Immunology 1976 30 7
The immune mechanism which expels the intestinal stage of Trichinella spiralis from rats
R. J. LOVE,* BRIDGET M. OGILVIE & DIANE J. McLAREN Division of Parasitology, National Institute for Medical Research, Mill Hill, London
Received 12 May 1975; acceptedfor publication 3 July 1975
been used extensively as a model system to study the development of immunity to nematode parasites (reviewed by Larsh, 1963, 1967, 1968). Nevertheless, the mechanism by which immunity causes expulsion of adult worms from the intestine remains undefined. There is evidence from serum transfer studies for participation of antibodies in immune expulsion of this parasite (Culbertson, 1942; Culbertson and Kaplan, 1938; Oliver-Gonzales, 1941; Hendricks, 1953; Mills and Kent, 1965) although some authors were unable to transfer immunity with antiserum (McCoy and Bond, 1941; Denham, 1969; Larsh, Goulson and Weatherley, 1964). Larsh et al. (1964, 1966) transferred a degree of immunity to mice with peritoneal cells, but it is difficult to draw conclusions from this work because of the unduly long time interval between giving the animals cells and the challenge infection. Apparently Nippostrongylus brasiliensis, another nematode of the gut, is expelled from both rats and mice by an immune mechanism which requires the participation of both antibodies and sensitized lymphocytes (reviewed by Ogilvie and Jones, 1973; Ogilvie and Love, 1974). The conflicting results with T. spiralis described above, the detailed histopathological studies and the suppressive effect of cortisone and X-irradiation on previously established immunity to this parasite (Larsh, 1963, 1967, 1968), all suggest that a similar compound mechanism is required for the immunological control of T. spiralis.
Summary. The immunological response of rats to the intestinal phase of Trichinella spiralis was assessed using criteria derived from previous studies with the nematode Nippostrongylus brasiliensis in rats and mice. In adult rats, the duration of infection with either parasite is similar and both infections are prolonged in young and lactating rats. As previously shown with N. brasiliensis, immunity to T. spiralis was transferred to recipients with antiserum or mesenteric lymph node cells from immune donors and antisera and cells given in combination had an additive effect. Signs of damage similar to that caused by antibodies in N. brasiliensis appeared in T. spiralis adult worms as the infection progressed and this damage occurred earlier in animals given antiserum or cells. On the basis of these results, it is concluded that the immunological control of adult T. spiralis requires both antibodies and cells, but the relative importance of these components and the way in which they affect T. spiralis requires further analysis. INTRODUCTION
Trichinella spiralis infection in laboratory animals has * Present address: Department of Veterinary Medicine, University of Sydney, Sydney, New South Wales 2006, Australia. Correspondence: Dr B. M. Ogilvie, Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA.
7
8
R. J. Love, Bridget M. Ogilvie & Diane J. McLaren
The present study is possibility.
an
initial investigation into this
MATERIALS AND METHODS Animals Syngeneic PVG/c rats were used in the experiments but allogeneic Sprague-Dawley rats were used for the preparation of antiserum. These rats were from closed colonies at the National Institute for Medical Research. Parasites and parasitological techniques The strain of T. spiralis used in these experiments was obtained from the London School of Hygiene and Tropical Medicine where it has been passaged in mice for several years. In this laboratory it was maintained by passage in Sprague-Dawley rats. The methods used for collecting larvae and adult worms have been adapted from methods developed by Dr D. A. Denham. Rats were killed, skinned and eviscerated, then minced twice in a domestic mincer. The mince was digested in 800 ml of tap water containing 1 per cent HCl and 1 per cent pepsin for about 3 h at 370 with intermittent shaking. The digest was passed through a wire mesh sieve (aperture 355 um) which retained non-digested material, then through a second sieve (aperture 63 pm) which retained the larvae. The larvae were washed from the second sieve and allowed to sediment in tap water. Rats were infected using a 2-ml syringe and wide-bore metal tube inserted well down the oesophagus. The worms were recovered for counting from the intestine using a Baerman-type apparatus. Rats were killed by a blow on the head and the intestine immediately removed, opened lengthwise and rinsed free of ingesta in warm water. The intestine was then supported on surgical gauze in a large crystallization dish filled with warm isotonic saline. After incubation at 370 for 5-6 h the worms in the dishes were concentrated by natural sedimentation in urine flasks. Formalin was then added to a final concentration of 10 per cent v/v. To facilitate counting, worms were stained with Lugols iodine, decolourizing with a few drops of 5 per cent sodium thiosulphate. More than 90 per cent of the infective larvae given to rats established as adult worms in the small intestine. This is quite different from the 53 per cent infectivity found by Gursch (1949) and probably reflects different methods of preparing infective larvae. If
muscle containing larvae was digested for long periods, their infectivity was reduced. Preparation of anti-T. spiralis serum Rats were given two infections of 2000 and 4000 larvae 3 weeks apart and blood collected for preparation of serum 7-14 days after the second infection. Serum (5 ml/100 g body weight) was administered by intraperitoneal injection. Preparation of immune mesenteric lymph node cell suspensions The method of cell preparation has been described previously (Love, 1975). Immune donors were given either one infection of 4000 larvae or two infections of 2000 and 4000 larvae 3 weeks apart. Cells were prepared 19-20 days after a single infection or 7 days after the second infection. 2-5 x 108 viable cells were injected intravenously into each recipient.
