repeated drawings with a wide mouth (10 ram. in diameter) syringe, the contents of which were discharged into a measuring cylinder. The size of the aliquot samples actually taken ranged from 118 to 121 ml. that is approximately 6 per cent. of the whole. These samples were extensively diluted and each was searched twice under a binocular dissecting microscope at a magnification of x 25. All the worms found were picked out with a fine pipette. The material was held in rectangular dishes in which the sides were sloped at 45 ° to facilitate observation of settled material right up to the edge of the dish. T h e glass base was marked out with lines spaced at 3 ram. to confine the search to the centre of the field of view and to ensure orderly progression through the material. The corners of the dish were fitted with hemispherical feet to give smooth motion over the stage of the microscope and so to avoid jerking of material from the unsearched to the searched area of the dish. In 7 of the protected lambs a further fraction comprising one half of the total stomach contents and washings was searched at a magnification of × 3 for adult worms surviving from the immunising treatment. The corresponding sample for the 8th lamb was accidentally lost after the 6 per cent. aliquot had been searched. ,Measurement of worms. The image of each worm was projected at a magnification of × 19 on to a roll of paper and a pencil line was drawn down the middle of the image. T h e length of the pencil line was measured with dividers. This information was transferred to punched cards and sorted and tabulated on standard punch card machinery. The number of worms examined and measured was 9,881. Reliable sexing at the magnification used is not possible until the worms have reached a size of 2 to 3 ram. As m a n y of the worms found were smaller than this, classification by sex was not attempted, RESULTS
Table I sets out the main details of the experiment. Table 2 shows the numbers of worms found in the 16 aliquot samples, the proportion of worms damaged and the mean body length of the undamaged worms. Table 3 shows the way in which these sizes were distributed and lists all the undamaged worms found in each aliquot sample classifiedin 0"4 turn. steps of the body length. Over 99 per cent. of the worms were found in the 4th stage.
TABLE 1 TIME TABLE OF MAIN EVENTS OF THE EXPERIMENT
Age (days)
1 45 205-214
Treatment
Taken from mothers. Reared by hand on cow's milk. Weaned Protected
10 daily doses 25~000 H, oontortus
215 219 223 230
90 mg./kg, thiabendazole 90 mg./kg, thiabendazole 50,000larvae of/-/, contortus Slaughtered
Control
Nil
M.
G.
CHRISTIE
AND
M.
101
R. B R A M B E L L
TABLE 2 MAIN OFIAI~AOTERISTIOBOF WORM I~OPULATIONfi IN THE 16 SHEEP
No. of wormsfound in aliquot (approx. 6per centof whole)
Sheep JYo.
Percentageof worms damaged
Mean lengthof undamagedworms mm.
Est. percentageo3,. dMlengedose established
1.0 0.9 1"2 1.6 0,8 1.1 1.0 1 "4
11 10 8 10 4 6 12 27
32 27 35 29 32 25 31 32
Protected P-1 P-2 P-3 P-4 P-5 P-6 P-7 P-8
325 285 251 307 117 180 355 782
6 2 1 1 2 4 5 0 Qontrol
C- i
956
l
3.9
0-2 G-3 C-4
801 1036 854 954 753 933 957
9 10 13 6 10 10 13
4,3 4,0 4,1 3.8 3.4 4.0 4.0
C-5 0-6 C-7 C-8
Some of the worms were damaged, either while being picked out for measuring or by spontaneous rupture. While counts are not affected by such damage, the apparent body length is affected by rupture of the body wall, and the damaged worms have, therefore, been omitted from the analysis of lengths. Damaged Fig. 1.
4.0 protected 0
'b .~ 20 control
u
I
3
body
5
mm.
length ot worms
Frequency distribution o£ worm lengths within the 2 groups, Each point represents the percentage of the group total falling in that class and is plotted at the mid point of the class range.
