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Dirofilaria immitis: Fate and immunogenicity of irradiated infective stage larvae in beagles
EXPERIMENTAL
PARASITOLOGY
Dirofilaria
MING California Veterinary
35,
465474
( 1974)
immitis: Fate and lmmunogenicity of Irradiated Infective Stage Larvae in Beagles
M. WONC, MARY F. GUEST,~ AND MICHEL
M. J. LAVOIPIERRE
Primate Research Center, and Departments of Physiological Microbiology, School of Veterinary Medicine, University Dads, California 95616 (Submitted
for publication,
September
Sciences of California,
and
24, 1973)
WONG, MING M., GUEST, MARY F., AND LAVOIPIERRE, MICHEL M. J. 1974. Dirofilaria immitis: Fate and immunogenicity of irradiated infective stage larvae in beagles. Experimental Parasitology 35, 465474. Experiments were conducted on the fate of irradiated infective larvae of Dirofilaria immtiis in dogs, and on the effect of these infections on a challenge dose of nonirradiated larvae administered at a later date. Six dogs were inoculated with 200 to 296 irradiated larvae; in no case was a patent infection established. No living worm was recovered beyond 66 days. Eight dogs inoculated with 200 to 2401 irradiated larvae over varying periods of time were exposed 57 to 190 days after the final inoculation of irradiated larvae, to a challenge infection of 200 to 250 nonirradiated (normal) larvae. The results showed that the number of worms which developed to maturity in these dogs was sharply reduced compared to that in the 5 controls (dogs inoculated with normal larvae only). The most striking effect was seen in “vaccinated” dogs which were challenged 3 months or more after the final administration of irradiated larvae, INDEX DESCRIPTORS: Dirof&ria immitis; Immunogenicity; Beagles; Vaccination; Immunity; Irradiation; Aedei aegypti.
crofilaremia, no effort was made in that experiment to determine the fate of these The use of irradiated infective larvae to worms. Since it is essential that the degree stimulate protective immunity, has been enof development of any living immunizing couraging in a number of helminthic infeclarvae be determined, the present experitions (Jarrett et al. 1959; Dow et al. 1959; ments were designed to trace the fate of Poynter et al. 1960; Hsii and Hsii 1961; living irradiated infective stage larvae of Radke and Sadun 1963; Miller 1964). Using Dirofilaria immitis in dogs, and to observe a ,filarial system, Brugiu malayi in rhesus the effect of these larvae on subsequent monkeys, Wong et al. (1969) found that homologous challenge infections. inoculation of the monkeys with X-irradiated third stage larvae afforded some proMATERIALS AND METHODS tection to subsequent homologous challenge infection. Although none of the con- Experimental Animals trol monkeys, i.e., those which received Nineteen laboratory-bred beagles ,aconly irradimated larvae, ever exhibited mi- quired from the Animal Resources Service of the University of California, Davis, were 1 Present address: Department of Biology, Clarke College. Dubuque, Iowa 52001. used. The animals were young adult males 465 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
466
WONG,
TABLE
GUEST
No.
of Day p.i. mf. deocutected lated
A l3eagles La of
Worms
recovered
No.
%
Days pi.
165 217 62 58 40
41 54 36 29 22
136 206 421 471 651
La in-
1 2 3 4 5 Q p.p.,
400 400 170 200 180 killed
PP..” 210 184 196 187 during
prepatent
LAVOIPIERRE
Inoculation
I
Worm Load and Microjila,remia in Group Experimentally Infected with Normal Dirofilaria immitis Dog
AND
period.