Preparation of worms for electron microscopic examination Infected rat intestines were slit open under isotonic saline and incubated at 370 for about 10 min until the nematodes had detached from the host tissue. The worms were then prepared for examination with the electron microscope according to the techniques described by McLaren (1974). To obtain adequately preserved sections of nematodes, it was essential that the time which elapsed between death of the host and fixation of the worms was minimal.
RESULTS Duration of T. spiralis infections in young, adult and lactating PVG/c rats
(a) Adult rats Thirty adult female PVG/c rats were infected with doses of approximately 1500 infective larvae and groups of these rats were killed for intestinal worm counts 6, 8, 10, 12, 14 or 16 days after infection. The resulting group mean worm counts are shown in Fig. 1. More than 90 per cent of the larvae established in the small intestine. Expulsion of adult worms from the intestine began 8-10 days after infection. Approximately a third of the nematodes were expelled from the small intestine during this period. By day 12, only 20 per cent of the worms were still present in
Immunity to T. spiralis
12
9
Complete expulsion of the infection in the young rats occurred between 21 and 28 days after infection. This experiment showed that, compared with the elimination of this parasite by adult rats, worm elimination from the young rats was considerably slower, but nevertheless continued until few if any parasites remained.
-
0
o
4D 6
10
8
Figure 1. Course of a
12
14
16
Days after infection primary infection with T. spiralis in
female PVG/c rats.
the small intestine, and expulsion was complete by day 16, leaving no residual worm population.
(b) Young rats Twenty young male PVG/c rats (21 days old) and ten adult male rats were infected with approximately 500 T. spiralis larvae. Groups of young and adult rats were killed for worm counts at various times after infection. The worm counts are shown in Table 1. The worm counts on day 6 after infection showed Table 1. Course of a primary T. spiralis infection in young and lactating rats Treatment group
Group mean intestinal worm count day of infection 6
Adult (9 weeks) Young(3weeks) Non-lactating Lactating
15
4+2 444+113 159+49 477+ 75 2+ 1
21
28
(c) Lactating rats Five lactating PVG/c rats were infected with 500 T. spiralis larvae within a few days of parturition. Matching non-lactating rats were similarly infected. Groups of control infected rats were killed for worm counts 6 and 15 days after infection and the lactating group 21 days post-infection. The mean worm counts are shown in Table 1. Non-lactating rats expelled the infection within 15 days as in previous experiments but the lactating rats still retained 65 per cent of the parasites when killed 21 days after infection. Rapidity of expulsion of a T. spiralis infection from previously infected rats Female PVGIc rats were infected with 4000 T. spiralis larvae and 8 weeks later reinfected with approximately 1000 larvae, together with groups of previously uninfected rats. Groups of reinfected rats and controls were killed for intestinal worm counts 24 and 48 h after infection. The group mean worm counts are given in Table 2. Within 24 h less than 10 Table 2. Course of a second T. spiralis infection in adult rats Treatment group
420+42
-
96+92 8+3
312+ 74
that equal numbers of larvae established in adult and young rats. The worm counts from the young rats were more variable than those from the adults, because it was more difficult to dose small rats accurately. The adult rats had expelled the infection by day 15 of the experiment, whereas young rats had expelled 64 per cent of the infection at this time.
