- Email: [email protected]
Oesophagostomum radiatum: Adult nematodes and intestinal hemorrhage
EXPERIhIENTAL
28, 416419
PARASITOLOGY
(1970)
Oesophagosfomum
radiafum: Adult Intestinal Hemorrhage K. C. Bremner
Division
of Animal
Health,
(Submitted
CSIRO, Brisbane,
Nematodes
and
and R. K. Keith Long Pocket
Laboratories,
lndooroopilly,
4068, Australia
for publication,
15 December
1969)
BREMNER, K. C., AND KEITH, R. K. 1970. Oesophagostomum radiatum: Adult Experimental Parasitology 28, 416419. nematodes and intestinal hemorrhage. Erythrocytes of six calves reared worm-free were labeled with radiochromium. Approximately 3000 adult Oesophagostomum radiatum then were transferred surgically into the ceca of four of these calves, and sham-operations were performed on two. The fecal output of WZr was measured during the subsequent 2 weeks, and the recipients were then necropsied. The mean fecal loss of WZr from the infected calves within 16 hours of the transfers was equivalent to 16.5 ml blood, rising to 84 ml on the following day. At the same time, the sham-operated calves lost ~1Cr equivalent to only I-3 ml blood daily. It was concluded that adult 0. radiatum caused intestinal bleeding in the host through a direct action, and that allergic phenomena were not involved initially in the genesis of the hemorrhage. INDEX
bovine;
DESCRIPTORS:
Helminthosis;
Pathogenesis; Oesophagostomum radiatum; Oesophagostomosis, SlCr-labeled ervthrocytes. Hemorrhage, intestinal;
When calves are infected with a single dose of 7500 infective larvae of Oesophagostomum radiatum, marked intestinal hemorrhage commences 19-20 days later, immediately following the fourth larval ecdysis (Bremner 1970). Elek and Durie (1967) consider that the ulceration occurring in the colon of infected calves at this time is probably associated with a hypersensitivity reaction of the host, rather than with the activity of adult worms. It is not known whether the hemorrhage is also associated with an immune response by the host. If it is, then a delay in the onset of bleeding should occur when adult parasites are transferred to a second host maintained worm-free from birth. The present experiment was performed to examine this possibility. 416
MATERIALS ASI) METHODS
Donor Calves Australian Illawarra Shorthorn calves, reared worm-free, were each given 10,000 infective larvae of 0. radiatum when 10 weeks old. Calves were killed as required 38 and 39 days later, after the infections became patent, to provide worms for transfer. Two worm transfers and one shamoperation were performed on each of these days. The contents of the cecum and colon of each calf were washed on a sieve with warm Locke’s solution, and the worms and coarse digesta remaining were placed in a bath of Locke’s solution at 39°C. Approximately 3000 active adult worms were picked out and counted for each transfer.
INTESTINAL
BLEEDING
Recipient Calves Six male calves were reared worm-free, and when 11 weeks old were prepared for estimation of fecal blood loss by labeling of erythrocytes with 300 $i of %hromium (Bremner 1970). Two days later their fecal output of Wr was determined over a 24hour control period, and the equivalent fecal blood losses were found to range from 2 to 5 ml. A known number of 0. radiatum suspended in 100 ml of Locke’s solution (Table I) was then transferred surgically to four of these calves (Nos. l4) through a small incision in the apex of the cecum. The sham-operated calves (Nos. 5 and 6), were given 100 ml of Locke’s solution through incisions into the cecum. Not more than 65 minutes elapsed between slaughter and transfer of the worms to the recipients. The calves were replaced in metabolism cages, and determinations of fecal blood loss were made daily for the following 7-8 days, and again between 11 and 15 days after the transfers. Fecal egg counts were performed daily until the infected calves were killed: Nos. 2 and 3 on the 14th day after transfer and Nos. 1 and 4 on the 15th day after transfer. The number of worms present in the cecum and anterior portion of the colon of each calf was then counted. The helminthological TABLE 1 of Adult Oesophagostomum Implanted in Worm-free Calves,
Numbers
IN’
417
OESOPHAGOSTOMOSIS
methods used were those given by Roberts et al. (1962). %WJLTS
The numbers of worms transferred to, and recovered from, the recipient calves are given in Table I, and the maximum egg counts recorded during the experiment are also shown. Examination of the feces of each recipient during the last 8 days of the trial revealed that between 20 and 60 worms were expelled daily by individual animals. Thus the numbers of worms ini-
radiatum
Resultant Maximum Egg Count, and Numbers of Worms Recovered at Necropsy Two Weeks Later
Calf No.