and females in good health, and were screened for microfilariae by Knott’s test (Knott 1939). All were vaccinated against distemper, hepatitis, leptospir,osis, and rabies. They were housed in pairs in mosquitoproofed, indoor runs. The room containing the runs had a single small window at one end, but the main source of illumination was provi,ded by a fluorescent light, automatically controlled to give a 12 hr light/ dark cycle. Parasite and Vector A beagle naturally infected with D. immitis was used to infect the mosquitoes. The species of mosquito used was a black eye, selected Liverpool strain of Aedes aegypti, obtained from Dr. A. Ewert of the University of Texas. The mosquitoes were infected by feeding on the beagle at a time when approximately 200 mf/20 cm” blood were circulating peripherally. Infective larvae (Ls) were collected from the mosquitoes 10 to 14 days after the infective meal. The larvae were suspended in Ephrussi-Beadle ( 1936) saline (a modified Ringer’s saline), only active third stage larvae being selected for inoculation. Irradiation of the Ls was carried out by exposing ,a given number of larvae to approximately 20 krads of irradiation from a cobalt source.
of Experimental
Animals
Inoculation of infective stage larvae (nonirradiated and irradiated) into the animals was accomplished by the following methods. In the first method a subcutaneous pocket was made in the inguinal area, and the larvae were introduced into it by means of a sterile Wintrobe pipette. After all the worms had been placed in the pocket, it was closed by a single suture. In the first 2 dogs inoculated by this method, novocaine was used to deaden the skin before the subcutaneous pocket was prepared; however, as observations of the activity of La in dilute solutions of the drug indicated that the larvae became permanently inactivated, its use was discontinued. Thereafter, ice was substituted for novocaine; the ice was held against the inguinal area of the thigh, both to numb the skin and to constrict local blood vessels. The larvae were picked up in the Wintrobe pipette in as small a quantity of fluid as possible. After they were placed in the inguinal pocket, the pipette was rinsed in order to ensure that all the larvae had been introduced. In the second method, the larvae were injected subcutaneously, with a 1 ml syringe and an 18 gauge needle, directly into the inguinal region. The same rinsing precautions as before were used with the syringe in order to make sure that all the larvae had been injected into the skin. For the purpose of the experiment, the 19 dogs were divided into 3 groups. The first group (A) contained 5 animals (4 males and 1 female). This group received only nonirradiated larvae and served as a control (Table I). E’ach of the 5 animals was inoculated with only a single dose of larvae, the number in each inoculum varying from 170 to 400. The second group ( B ), composed of 6 animals (1 male and 5 females), was designed to follow the fate of irradiated larvae in the dogs (Table II). Each of the 6
200
21
200
200
28 P
26 d
Left
Left
Left
Left Right
425b
1376
88b
87” 66
66
15
Right
Right
36
Left
Side
Days p.i. dogs sacrificed
of Irradiated
inoculated into the inguinal area. from heart or pulmonary artery.
200
29 0
= In all cases LI were b No worms recovered
200 140
25 Q
Q
196
Q
of L3
200
No.
No. and location” of La inoculation
6788
Dog
Fate
Left Right
Left Right
0 0
0 0
0 0
0 0
0
Right
Left Right
Leg
(1.4!2.0) 0
Hind
0
Left Right
in Group
Number (measurements
immitis
II
Left
Right
Left
Side
LB of Dirofilaria
TABLE
0 0
0 0
0 0
0 0
0
0
(2.OY2.9)
(1.622.2)
Abdomen
Skin
0 0
0 0
0 0
0 0
0 0
(i.5)
(A) 0 0
0
(5.228.7)
1
0 0
0 0
0 0
0 0
0
I
(2.574.8)
Thoracic
fascia
Lumbar
Dorsal
0
1
0
(4Tl)
0
Chest
and location of worms recovered of worms in mm given in parenthesis)
H Beagles
b z
E
200 201 1000 1000
31
200 200 1000 1000
24
0
200 1000 1000
36 0
c7
1000 1000
NO.
Load
Irradiated
730D c?
kz
Worm
21 420 457
435 456
19 384 405
28
Days after 1st inoculation
L3
200
200
250
200
III
89
80
57
57
Days after final immunizing inoculation
L,
never
never
188
?a
Day mfac detected
C Beagles Challenged with Normal L, of Dirofilaria of Irradiated L3 of Dirofilaria immitis Challenge
No.
and Microfilaremia in Group 1 to 4 Inoculations
TABLE immitis
0
0
26
34
NO.
ajter
Worms
0
0
10.4
17
%
recovered
480
480
204
216
Days after challenge
8 !m
a
8 2 9
200 160
200 159
200
48 0
45 0
47 0
a ?, no circulating mf. b p.p., prepatent period. c mfa, microfilaremia.