Primary infection Secondary infection
Mean intestinal worm count time after infection (h)
24
48
1086+ 96
1105+ 117 41 + 14
80+ 60
per cent of the worms were recovered from the previously infected rats compared to the number which had established in the controls. There was a further reduction in the worm numbers recovered 48 h after infection. Therefore, infective larvae were expelled extremely rapidly from rats immunized by a previous infection.
10
R. J. Love, Bridget M. Ogilvie & Diane J. McLaren
Transfer of immunity to T. spiralis with antiserum and lymph node cells from immune rats A number of experiments were carried out in order to determine the relative importance of antibodies and sensitized cells in the expulsion of T. spiralis infection. In all these experiments, rats were infected on the same day that they were given antiserum and/ or cells. For the first experiment, immune rats used as cell and antiserum donors were prepared by giving two infections, the first of 2000 and the second of 4000 larvae 3 weeks later. Cells and antiserum were prepared 7 days after the second infection. Groups of male PVG/c rats were given either immune cells or antiserum (pool 1) and then infected, along with a matching control group, with approximately 800 T. spiralis larvae. All rats were killed for
In a second experiment, the cell transfer was repeated using female PVG/c rats infected with approximately 1000 larvae, but the rats were killed for worm counts on day 6 rather than day 10. The group mean worm counts are shown in Table 3 and show that the transfer of immune cells into rats resulted in the expulsion of 40 per cent of the infection in the 6-day period. Because cells caused expulsion of worms from recipients within 6 days, the number of worms present 4 and 6 days after infection of cell recipients was studied in a third experiment. Cell donors were prepared by infecting rats with 4000 T. spiralis larvae 19 days before they were killed and used for the preparation of a mesenteric lymph node cell suspension. Cells were transferred to groups of PVG/c female rats which, together with control groups, were infected with approximately
Table 3. Transfer of immunity to T. spiralis infection with immune cells and antiserum Treatment group
Experiment 1 Controls Antiserum Immune cells
Day of worm recovery
Group mean intestinal worm count
Statistical significance
10 10 10
451+ 105a 253+42b 2+ 2C
a V b P
The immune mechanism which expels the intestinal stage of Trichinella spiralis from rats
R. J. LOVE,* BRIDGET M. OGILVIE & DIANE J. McLAREN Division of Parasitology, National Institute for Medical Research, Mill Hill, London
Received 12 May 1975; acceptedfor publication 3 July 1975
been used extensively as a model system to study the development of immunity to nematode parasites (reviewed by Larsh, 1963, 1967, 1968). Nevertheless, the mechanism by which immunity causes expulsion of adult worms from the intestine remains undefined. There is evidence from serum transfer studies for participation of antibodies in immune expulsion of this parasite (Culbertson, 1942; Culbertson and Kaplan, 1938; Oliver-Gonzales, 1941; Hendricks, 1953; Mills and Kent, 1965) although some authors were unable to transfer immunity with antiserum (McCoy and Bond, 1941; Denham, 1969; Larsh, Goulson and Weatherley, 1964). Larsh et al. (1964, 1966) transferred a degree of immunity to mice with peritoneal cells, but it is difficult to draw conclusions from this work because of the unduly long time interval between giving the animals cells and the challenge infection. Apparently Nippostrongylus brasiliensis, another nematode of the gut, is expelled from both rats and mice by an immune mechanism which requires the participation of both antibodies and sensitized lymphocytes (reviewed by Ogilvie and Jones, 1973; Ogilvie and Love, 1974). The conflicting results with T. spiralis described above, the detailed histopathological studies and the suppressive effect of cortisone and X-irradiation on previously established immunity to this parasite (Larsh, 1963, 1967, 1968), all suggest that a similar compound mechanism is required for the immunological control of T. spiralis.