102
Haemonchus
contortus
nq SHEEP
9"9 .~a~,.o
t l l l J ] l S
~'9-g'9
I
]
T'9-R'9
[
I~
I
I
~
I
]
I
I
•.¢-k "g O
g'9-O'r2
6"~-9"t, ¢1
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~.~-~.~ 0
~.~--~.~
i! •
I
"4" v,.,. ~
~
[
g'g-O'g 0
6"~-9"~
9'8-~'~
7~ 0
~c'~ It'-.
Ice')
f "g-O"~
£'f-O'[
6,0-..a.O 9.0>
~1111~
dd~dd~
M.
G.
CHRISTIE
AND
M. R. BRAMBE, L L
103
specimens appeared to have been representative of the aliquot sample in which they were found, but even if all damaged worms in each group of sheep had the average characteristics of undamaged worms in the other group, the ratio of the mean worm body length protected group: control group would only be altered from 3"9 : 1 to 3"3 : 1. Figure 1 gives a graphical comparison of the body size composition of the worm populations of the two groups of lambs. All the undamaged worms in each 0"4 ram. body length class in the 8 lambs have been totalled and expressed as a percentage of the total number of undamaged worms in the samples for that group. These totals are: protected, 2,547; control, 6,608. The calculated percentages have been plotted at the mid-point of each size-class. For example, in the 5th column of the upper half of Table 3 there is a total of 121 worms of the 1"95 ram. size class and this constiutes 121/2547 x 10O = 4"8 per cent. of the total for the protected group. The estimated total numbers of aduk worms surviving from the thiabendazole treatment in 7 of the protected sheep were 90, 26, 4, 0, 0, 0, 0. In the case of the 8th lamb no adults were found in the 1/20th aliquot searched for larval stages before the material was lost. DISCUSSION
The nature of the worm populations that are under consideration in this experiment must be clearly defined. These populations comprise the H. contortus larvae which are free in the stomach contents or which can be removed from the gastric mucosal surfaces of freshly killed sheep by gentle rubbing in 1 per cent. NaHCO~ at 39°C. Because some third ,stage larvae lie with their whole body concealed within a gland, and some early fourth stage larvae may be anchored in a gland by one or more turns of the body with only the tail projeering into the lumen of the abomasum, the populations described here may not have included all the larvae in the earliest phases of their parasitic development. The probability of there having been such hidden larvae as late as 7 days after challenge is considered below. I f the worm populations so defined which were observed in the control and protected sheep are compared, the populations in the protected sheep have the following relative characteristics : (1) the worms have made less progress in development being much shorter (group mean body length 1"0 ram.; in controls 3"9 mm.); (2) there are fewer worms (group mean worm burden 5,473; in controls 15,160); (3) the numbers of larvae in the individual sheep are more widely dispersed about the group mean (coefficient of variation 62 per cent., in controls 10 per cent.). These figures show conclusively that conditions are less favourable for the early parasitic stages of H. contortus in the stomachs of sheep with recent previous experience of this species, than in the stomachs of sheep with no previous exposure to Trichostrongylid worms. Almost all the worms in the protected sheep were retarded in relation to those in the controls : nevertheless the uniformity of development of the worms in each of the two groups of sheep is also remarkable. One explanation of .this result could be the absence of some signal which initiates the phase of rapid growth and development of the fourth stage larvae or the absence of some essential nutrient. The effect on the numbers was I-I
104
Haemonchus contortus IN
SHEEP
equally definite but less uniform. Although these two effects observed in the protected sheep--the delay in development and the reduction in the proportion of the challenge dose surviving--were associated in the sense that both were observable at an early stage of the development of the worms, there is no evidence by which we can decide whether they are different expressions of the same alteration in the abomasal environment, or quite unrelated phenomena. We have already emphasised that our observations concerned ofily those larvae which could be detached from the mucosa without disrupting the tissue. If larvae remained embedded in the mucosa more than, say, 48 hours after dosing, then the larvae of the challenge doses administered to the protected sheep could have had one of these 3 possible histories: (1) the larvae were delayed several days in the mucosa, but had begun their normal development on the mucosa shortly before slaughter; (2) the larvae emerged from the mucosa at the same time as in