200 215
No.
L,
23
29
16
Days after 1st inoculation
Irradiated
688 3
Dog
III
234
225
200
200
No.
(Continued)
190
160
127
120
Days after final immunizing inoculation
Challenge L3
TABLE
never
203
P.P.b
never
Day mfac detected
1
6
7
0
No.
0.5
2.7
3.5
0
%
381
317
140
41
Days after challenge
Worms recovered
E R F m
5
[ C. r/
P
470
WONG,
GUEST
AND
LAVOIPIERRE
The aorta, the carotids, the posterior vena cava, and the jugulars were exposed by blunt dissection. They were clamped and removed with the heart and lungs and transferred to a pan. After transfer the clamps were removed and the blood collected in the pan for later examination. The heart and lungs were examined thoroughly with the aid of a dissecting microscope. Blood vesselswere followed into the lungs, opened, and examined. Larger veins of the liver were also dissected open and searched. Gross lesions or nodules were removed for histopathological examination. In group B, euthanization was based on times at which the fourth larval stage could be expected to be found under the skin, and the fifth stage in the heart (Orihel 1961). Dog 678A died before it was due t’o be euthanized, but is included in Table II. Dogs in group C were euthanized only at times (140 to 480 days postchallenge infection) when challenge larvae had had sufficient time to migrate to the heart and become sexually mature. An exception was made for Dog 688, in which a search was made for worms on the 41st day after the challenge infection was administered. Recovery of Larvae and Adult Worms The procedure used in groups B and C In order to determine the fate of the in- was somewhat different to that in group A. oculated larvae, all 19 dogs were eutha- After euthanization with sodium pentobarnized at appropriate intervals of time fol- bital, the hair was clipped and a depilatory lowing the last inoculation of L3. was used to remove the remaining hair. The dogs in group A (the control group), After being rinsed, the skin was removed with one exception, were killed and ex- in sections, and each section was placed in amined well ,after the infection had become physiol’ogical saline. An inspection was patent, as evidenced by the appearance of made of the viscera. Evidence of host remicrofilariae in the blood stream. Followaction to the presence of larvae, as well as ing euthanization with sodium pentobarbideveloping worms was sought. Gross letal, the skin was removed and its under- sions or nodules were preserved. The heart surface examined for lesions and/or larvae, and lungs were removed for more detailed and the viscera were inspected for gross examination, and portions of the right lesions. Only the heart and lungs were re- atrium and ventricle, the pulmonary artery, moved for closer study using the following and the lobes of the lungs were taken for method. subsequent histological study. The chest was opened and the sternum The carcass, with the exception of the removed so that the heart could be seen. head, feet, and tail, was dismembered and
animals received an initial dose of 200 larvae. Subsequently, 2 of the animals were given an additional dose of 140 and 196 larvae, respectively, 19 to 21 days after the first inoculation. The third group (C) consisted of 8 animals (3 males and 5 females). The purpose of this experiment was to investigate the effect of a single challenge dose of nonirradiated infective larvae (normal larvae ) , in dogs which had been exposed previously to varying doses of irradiated L3. The irradiated larvae were administered to each animal in the dosages shown in Table III. The total number of larvae given varied from 200, as in the case of Dog 47, to as many as 2401 in Dog 31. The regimen under which the LB were administered varied from a single inoculum of 200 worms in Dog 47 to 4 separate inocula of 200, 200, 1000, and 1000, and 200, 201, 1000, and 1000 in Dogs 24 and 31, respectively. The single challenge dose of nonirradiated larvae varied from 200 to 250 LB, the doses being injected 57 to 190 days after the final inoculation of irradiated larvae (see Table III).