Summary. The immunological response of rats to the intestinal phase of Trichinella spiralis was assessed using criteria derived from previous studies with the nematode Nippostrongylus brasiliensis in rats and mice. In adult rats, the duration of infection with either parasite is similar and both infections are prolonged in young and lactating rats. As previously shown with N. brasiliensis, immunity to T. spiralis was transferred to recipients with antiserum or mesenteric lymph node cells from immune donors and antisera and cells given in combination had an additive effect. Signs of damage similar to that caused by antibodies in N. brasiliensis appeared in T. spiralis adult worms as the infection progressed and this damage occurred earlier in animals given antiserum or cells. On the basis of these results, it is concluded that the immunological control of adult T. spiralis requires both antibodies and cells, but the relative importance of these components and the way in which they affect T. spiralis requires further analysis. INTRODUCTION
Trichinella spiralis infection in laboratory animals has * Present address: Department of Veterinary Medicine, University of Sydney, Sydney, New South Wales 2006, Australia. Correspondence: Dr B. M. Ogilvie, Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA.
7
8
R. J. Love, Bridget M. Ogilvie & Diane J. McLaren
The present study is possibility.
an
initial investigation into this
MATERIALS AND METHODS Animals Syngeneic PVG/c rats were used in the experiments but allogeneic Sprague-Dawley rats were used for the preparation of antiserum. These rats were from closed colonies at the National Institute for Medical Research. Parasites and parasitological techniques The strain of T. spiralis used in these experiments was obtained from the London School of Hygiene and Tropical Medicine where it has been passaged in mice for several years. In this laboratory it was maintained by passage in Sprague-Dawley rats. The methods used for collecting larvae and adult worms have been adapted from methods developed by Dr D. A. Denham. Rats were killed, skinned and eviscerated, then minced twice in a domestic mincer. The mince was digested in 800 ml of tap water containing 1 per cent HCl and 1 per cent pepsin for about 3 h at 370 with intermittent shaking. The digest was passed through a wire mesh sieve (aperture 355 um) which retained non-digested material, then through a second sieve (aperture 63 pm) which retained the larvae. The larvae were washed from the second sieve and allowed to sediment in tap water. Rats were infected using a 2-ml syringe and wide-bore metal tube inserted well down the oesophagus. The worms were recovered for counting from the intestine using a Baerman-type apparatus. Rats were killed by a blow on the head and the intestine immediately removed, opened lengthwise and rinsed free of ingesta in warm water. The intestine was then supported on surgical gauze in a large crystallization dish filled with warm isotonic saline. After incubation at 370 for 5-6 h the worms in the dishes were concentrated by natural sedimentation in urine flasks. Formalin was then added to a final concentration of 10 per cent v/v. To facilitate counting, worms were stained with Lugols iodine, decolourizing with a few drops of 5 per cent sodium thiosulphate. More than 90 per cent of the infective larvae given to rats established as adult worms in the small intestine. This is quite different from the 53 per cent infectivity found by Gursch (1949) and probably reflects different methods of preparing infective larvae. If
muscle containing larvae was digested for long periods, their infectivity was reduced. Preparation of anti-T. spiralis serum Rats were given two infections of 2000 and 4000 larvae 3 weeks apart and blood collected for preparation of serum 7-14 days after the second infection. Serum (5 ml/100 g body weight) was administered by intraperitoneal injection. Preparation of immune mesenteric lymph node cell suspensions The method of cell preparation has been described previously (Love, 1975). Immune donors were given either one infection of 4000 larvae or two infections of 2000 and 4000 larvae 3 weeks apart. Cells were prepared 19-20 days after a single infection or 7 days after the second infection. 2-5 x 108 viable cells were injected intravenously into each recipient.
Preparation of worms for electron microscopic examination Infected rat intestines were slit open under isotonic saline and incubated at 370 for about 10 min until the nematodes had detached from the host tissue. The worms were then prepared for examination with the electron microscope according to the techniques described by McLaren (1974). To obtain adequately preserved sections of nematodes, it was essential that the time which elapsed between death of the host and fixation of the worms was minimal.
RESULTS Duration of T. spiralis infections in young, adult and lactating PVG/c rats
(a) Adult rats Thirty adult female PVG/c rats were infected with doses of approximately 1500 infective larvae and groups of these rats were killed for intestinal worm counts 6, 8, 10, 12, 14 or 16 days after infection. The resulting group mean worm counts are shown in Fig. 1. More than 90 per cent of the larvae established in the small intestine. Expulsion of adult worms from the intestine began 8-10 days after infection. Approximately a third of the nematodes were expelled from the small intestine during this period. By day 12, only 20 per cent of the worms were still present in
Immunity to T. spiralis
12
9
Complete expulsion of the infection in the young rats occurred between 21 and 28 days after infection. This experiment showed that, compared with the elimination of this parasite by adult rats, worm elimination from the young rats was considerably slower, but nevertheless continued until few if any parasites remained.