the control sheep, b u t were developing more slowly; (3) some of the worms were delayed in one of these two ways, while oflaers remained in the mucosa and were not seen by us. However, Stoll (1943) who appears to be the only author to have published direct observations on the actual location-of Haemonchus, concluded that the phase of the life cycle spent in the mucosa w ~ always brief. Assessment of the problem may be confused by use of the word 'histotropic' which may be used either in an unconfirmed analogy with other genera such as Ostertagia or Oesophagostomum or to distinguish between the closer association between worm and tissue observed in the fourth stage of Haemonchus when this stage is compared with the highly motile adult worm. A comparison of our results with other published work on H. contortus in sheep poses two major questions. Firstly, what would have been the probable fate of the larvae which were retarded i n the protected sheep? Secondly, are these results comparable with other accounts of retardation of this species in sheep ? A likely answer to the first question is suggested by comparing the resuks of the present experiment with one reported by us earlier (Christie et al., 1964). All conditions were similar in the two experiments except that the sheep were slaughtered 26 days after challenge in the 1964 experiment. At this time there was no difference in the degree of development of the worms, and both populations were reproductively active, but there were fewer worms in the protected group. This suggests that the retardation observed in the present experiment was a temporary one of only a few days duration which left many of the worms With an unimpaired capacity to complete development to rill sexual maturity. In the other cases of retardation reported in the literature, its duration was very much greater. Field; Brambell and Campbell (1960) and Gibbs (1964) reported haemonchosis in housed sheep which had been denied access to infective larvae for many weeks. Extensive retardation of H. contortus at the 4th stage was found by Dineen, Donald, Wagland and Offner (1965) in a proportion of the larvae administered in 30 daily doses of 100 larvae each to 3 month old Merino lambs. They found some larvae were still i n the 4th stage 100 days after the last dose of infective larvae. None of these three papers gives any evidence of larvae being sited in the mucosae. Although Dineen et al. (1965) treated the abomasa by incubating them in 1 per cent. HC1 for l hour immediately after slaughter which would bring
M.
O. CHRISTIE AND M. R. BRAMBELL
105
about digestion of the tissue by the pepsin released from the mucosa, they did not distinguish between larvae present in the washings and in the digest. There seems to be no reason, therefore, for supposing that the relation between the retarded larvae observed, or postulated, in these three papers and the abomasal mucosa was any different from that of the retarded larvae observed by us. SUMMARY
Eight sheep aged 7 months Were treated by giving them 10 daffy doses of 20,000 larvae of H. contortus and removing these with thiabendazole given on the 11th and 16th days. On the 25th day the 8 protected lambs, a n d another 8 control lambs which had been raised worm free, were challenged w i t h 50,000 H. contortus larvae. On the 32nd day aU 16 sheep were killed, and the stomachs removed and washed out in 3 changes of I per cent. NaHCO~. All t h e worms in 6 per cent. of these washings were counted and measured. There were more worms in the control than in the immunlsed s h e e p : moreover, the worms in the controls had developed further than the worms in the protected sheep. The worm populations had the following characteristics (group means): in the immunised group the proportion of dose recovered was 11 per cent. and the mean body length was 1"i ram.; in the control group the proportion of dose recovered was 30 per cent. and the mean body length was 3"9 mm. Comparison with published observations on retardation of H. contortus in sheep suggests that under these experimental conditions the retardation would have lasted only a few days, but there is no evidence for supposing it to be different in character from the very much longer delays in sheep housed over winter or in young merino lambs. REFERENCES
Christie, M. G., and Patterson, J. E. (1963). ]. HelminthoI., 37, 251. Christie, M. G., Brambell, M. R., and Charleston, W. A. G. (1964). ]. comp. Path., 74, 338. Dineen, J. K., Donald, A. D., Wagland, B. M., and Offner, J. (1965). Parasitology, 55, 515. Field, A. C., Brambell, M. R., and Campbell, J. A. (1960). Ibid., SO, 387. Gibbs, H. C. (1964). Canadian vet. J., S, 8. Stoll, N. R. (1943). ]. Parasitol., 29, 407.