D. immitis placed in saline. After incubation at 37°C for approximately 3 hr, the skin portions, the heart, the lungs, and the saline in which these had been soaked were examined with a ‘dissecting microscope. After being inspected, various portions ‘of the skin and the carcass were placed separately in fresh saline and incubated overnight. The tissues were discarded the following morning and the saline allowed to stand for sedimentation. Excess fluid was then decanted, and the sediment concentrated by centrifugation before it was examined for the presence of larvae. Larvae recovered were examined microscopically and then fixed for detailed morphological study.
IN BEAGLES
471
Irradiated L3 of D. immitis in Beagles
The fate of the irradiated L3 in the dogs was studied by necropsy examination of 6 euthanized dogs at varying intervals p.i., corresponding to developmental times of the postinfective stages as given by Orihel (1961) for normal worms. All the worms which were recovered were found during the soaking of subcutaneous tissues taken from the same side of the body as that into which the Ls had been inoculated. With few exceptions, the helminths recovered were entire and living. The rate of recoveries of inoculated worms ,decreased as the intervals between inoculation and examination increased Blood Collection (Table II). The greatest number of worms Midafternoon samples of venous blood (20) was recovered from Dog 678A. This were collected routinely from each dog at dog had received 200 and 196 irradiated fortnightly intervals. One milliliter portions L3, 36 and 15 days respectively prior to were oxylated for examination fcor micro- euthanization. Included in this number are filariae by Knott’s technique; the remainder portions of 2 worms. One of these was found in the dorsal lumbar fascia, the other provided sera for use in IFA tests. in the dorsal thoracic fascia. Living worms IFA Tests for L3 Antibodies varied in length from 1.4 to 6.1 mm. Five IFA tests were done according to the worms, all 4th stage larvae ( L4), were remethod of W’ong and Guest ( 1969), using covered from Dog 21 into which a single infective larvae of D. immitis as the anti- inoculum of 200 irradiated LB had been gen, Sera from dogs in groups B and C introduced 66 days prior to euthanization. were tested in double blind series over a The length ‘of these worms, one of which was dead, ranged from 5.2 to 8.7 mm. The period of one year. last recovery of living worms (2) was from Dog 25, which had been inoculated 87 and RFXJLTS 66 days pre-euthanization, with 200 and 140 Normal (Nonirradiated) L3 of D. immitis irradiated LB in the respective inocula. in Beagles These worms were also L* and measured The results of experimental infections 6.1 and 6.5 mm in length. Compared to using normal L3 in 5 beagles are summa- normal Lq (Orihel 1961), they appeared rized in Table I. With the exception of less developed. No worms were recovered Dog 1, which was killed 136 days postinoc- from Dogs 29, 28, and 26, although the ulation ( p.i. ) during the prepatent period, skins and carcasses as well as the hearts infections in all dogs resulted in the pro- and lungs were examined thoroughly. duction of microfilaremia. Prepatent periods These dogs had each been inoculated once detected ranged from 184 to 210 days. Per- with 260 irradiated L3 at 88, 137, and 425 centage recovery of adult worms ranged days respectively, prior to euthanization. from 22% in Dog 5, killed 651 days p.i., The maximum longevity of the irradiated to 54% in Dog 2, killed 266 days p.i. larvae was thus found to be about 66 days.