-
0
o
4D 6
10
8
Figure 1. Course of a
12
14
16
Days after infection primary infection with T. spiralis in
female PVG/c rats.
the small intestine, and expulsion was complete by day 16, leaving no residual worm population.
(b) Young rats Twenty young male PVG/c rats (21 days old) and ten adult male rats were infected with approximately 500 T. spiralis larvae. Groups of young and adult rats were killed for worm counts at various times after infection. The worm counts are shown in Table 1. The worm counts on day 6 after infection showed Table 1. Course of a primary T. spiralis infection in young and lactating rats Treatment group
Group mean intestinal worm count day of infection 6
Adult (9 weeks) Young(3weeks) Non-lactating Lactating
15
4+2 444+113 159+49 477+ 75 2+ 1
21
28
(c) Lactating rats Five lactating PVG/c rats were infected with 500 T. spiralis larvae within a few days of parturition. Matching non-lactating rats were similarly infected. Groups of control infected rats were killed for worm counts 6 and 15 days after infection and the lactating group 21 days post-infection. The mean worm counts are shown in Table 1. Non-lactating rats expelled the infection within 15 days as in previous experiments but the lactating rats still retained 65 per cent of the parasites when killed 21 days after infection. Rapidity of expulsion of a T. spiralis infection from previously infected rats Female PVGIc rats were infected with 4000 T. spiralis larvae and 8 weeks later reinfected with approximately 1000 larvae, together with groups of previously uninfected rats. Groups of reinfected rats and controls were killed for intestinal worm counts 24 and 48 h after infection. The group mean worm counts are given in Table 2. Within 24 h less than 10 Table 2. Course of a second T. spiralis infection in adult rats Treatment group
420+42
-
96+92 8+3
312+ 74
that equal numbers of larvae established in adult and young rats. The worm counts from the young rats were more variable than those from the adults, because it was more difficult to dose small rats accurately. The adult rats had expelled the infection by day 15 of the experiment, whereas young rats had expelled 64 per cent of the infection at this time.
Primary infection Secondary infection
Mean intestinal worm count time after infection (h)
24
48
1086+ 96
1105+ 117 41 + 14
80+ 60
per cent of the worms were recovered from the previously infected rats compared to the number which had established in the controls. There was a further reduction in the worm numbers recovered 48 h after infection. Therefore, infective larvae were expelled extremely rapidly from rats immunized by a previous infection.
10
R. J. Love, Bridget M. Ogilvie & Diane J. McLaren
Transfer of immunity to T. spiralis with antiserum and lymph node cells from immune rats A number of experiments were carried out in order to determine the relative importance of antibodies and sensitized cells in the expulsion of T. spiralis infection. In all these experiments, rats were infected on the same day that they were given antiserum and/ or cells. For the first experiment, immune rats used as cell and antiserum donors were prepared by giving two infections, the first of 2000 and the second of 4000 larvae 3 weeks later. Cells and antiserum were prepared 7 days after the second infection. Groups of male PVG/c rats were given either immune cells or antiserum (pool 1) and then infected, along with a matching control group, with approximately 800 T. spiralis larvae. All rats were killed for
In a second experiment, the cell transfer was repeated using female PVG/c rats infected with approximately 1000 larvae, but the rats were killed for worm counts on day 6 rather than day 10. The group mean worm counts are shown in Table 3 and show that the transfer of immune cells into rats resulted in the expulsion of 40 per cent of the infection in the 6-day period. Because cells caused expulsion of worms from recipients within 6 days, the number of worms present 4 and 6 days after infection of cell recipients was studied in a third experiment. Cell donors were prepared by infecting rats with 4000 T. spiralis larvae 19 days before they were killed and used for the preparation of a mesenteric lymph node cell suspension. Cells were transferred to groups of PVG/c female rats which, together with control groups, were infected with approximately
Table 3. Transfer of immunity to T. spiralis infection with immune cells and antiserum Treatment group
Experiment 1 Controls Antiserum Immune cells
Day of worm recovery
Group mean intestinal worm count
Statistical significance
10 10 10
451+ 105a 253+42b 2+ 2C
a V b P