No. worms implanted
Maximum fecal egg count (e.p.g. 1”
1 2 3 4
3010 2830 3220 3020
730 720 580 560
a Eggs per gram of feces.
e--..
No. worms recovered at necropsy 940 1000 530 550
___..-- -As 2 4 DAYS
10 12 TRANSFER
14
Fecal blood loss from four calves ( 0) surgical transfer of adult Oesophagostomum mdiatum into the large intestine. Losses from two sham-operated calves ( 0) are also shown. Bleeding was measured with srCr-labeled erythrocytes. FIG.
following
1.
6 8 AFTER
418
6.
C. BRJiMNER
tially established in the recipients were greater than those found at necropsy. The first post-transfer determinations of 51Cr were made on the feces passed over a period of 16 hours, and showed a mean fecal blood loss of 16.5 ml from the four recipients, while each sham-operated calf passed “lCr equivalent to 2 ml blood. Over the following 24 hours, the mean blood loss from the infected calves was 84 ml, as compared with losses of 1 and 3 ml, respectively, from the two control calves. The results of the individual determinations of fecal blood loss are given in Fig. 1. At necropsy, no evidence of bleeding from the sites of incision could be seen, and all the wounds had healed cleanly. DISCUSSION
Fecal loss of blood was evident within 16 hours of the transfers, and had increased considerably a day later ( Fig. 1) . It is unlikely that the colon and rectum of each calf were empty at the time of the exchanges, and the fecal output from the calves during the first 16 hours probably was comprised largely of digesta which, at transfer, was distal to the site of implantation. Thus there seems little doubt that the intestinal bleeding commenced within a few hours of the establishment of the parasites in their new environment. As all calves which received worms showed a similar hemorrhagic response, and as the fecal output of 51Cr by the sham-operated calves was negligible throughout the experiment, it is improbable that the 51Cr excreted in the feces originated from seepage of blood from the cecal incisions. Figure 1 shows that there was a tendency for the bleeding to lessen on days 3 and 4 after transfer, but this trend was subsequently reversed. When compared with the blood losses reported from calves infected experimentally with 0. radiatum
ANII
H. IL
KEITfI
(Bremner 1970), the magnitude of the bleeding recorded terminally from the recipients was in accord with the numbers of parasites recovered at necropsy (Table I). The secondary increase in the rate of blood loss occurred while worms were being expelled in the feces, and may have resulted from some host reaction to the presence of parasites. As the anemia associated with moderate parasitism by 0. radiatum does not become manifest during the larval stages of the parasitic life cycle (Bremner 1970), cross reactions of worm antigens with “naturally occurring” host antibodies ( Soulsby and Coombs 1959; Hogarth-Scott 1968) apparently are not involved in the genesis of the hemorrhage. Also, it has been shown in mammals that a detectable increase in antibody levels does not occur before the third day after primary stimulation by antigen (Miller 1968). As the present results revealed that the transferred worms induced intestinal hemorrhage within a day of their establishment in hosts previously worm-free, it would seem that this bleeding resulted from a direct effect of the parasite on the host tissue, rather than from an allergic reaction. Whether the bleeding is caused by secretions from the worms, or mechanical abrasion, remains to be determined. ACKXOWLEDGMENTS The technical assistance of Messrs. G. B. Mirre, R. Fridemanis, D. A. Berrie, T. W. Henderson, and R. P. Bryan is gratefully acknowledged. This study was supported in part by funds from the Australian Meat Research Committee, and the Dairy Research Fund. REFERENCES factors in BREMNER, K. C. 1970. Pathogenetic experimental bovine orsophagostomosis. 1’. Intestinal bleeding as the callse of anemia. Experimental Parasitology 27, 236-245.
INTESTINAL
BLEEDING
ELEK, P., AND Durun, P. H. 1967. The histopathology of the reactions of calves to experimental infection with the nodular worm, Oesophagostomum radiatum. II. Reaction of the susceptible host to infection with a single dose of larvae. Australian Journal of Agricultural Research 18, 549-559. HOGARTH~COTT, R. S. 1968. Naturally cccurring antibodies to the cuticle of nematodes. Parasitology 58, 221-226. MILLER, J. F. A. P. 1968. Biology of the imAspects of Immune response. In “Clinical
IN OESOPHAGOSTOMOSIS
419
munology” (P. G. H. Gel1 and R. R. A. Coombs, eds.), pp. 289-333. Blackwell, Oxford. R. K. ROBERTS, F. H. S., ELEK, P., AND l&m, 1962. Studies on resistance in calves to experimental infections with the nodular worm, Oesophagostomum radiatum. Australian Journal of Agricultural Research 13, 551-573. .%XJLSBY, E. J. L., AND cooms, R. R. A. 1959. Studies on blood group substances associated with Ascaris lumbricoides. Parasitology 49, 505-510.