472 Challenge
WONG,
GUEST
AND
Infections
Eight dogs which had received various regimens of irradiated larvae were each given a single challenge dose of approximately the same number of Ls ( Table III ) . Depending on the individual dog, the challenge was delivered at approximately 2, 2, 3, 3, 4, 4, 5, and 6 months respectively after the final immunizing inoculum. Adult worms ranging in number from a single individual (a female) in the case of Dog 47 to 34 worms (males and females ) in the case ‘of Dog 730D were recovered from 5 of the 8 d’ogs challenged. These represented from 0.5 to 17% recovery. Microfilaremia was detected in 2 of the 5 dogs within the normal prepatent period. Of the 3 remaining dogs, 1 (Dog 48) was sacrificed during prepatency, 1 (Dog 47) had only a single unfertilized female worm as previously noted, and 1 (Dog 730D) did not have any circulating microfilariae although 34 mature worms were recovered from it. No worms were found in 3 dogs: 24, 36, and 688. Dogs 24 and 36, which had received 200 irradiated Ls over a period of 1 year and 3 months, and which were challenged 3 months after the final inoculations, were sacrificed 480 days later. Dog 688 received 2 inoculations totaling 415 irradiated Ls; it was challenged 4 months later and sacrificed 41 days postchallenge. IFA Antibodies
against L3
With the exception of 1 dog (678A), which was necropsied 36 days after the first inoculation of irradiated La, all dogs which had received irradiated worms developed antibodies to Ls detectable by the IFA method. The titers were generally low, with the highest being 1: 128. Antibodies became detectable in the unchallenged dogs generally about 3 weeks after the first inoculation, and titers peaked around 4 months postinoculation. Increased titers were not consistently seen following subsequent inoculations (boosters) of irradiated L3, but increased titers were observed in
LAVOIPIEFtRE
5 of 8 dogs which had received multiple injections of irradiated Ls and/or challenge inocula. Peak titers in challenged dogs usually occurred after challenge inoculation. As a whole, higher titers were observed in dogs with a lower percentage of worm recoveries and vice versa. DISCUSSION
Although a small number of dogs was used in this experiment, the results obtained from this preliminary study appear to provide useful guidelines for future investigations in the utilization of irradiated larvae ,as live vaccine against D. immitis infection. Since no worm was ever found in the heart ‘or lung of the dogs which were given irradiated larvae only, it is reasonable to conclude that the infective larva of D. immitis when irradiated at 20 krads loses its ability to establish patent infection in its host. It is noteworthy that the last day on which irradiated live worms were recoverable was 66 days pi. As no live worms were found 87 days or later, it would seem reasonable to assume that the longevity of irradiated larvae of D. immitis, when exposed to 20 krads, is somewhere between 2 and 3 months. According to Orihel ( 1!361), the normal L4 ‘of D. immitis ,develops for about 70 days before moulting to the 5th stage, at which point it begins its migration to the heart. Our experiments indicate that irradiated worms died bedore moulting to the 5th stage. Whether this failure of irradiated D. immitb to become established in the host is due solely to factors inherent in the irradiated worm, or is due in part also to adverse host response, can only be speculative at present. That host factors may also play a part in limiting development of the worms is shown by the finding that normal D. immitis in an abnormal host (the macaque monkey) meets with a similar fate as irradiated larvae in the dog ( Wong, unpublished data).
D. immitis The effect produced by multi-inoculations ,of irradiated larvae remains an open question. It is not cIear whether the worms in the booster doses had an equal chance to develop, ‘or were eliminated by the already immunized/sensitized host. Judging by the results of the IFA tests as well as resulting worm burdens after challenge inocula, there did not appear to be a very obvious advantage in administering multiinoculations of irradiated larvae. The results of the tests did show, however, that the highest titers ‘of anti-L3 antibodies appeared after the death of all immunizing larvae. The role played by the humoral antibodies to LI as detected by the IFA technique is not clear at present. Although there appears to be some evidence of correlation between LB antibody levels and the degree of resistance achieved in dogs which received inocula of irradiated L3, the levels as a whole were rather low. Similar or even higher levels of antibodies to L3 have been noted in infected dogs which were found to harbor many ad& worms. Therefore, it remains a matter for conjecture as to whether these humoral antibodies indicate the presence of functional antibodies leading to immunity. By far the best results of protective immunity appeared in “vaccinated” dogs which were challenged at least 3 months after receiving irradiated larval vaccines. The least effect was seen in 2 dogs challenged only 57 ,days after final inoculation. In one of these dogs, the worm recovery rate of 17% was only slightly lower than those in nonvaccinated controls (22 to 54% ). It thus appears that the timing of the challenge exposure after all the immunizing larvae were dead was significant. Numbers of immunizing larvae alone did not seem important, as seen in Dog 31, but large numbers of larvae combined with proper timing gave the best results, as seen in Dogs 24 and 36. Based on these data, we are repeating this part of the experi-
473
IN BEAGLES
ment using larger numbers and other breeds of ,dogs. In conclusion, our study suggests that the use of live vaccine for protection against D. immitis is possible. However, commercial production of such a vaccine has many inherent problems, such as the high cost of maintaining populations of live worms in experimental dogs and large colonies of vector mosquitoes. Much still remains to be learned of the mechanism of protective immunity to D. immitis and the nature of the functional antigens. Before vaccine production becomes a practical proposition, the isolation, characterization, concentration, preservation, and standardization of these antigens, be they exsheathing fluid, tissue migratory enzymes, or metabolic products, must be undertaken. ACKNOWLEDGMENTS We take pleasure in acknowledging technical assistance ‘received from S. Mullin, D. Evans, K. Brown, and I. Sakaizi; all were veterinary students at the time of this study. Appreciation is due to Professor E. Maxie, Department of Pomology, School of Agricultural and Environmental Sciences, University of California at Davis, for excellent services rendered in irradiation of the infective larvae used in this experiment. This investigation was supported by GRS grant 69-23, University of California at Davis, and by the U.S.-Japan Cooperative Medical Science Program, ‘administered by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, grants R22 AI09441 and A109741, and partially by grant RRO-0169 from the National Institutes of Health.