[Received [or publication, April 29th, 1966]
g9
T H E S E V E N - D A Y G R O W T H OF HAEMONCHUS CONTORTUS C O M P A R E D IN W O R M - F R E E AND EXPERIENCED
SHEEP
By
M. G. CHRISTIE and M. R. BRAMBEL5 Parasitology Department, Moredun Research Institute, Edinburgh INTRODUCTION
In an earlier experiment (Christie, Brambell and Charleston, 1964) we showed that a group of lambs dosed first with infective larvae of Haemonchus contortus and then, after 10 days, with an anthelmintic to cut short the parasitic career of the larvae, acquired a measure of resistance to a further challenge with this worm. This was shown by comparing the numbers of adult worms surviving from a challenge dose given 26 days previously in a protected and a control group. The experiment described in this paper followed a similar pattern up to the time of challenge, but the sheep were slaughtered 7 days after challenge, in order to see whether any difference was discernible early in the life of the worms of the challenge dose. This experimental design has the advantage that, while worms from the challenge were 7 days old at slaughter, any persisting from the immunising course were 24 days old and, therefore, easily recognised. METHODS
Experimental animals. The lambs were ¼ Scottish Hill Blackface all born over a period of l0 days. They were taken from their mothers 24 hours after birth and reared by hand on cows' milk under worm free conditions. Weaning was completed by the age of 6 weeks. The lambs were 7½ months old at the time of challenge. Allocation of animals to groups. Two groups, each of 3 males and 5 females, were made up with a table of random numbers from the 20 female and 12 male lambs which were available in the weight range 85 to 95 lb. A further random choice designated one as the protected group. Infective larvae. Both for immunisation and challenge, larvae were used which had been stored for 5 to 11 days at 5°C. after harvesting from the cultures. They were grown and harvested by the method of Christie and Patterson (1963). For administration to the lambs they were taken up on fragments of paper tissue packed into No. 0 gelatin capsules. Immunising treatment. Lambs in the protected group were each given 10 daily doses of 25,000 larvae of H. contortus. On the 11th and 16th day after the first immunising dose of larvae, both protected and control animals were dosed with thiabendazole suspension at the rate of 90 mg./kg, of body weight. Both groups were challenged with 50,000 larvae 9 days after the second dose of thiabendazole. Examination of worm populations. The lambs were shot with a humane killer 7 days after receiving the challenge dose. The abdomen was opened within 5 minutes of death, and ligatures and a label were tied round the omasum and the proximal end of the duodenum. The abomasum was removed and taken to the laboratory where it was opened and washed in 3 changes of 2,000 ml. of 1 per cent. NaHCO~, all the mucosal surfaces being rubbed gently. The worms were left to relax and die overnight at 5°C. The next morning about 4,500 ml. of supernatant liquid was
100
H a e m o n c h u s contortus IN SHEEP