28, 416419
PARASITOLOGY
(1970)
Oesophagosfomum
radiafum: Adult Intestinal Hemorrhage K. C. Bremner
Division
of Animal
Health,
(Submitted
CSIRO, Brisbane,
Nematodes
and
and R. K. Keith Long Pocket
Laboratories,
lndooroopilly,
4068, Australia
for publication,
15 December
1969)
BREMNER, K. C., AND KEITH, R. K. 1970. Oesophagostomum radiatum: Adult Experimental Parasitology 28, 416419. nematodes and intestinal hemorrhage. Erythrocytes of six calves reared worm-free were labeled with radiochromium. Approximately 3000 adult Oesophagostomum radiatum then were transferred surgically into the ceca of four of these calves, and sham-operations were performed on two. The fecal output of WZr was measured during the subsequent 2 weeks, and the recipients were then necropsied. The mean fecal loss of WZr from the infected calves within 16 hours of the transfers was equivalent to 16.5 ml blood, rising to 84 ml on the following day. At the same time, the sham-operated calves lost ~1Cr equivalent to only I-3 ml blood daily. It was concluded that adult 0. radiatum caused intestinal bleeding in the host through a direct action, and that allergic phenomena were not involved initially in the genesis of the hemorrhage. INDEX
bovine;
DESCRIPTORS:
Helminthosis;
Pathogenesis; Oesophagostomum radiatum; Oesophagostomosis, SlCr-labeled ervthrocytes. Hemorrhage, intestinal;
When calves are infected with a single dose of 7500 infective larvae of Oesophagostomum radiatum, marked intestinal hemorrhage commences 19-20 days later, immediately following the fourth larval ecdysis (Bremner 1970). Elek and Durie (1967) consider that the ulceration occurring in the colon of infected calves at this time is probably associated with a hypersensitivity reaction of the host, rather than with the activity of adult worms. It is not known whether the hemorrhage is also associated with an immune response by the host. If it is, then a delay in the onset of bleeding should occur when adult parasites are transferred to a second host maintained worm-free from birth. The present experiment was performed to examine this possibility. 416
MATERIALS ASI) METHODS
Donor Calves Australian Illawarra Shorthorn calves, reared worm-free, were each given 10,000 infective larvae of 0. radiatum when 10 weeks old. Calves were killed as required 38 and 39 days later, after the infections became patent, to provide worms for transfer. Two worm transfers and one shamoperation were performed on each of these days. The contents of the cecum and colon of each calf were washed on a sieve with warm Locke’s solution, and the worms and coarse digesta remaining were placed in a bath of Locke’s solution at 39°C. Approximately 3000 active adult worms were picked out and counted for each transfer.
INTESTINAL
BLEEDING
Recipient Calves Six male calves were reared worm-free, and when 11 weeks old were prepared for estimation of fecal blood loss by labeling of erythrocytes with 300 $i of %hromium (Bremner 1970). Two days later their fecal output of Wr was determined over a 24hour control period, and the equivalent fecal blood losses were found to range from 2 to 5 ml. A known number of 0. radiatum suspended in 100 ml of Locke’s solution (Table I) was then transferred surgically to four of these calves (Nos. l4) through a small incision in the apex of the cecum. The sham-operated calves (Nos. 5 and 6), were given 100 ml of Locke’s solution through incisions into the cecum. Not more than 65 minutes elapsed between slaughter and transfer of the worms to the recipients. The calves were replaced in metabolism cages, and determinations of fecal blood loss were made daily for the following 7-8 days, and again between 11 and 15 days after the transfers. Fecal egg counts were performed daily until the infected calves were killed: Nos. 2 and 3 on the 14th day after transfer and Nos. 1 and 4 on the 15th day after transfer. The number of worms present in the cecum and anterior portion of the colon of each calf was then counted. The helminthological TABLE 1 of Adult Oesophagostomum Implanted in Worm-free Calves,
Numbers
IN’
417
OESOPHAGOSTOMOSIS
methods used were those given by Roberts et al. (1962). %WJLTS
The numbers of worms transferred to, and recovered from, the recipient calves are given in Table I, and the maximum egg counts recorded during the experiment are also shown. Examination of the feces of each recipient during the last 8 days of the trial revealed that between 20 and 60 worms were expelled daily by individual animals. Thus the numbers of worms ini-
radiatum
Resultant Maximum Egg Count, and Numbers of Worms Recovered at Necropsy Two Weeks Later
Calf No.
No. worms implanted
Maximum fecal egg count (e.p.g. 1”
1 2 3 4
3010 2830 3220 3020
730 720 580 560
a Eggs per gram of feces.
e--..