REFERENCES Dow,
C., JARRETT, W. F. H., JENNINGS, F. W., MCINTYRE, W. I. M., AND MULLIGAN, W. 1959. Studies on immunity to Uncinaria stenocephala infection in the dog-double vaccination with irradiated larvae. Journal of the Veterinary Medical Association 135, 407-411. EPHRUSSI, B., AND BEADLE, G. W. 1936. A technique of transplantation for Drosophiliu. American Naturalist 70, 21?+225. Hsii, S. Y., AND Hsti, H. F. 1961. New approach ,to immunization against Schistosoma japonicum. Science 133, 766.
474
WONG,
GUEST
AND
JARRETT, W. F. H., JENNINGS, F. W., MCINTYRE, W. I. M., MULLIGAN, W., SHARP, N. C. C., AND URQUHART, G. M. 1959. Immunological studies on Dictyocaulus viviparus infection in calves: Double vaccination with irradiated larvae. American Journul of Veterinary Research 20, 522-526. KNOTT, J. 1939. Method for making microfilarial surveys on day blood. Transactions of the Royal Society of Tropical Medicine and Hygiene 33, 191-196. MILLER, T. A. 1964. Effect of X-irradiation upon the infective larvae of Ancylostoma caninum and the immunogenic effect in dogs of a single infection with 40 kr-irradiated larvae. Journal of Parasitology 50, 735-742. ORIHEL, T. C. 1961. Morphology of the larval stages of DiToflaTia immitis in the dog. JOUTnal of Parasitology 47, 251-262.
LAVOIPIERRE
POYNTER, D., JONES, B. V., NELSON, A. M. Il., PEACOCK, R., ROBINSON, J., SILVERMAN, P. H., AND TERRY, R. J. 1960. Recent experiences with vaccination. Veterinary RKOTCZ 72, 10781086. RADKE, M. G., AND SADUN, E. H. 1963. Resistance produced in mice by exposure to irradiated Schistosoma munsoni cercariae. Experimental Parasitology 13, 134-142. WONC, M. M., FREDERICKS, H. J., AND RAMACHANDRAN, C. P. 1969. Studies on immunization against Brugia mulayi infection in the rhesus monkey. Bulletin of the World Health Organization 40, 493-501. WONC, M. M., AND GUEST, M. F. 1969. Filarial a#ntibodies and eosinophilia in human subjects in an endemic area. Transactions of the Royal Society of Tropical Medicine and Hygiene 63, 796-800.