drawn off from the surface and the remaining material was treated with formalin and made up to 2,000 ml. This suspension was thoroughly mixed and an aliquot was taken by repeated drawings with a wide mouth (10 ram. in diameter) syringe, the contents of which were discharged into a measuring cylinder. The size of the aliquot samples actually taken ranged from 118 to 121 ml. that is approximately 6 per cent. of the whole. These samples were extensively diluted and each was searched twice under a binocular dissecting microscope at a magnification of x 25. All the worms found were picked out with a fine pipette. The material was held in rectangular dishes in which the sides were sloped at 45 ° to facilitate observation of settled material right up to the edge of the dish. T h e glass base was marked out with lines spaced at 3 ram. to confine the search to the centre of the field of view and to ensure orderly progression through the material. The corners of the dish were fitted with hemispherical feet to give smooth motion over the stage of the microscope and so to avoid jerking of material from the unsearched to the searched area of the dish. In 7 of the protected lambs a further fraction comprising one half of the total stomach contents and washings was searched at a magnification of × 3 for adult worms surviving from the immunising treatment. The corresponding sample for the 8th lamb was accidentally lost after the 6 per cent. aliquot had been searched. ,Measurement of worms. The image of each worm was projected at a magnification of × 19 on to a roll of paper and a pencil line was drawn down the middle of the image. T h e length of the pencil line was measured with dividers. This information was transferred to punched cards and sorted and tabulated on standard punch card machinery. The number of worms examined and measured was 9,881. Reliable sexing at the magnification used is not possible until the worms have reached a size of 2 to 3 ram. As m a n y of the worms found were smaller than this, classification by sex was not attempted, RESULTS
Table I sets out the main details of the experiment. Table 2 shows the numbers of worms found in the 16 aliquot samples, the proportion of worms damaged and the mean body length of the undamaged worms. Table 3 shows the way in which these sizes were distributed and lists all the undamaged worms found in each aliquot sample classifiedin 0"4 turn. steps of the body length. Over 99 per cent. of the worms were found in the 4th stage.
TABLE 1 TIME TABLE OF MAIN EVENTS OF THE EXPERIMENT
Age (days)
1 45 205-214
Treatment
Taken from mothers. Reared by hand on cow's milk. Weaned Protected
10 daily doses 25~000 H, oontortus
215 219 223 230
90 mg./kg, thiabendazole 90 mg./kg, thiabendazole 50,000larvae of/-/, contortus Slaughtered
Control
Nil
M.
G.
CHRISTIE
AND
M.
101
R. B R A M B E L L
TABLE 2 MAIN OFIAI~AOTERISTIOBOF WORM I~OPULATIONfi IN THE 16 SHEEP
No. of wormsfound in aliquot (approx. 6per centof whole)
Sheep JYo.
Percentageof worms damaged
Mean lengthof undamagedworms mm.
Est. percentageo3,. dMlengedose established
1.0 0.9 1"2 1.6 0,8 1.1 1.0 1 "4
11 10 8 10 4 6 12 27
32 27 35 29 32 25 31 32
Protected P-1 P-2 P-3 P-4 P-5 P-6 P-7 P-8
325 285 251 307 117 180 355 782
6 2 1 1 2 4 5 0 Qontrol
C- i
956
l
3.9
0-2 G-3 C-4
801 1036 854 954 753 933 957
9 10 13 6 10 10 13
4,3 4,0 4,1 3.8 3.4 4.0 4.0
C-5 0-6 C-7 C-8
Some of the worms were damaged, either while being picked out for measuring or by spontaneous rupture. While counts are not affected by such damage, the apparent body length is affected by rupture of the body wall, and the damaged worms have, therefore, been omitted from the analysis of lengths. Damaged Fig. 1.
4.0 protected 0
'b .~ 20 control
u
I
3
body
5
mm.
length ot worms
Frequency distribution o£ worm lengths within the 2 groups, Each point represents the percentage of the group total falling in that class and is plotted at the mid point of the class range.
102
Haemonchus
contortus
nq SHEEP
9"9 .~a~,.o
t l l l J ] l S
~'9-g'9
I
]
T'9-R'9
[
I~
I
I
~
I
]
I
I
•.¢-k "g O
g'9-O'r2
6"~-9"t, ¢1
0
~.~-~.~ 0
~.~--~.~
i! •
I
"4" v,.,. ~
~
[
g'g-O'g 0
6"~-9"~
9'8-~'~
7~ 0
~c'~ It'-.