No. worms recovered at necropsy 940 1000 530 550
___..-- -As 2 4 DAYS
10 12 TRANSFER
14
Fecal blood loss from four calves ( 0) surgical transfer of adult Oesophagostomum mdiatum into the large intestine. Losses from two sham-operated calves ( 0) are also shown. Bleeding was measured with srCr-labeled erythrocytes. FIG.
following
1.
6 8 AFTER
418
6.
C. BRJiMNER
tially established in the recipients were greater than those found at necropsy. The first post-transfer determinations of 51Cr were made on the feces passed over a period of 16 hours, and showed a mean fecal blood loss of 16.5 ml from the four recipients, while each sham-operated calf passed “lCr equivalent to 2 ml blood. Over the following 24 hours, the mean blood loss from the infected calves was 84 ml, as compared with losses of 1 and 3 ml, respectively, from the two control calves. The results of the individual determinations of fecal blood loss are given in Fig. 1. At necropsy, no evidence of bleeding from the sites of incision could be seen, and all the wounds had healed cleanly. DISCUSSION
Fecal loss of blood was evident within 16 hours of the transfers, and had increased considerably a day later ( Fig. 1) . It is unlikely that the colon and rectum of each calf were empty at the time of the exchanges, and the fecal output from the calves during the first 16 hours probably was comprised largely of digesta which, at transfer, was distal to the site of implantation. Thus there seems little doubt that the intestinal bleeding commenced within a few hours of the establishment of the parasites in their new environment. As all calves which received worms showed a similar hemorrhagic response, and as the fecal output of 51Cr by the sham-operated calves was negligible throughout the experiment, it is improbable that the 51Cr excreted in the feces originated from seepage of blood from the cecal incisions. Figure 1 shows that there was a tendency for the bleeding to lessen on days 3 and 4 after transfer, but this trend was subsequently reversed. When compared with the blood losses reported from calves infected experimentally with 0. radiatum
ANII
H. IL
KEITfI
(Bremner 1970), the magnitude of the bleeding recorded terminally from the recipients was in accord with the numbers of parasites recovered at necropsy (Table I). The secondary increase in the rate of blood loss occurred while worms were being expelled in the feces, and may have resulted from some host reaction to the presence of parasites. As the anemia associated with moderate parasitism by 0. radiatum does not become manifest during the larval stages of the parasitic life cycle (Bremner 1970), cross reactions of worm antigens with “naturally occurring” host antibodies ( Soulsby and Coombs 1959; Hogarth-Scott 1968) apparently are not involved in the genesis of the hemorrhage. Also, it has been shown in mammals that a detectable increase in antibody levels does not occur before the third day after primary stimulation by antigen (Miller 1968). As the present results revealed that the transferred worms induced intestinal hemorrhage within a day of their establishment in hosts previously worm-free, it would seem that this bleeding resulted from a direct effect of the parasite on the host tissue, rather than from an allergic reaction. Whether the bleeding is caused by secretions from the worms, or mechanical abrasion, remains to be determined. ACKXOWLEDGMENTS The technical assistance of Messrs. G. B. Mirre, R. Fridemanis, D. A. Berrie, T. W. Henderson, and R. P. Bryan is gratefully acknowledged. This study was supported in part by funds from the Australian Meat Research Committee, and the Dairy Research Fund. REFERENCES factors in BREMNER, K. C. 1970. Pathogenetic experimental bovine orsophagostomosis. 1’. Intestinal bleeding as the callse of anemia. Experimental Parasitology 27, 236-245.
INTESTINAL
BLEEDING
ELEK, P., AND Durun, P. H. 1967. The histopathology of the reactions of calves to experimental infection with the nodular worm, Oesophagostomum radiatum. II. Reaction of the susceptible host to infection with a single dose of larvae. Australian Journal of Agricultural Research 18, 549-559. HOGARTH~COTT, R. S. 1968. Naturally cccurring antibodies to the cuticle of nematodes. Parasitology 58, 221-226. MILLER, J. F. A. P. 1968. Biology of the imAspects of Immune response. In “Clinical
IN OESOPHAGOSTOMOSIS
419
munology” (P. G. H. Gel1 and R. R. A. Coombs, eds.), pp. 289-333. Blackwell, Oxford. R. K. ROBERTS, F. H. S., ELEK, P., AND l&m, 1962. Studies on resistance in calves to experimental infections with the nodular worm, Oesophagostomum radiatum. Australian Journal of Agricultural Research 13, 551-573. .%XJLSBY, E. J. L., AND cooms, R. R. A. 1959. Studies on blood group substances associated with Ascaris lumbricoides. Parasitology 49, 505-510.