PARASITOLOGY
Dirofilaria
MING California Veterinary
35,
465474
( 1974)
immitis: Fate and lmmunogenicity of Irradiated Infective Stage Larvae in Beagles
M. WONC, MARY F. GUEST,~ AND MICHEL
M. J. LAVOIPIERRE
Primate Research Center, and Departments of Physiological Microbiology, School of Veterinary Medicine, University Dads, California 95616 (Submitted
for publication,
September
Sciences of California,
and
24, 1973)
WONG, MING M., GUEST, MARY F., AND LAVOIPIERRE, MICHEL M. J. 1974. Dirofilaria immitis: Fate and immunogenicity of irradiated infective stage larvae in beagles. Experimental Parasitology 35, 465474. Experiments were conducted on the fate of irradiated infective larvae of Dirofilaria immtiis in dogs, and on the effect of these infections on a challenge dose of nonirradiated larvae administered at a later date. Six dogs were inoculated with 200 to 296 irradiated larvae; in no case was a patent infection established. No living worm was recovered beyond 66 days. Eight dogs inoculated with 200 to 2401 irradiated larvae over varying periods of time were exposed 57 to 190 days after the final inoculation of irradiated larvae, to a challenge infection of 200 to 250 nonirradiated (normal) larvae. The results showed that the number of worms which developed to maturity in these dogs was sharply reduced compared to that in the 5 controls (dogs inoculated with normal larvae only). The most striking effect was seen in “vaccinated” dogs which were challenged 3 months or more after the final administration of irradiated larvae, INDEX DESCRIPTORS: Dirof&ria immitis; Immunogenicity; Beagles; Vaccination; Immunity; Irradiation; Aedei aegypti.
crofilaremia, no effort was made in that experiment to determine the fate of these The use of irradiated infective larvae to worms. Since it is essential that the degree stimulate protective immunity, has been enof development of any living immunizing couraging in a number of helminthic infeclarvae be determined, the present experitions (Jarrett et al. 1959; Dow et al. 1959; ments were designed to trace the fate of Poynter et al. 1960; Hsii and Hsii 1961; living irradiated infective stage larvae of Radke and Sadun 1963; Miller 1964). Using Dirofilaria immitis in dogs, and to observe a ,filarial system, Brugiu malayi in rhesus the effect of these larvae on subsequent monkeys, Wong et al. (1969) found that homologous challenge infections. inoculation of the monkeys with X-irradiated third stage larvae afforded some proMATERIALS AND METHODS tection to subsequent homologous challenge infection. Although none of the con- Experimental Animals trol monkeys, i.e., those which received Nineteen laboratory-bred beagles ,aconly irradimated larvae, ever exhibited mi- quired from the Animal Resources Service of the University of California, Davis, were 1 Present address: Department of Biology, Clarke College. Dubuque, Iowa 52001. used. The animals were young adult males 465 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
466
WONG,
TABLE
GUEST
No.
of Day p.i. mf. deocutected lated
A l3eagles La of
Worms
recovered
No.
%
Days pi.
165 217 62 58 40
41 54 36 29 22
136 206 421 471 651
La in-
1 2 3 4 5 Q p.p.,
400 400 170 200 180 killed
PP..” 210 184 196 187 during
prepatent
LAVOIPIERRE
Inoculation
I
Worm Load and Microjila,remia in Group Experimentally Infected with Normal Dirofilaria immitis Dog
AND
period.
and females in good health, and were screened for microfilariae by Knott’s test (Knott 1939). All were vaccinated against distemper, hepatitis, leptospir,osis, and rabies. They were housed in pairs in mosquitoproofed, indoor runs. The room containing the runs had a single small window at one end, but the main source of illumination was provi,ded by a fluorescent light, automatically controlled to give a 12 hr light/ dark cycle. Parasite and Vector A beagle naturally infected with D. immitis was used to infect the mosquitoes. The species of mosquito used was a black eye, selected Liverpool strain of Aedes aegypti, obtained from Dr. A. Ewert of the University of Texas. The mosquitoes were infected by feeding on the beagle at a time when approximately 200 mf/20 cm” blood were circulating peripherally. Infective larvae (Ls) were collected from the mosquitoes 10 to 14 days after the infective meal. The larvae were suspended in Ephrussi-Beadle ( 1936) saline (a modified Ringer’s saline), only active third stage larvae being selected for inoculation. Irradiation of the Ls was carried out by exposing ,a given number of larvae to approximately 20 krads of irradiation from a cobalt source.