Ice')
f "g-O"~
£'f-O'[
6,0-..a.O 9.0>
~1111~
dd~dd~
M.
G.
CHRISTIE
AND
M. R. BRAMBE, L L
103
specimens appeared to have been representative of the aliquot sample in which they were found, but even if all damaged worms in each group of sheep had the average characteristics of undamaged worms in the other group, the ratio of the mean worm body length protected group: control group would only be altered from 3"9 : 1 to 3"3 : 1. Figure 1 gives a graphical comparison of the body size composition of the worm populations of the two groups of lambs. All the undamaged worms in each 0"4 ram. body length class in the 8 lambs have been totalled and expressed as a percentage of the total number of undamaged worms in the samples for that group. These totals are: protected, 2,547; control, 6,608. The calculated percentages have been plotted at the mid-point of each size-class. For example, in the 5th column of the upper half of Table 3 there is a total of 121 worms of the 1"95 ram. size class and this constiutes 121/2547 x 10O = 4"8 per cent. of the total for the protected group. The estimated total numbers of aduk worms surviving from the thiabendazole treatment in 7 of the protected sheep were 90, 26, 4, 0, 0, 0, 0. In the case of the 8th lamb no adults were found in the 1/20th aliquot searched for larval stages before the material was lost. DISCUSSION
The nature of the worm populations that are under consideration in this experiment must be clearly defined. These populations comprise the H. contortus larvae which are free in the stomach contents or which can be removed from the gastric mucosal surfaces of freshly killed sheep by gentle rubbing in 1 per cent. NaHCO~ at 39°C. Because some third ,stage larvae lie with their whole body concealed within a gland, and some early fourth stage larvae may be anchored in a gland by one or more turns of the body with only the tail projeering into the lumen of the abomasum, the populations described here may not have included all the larvae in the earliest phases of their parasitic development. The probability of there having been such hidden larvae as late as 7 days after challenge is considered below. I f the worm populations so defined which were observed in the control and protected sheep are compared, the populations in the protected sheep have the following relative characteristics : (1) the worms have made less progress in development being much shorter (group mean body length 1"0 ram.; in controls 3"9 mm.); (2) there are fewer worms (group mean worm burden 5,473; in controls 15,160); (3) the numbers of larvae in the individual sheep are more widely dispersed about the group mean (coefficient of variation 62 per cent., in controls 10 per cent.). These figures show conclusively that conditions are less favourable for the early parasitic stages of H. contortus in the stomachs of sheep with recent previous experience of this species, than in the stomachs of sheep with no previous exposure to Trichostrongylid worms. Almost all the worms in the protected sheep were retarded in relation to those in the controls : nevertheless the uniformity of development of the worms in each of the two groups of sheep is also remarkable. One explanation of .this result could be the absence of some signal which initiates the phase of rapid growth and development of the fourth stage larvae or the absence of some essential nutrient. The effect on the numbers was I-I
104
Haemonchus contortus IN
SHEEP
equally definite but less uniform. Although these two effects observed in the protected sheep--the delay in development and the reduction in the proportion of the challenge dose surviving--were associated in the sense that both were observable at an early stage of the development of the worms, there is no evidence by which we can decide whether they are different expressions of the same alteration in the abomasal environment, or quite unrelated phenomena. We have already emphasised that our observations concerned ofily those larvae which could be detached from the mucosa without disrupting the tissue. If larvae remained embedded in the mucosa more than, say, 48 hours after dosing, then the larvae of the challenge doses administered to the protected sheep could have had one of these 3 possible histories: (1) the larvae were delayed several days in the mucosa, but had begun their normal development on the mucosa shortly before slaughter; (2) the larvae emerged from the mucosa at the same time as in the control sheep, b u t were developing more slowly; (3) some of the worms were delayed in one of these two ways, while oflaers remained in the mucosa and were not seen by us. However, Stoll (1943) who appears to be the only author to have published direct observations on the actual location-of Haemonchus, concluded that the phase of the life cycle spent in the mucosa w ~ always brief. Assessment of the problem may be confused by use of the word 'histotropic' which may be used either in an unconfirmed analogy with other genera such as Ostertagia or Oesophagostomum or to distinguish between the closer association between worm and tissue observed in the fourth stage of Haemonchus when this stage is compared with the highly motile adult worm. A comparison of our results with other published work on H. contortus in sheep poses two major questions. Firstly, what would have been the probable fate of the larvae which were retarded i n the protected sheep? Secondly, are these results comparable with other accounts of retardation of this species in sheep ? A likely answer to the first question is suggested by comparing the resuks of the present experiment with one reported by us earlier (Christie et al., 1964). All conditions were similar in the two experiments except that the sheep were slaughtered 26 days after challenge in the 1964 experiment. At this time there was no difference in the degree of development of the worms, and both populations were reproductively active, but there were fewer worms in the protected group. This suggests that the retardation observed in the present experiment was a temporary one of only a few days duration which left many of the worms With an unimpaired capacity to complete development to rill sexual maturity. In the other cases of retardation reported in the literature, its duration was very much greater. Field; Brambell and Campbell (1960) and Gibbs (1964) reported haemonchosis in housed sheep which had been denied access to infective larvae for many weeks. Extensive retardation of H. contortus at the 4th stage was found by Dineen, Donald, Wagland and Offner (1965) in a proportion of the larvae administered in 30 daily doses of 100 larvae each to 3 month old Merino lambs. They found some larvae were still i n the 4th stage 100 days after the last dose of infective larvae. None of these three papers gives any evidence of larvae being sited in the mucosae. Although Dineen et al. (1965) treated the abomasa by incubating them in 1 per cent. HC1 for l hour immediately after slaughter which would bring
M.
O. CHRISTIE AND M. R. BRAMBELL
105
about digestion of the tissue by the pepsin released from the mucosa, they did not distinguish between larvae present in the washings and in the digest. There seems to be no reason, therefore, for supposing that the relation between the retarded larvae observed, or postulated, in these three papers and the abomasal mucosa was any different from that of the retarded larvae observed by us. SUMMARY
Eight sheep aged 7 months Were treated by giving them 10 daffy doses of 20,000 larvae of H. contortus and removing these with thiabendazole given on the 11th and 16th days. On the 25th day the 8 protected lambs, a n d another 8 control lambs which had been raised worm free, were challenged w i t h 50,000 H. contortus larvae. On the 32nd day aU 16 sheep were killed, and the stomachs removed and washed out in 3 changes of I per cent. NaHCO~. All t h e worms in 6 per cent. of these washings were counted and measured. There were more worms in the control than in the immunlsed s h e e p : moreover, the worms in the controls had developed further than the worms in the protected sheep. The worm populations had the following characteristics (group means): in the immunised group the proportion of dose recovered was 11 per cent. and the mean body length was 1"i ram.; in the control group the proportion of dose recovered was 30 per cent. and the mean body length was 3"9 mm. Comparison with published observations on retardation of H. contortus in sheep suggests that under these experimental conditions the retardation would have lasted only a few days, but there is no evidence for supposing it to be different in character from the very much longer delays in sheep housed over winter or in young merino lambs. REFERENCES
Christie, M. G., and Patterson, J. E. (1963). ]. HelminthoI., 37, 251. Christie, M. G., Brambell, M. R., and Charleston, W. A. G. (1964). ]. comp. Path., 74, 338. Dineen, J. K., Donald, A. D., Wagland, B. M., and Offner, J. (1965). Parasitology, 55, 515. Field, A. C., Brambell, M. R., and Campbell, J. A. (1960). Ibid., SO, 387. Gibbs, H. C. (1964). Canadian vet. J., S, 8. Stoll, N. R. (1943). ]. Parasitol., 29, 407.
[Received [or publication, April 29th, 1966]