of Experimental
Animals
Inoculation of infective stage larvae (nonirradiated and irradiated) into the animals was accomplished by the following methods. In the first method a subcutaneous pocket was made in the inguinal area, and the larvae were introduced into it by means of a sterile Wintrobe pipette. After all the worms had been placed in the pocket, it was closed by a single suture. In the first 2 dogs inoculated by this method, novocaine was used to deaden the skin before the subcutaneous pocket was prepared; however, as observations of the activity of La in dilute solutions of the drug indicated that the larvae became permanently inactivated, its use was discontinued. Thereafter, ice was substituted for novocaine; the ice was held against the inguinal area of the thigh, both to numb the skin and to constrict local blood vessels. The larvae were picked up in the Wintrobe pipette in as small a quantity of fluid as possible. After they were placed in the inguinal pocket, the pipette was rinsed in order to ensure that all the larvae had been introduced. In the second method, the larvae were injected subcutaneously, with a 1 ml syringe and an 18 gauge needle, directly into the inguinal region. The same rinsing precautions as before were used with the syringe in order to make sure that all the larvae had been injected into the skin. For the purpose of the experiment, the 19 dogs were divided into 3 groups. The first group (A) contained 5 animals (4 males and 1 female). This group received only nonirradiated larvae and served as a control (Table I). E’ach of the 5 animals was inoculated with only a single dose of larvae, the number in each inoculum varying from 170 to 400. The second group ( B ), composed of 6 animals (1 male and 5 females), was designed to follow the fate of irradiated larvae in the dogs (Table II). Each of the 6
200
21
200
200
28 P
26 d
Left
Left
Left
Left Right
425b
1376
88b
87” 66
66
15
Right
Right
36
Left
Side
Days p.i. dogs sacrificed
of Irradiated
inoculated into the inguinal area. from heart or pulmonary artery.
200
29 0
= In all cases LI were b No worms recovered
200 140
25 Q
Q
196
Q
of L3
200
No.
No. and location” of La inoculation
6788
Dog
Fate
Left Right
Left Right
0 0
0 0
0 0
0 0
0
Right
Left Right
Leg
(1.4!2.0) 0
Hind
0
Left Right
in Group
Number (measurements
immitis
II
Left
Right
Left
Side
LB of Dirofilaria
TABLE
0 0
0 0
0 0
0 0
0
0
(2.OY2.9)
(1.622.2)
Abdomen
Skin
0 0
0 0
0 0
0 0
0 0
(i.5)
(A) 0 0
0
(5.228.7)
1
0 0
0 0
0 0
0 0
0
I
(2.574.8)
Thoracic
fascia
Lumbar
Dorsal
0
1
0
(4Tl)
0
Chest
and location of worms recovered of worms in mm given in parenthesis)
H Beagles
b z
E
200 201 1000 1000
31
200 200 1000 1000
24
0
200 1000 1000
36 0
c7
1000 1000
NO.
Load
Irradiated
730D c?
kz
Worm
21 420 457
435 456
19 384 405
28
Days after 1st inoculation
L3
200
200
250
200
III
89
80
57
57
Days after final immunizing inoculation
L,
never
never
188
?a
Day mfac detected
C Beagles Challenged with Normal L, of Dirofilaria of Irradiated L3 of Dirofilaria immitis Challenge
No.
and Microfilaremia in Group 1 to 4 Inoculations
TABLE immitis
0
0
26
34
NO.
ajter
Worms
0
0
10.4
17
%
recovered
480
480
204
216
Days after challenge
8 !m
a
8 2 9
200 160
200 159
200
48 0
45 0
47 0
a ?, no circulating mf. b p.p., prepatent period. c mfa, microfilaremia.
200 215
No.
L,
23
29
16
Days after 1st inoculation
Irradiated
688 3
Dog
III
234
225
200
200
No.
(Continued)
190
160
127
120
Days after final immunizing inoculation
Challenge L3
TABLE
never
203
P.P.b
never
Day mfac detected
1
6
7
0
No.
0.5
2.7
3.5
0
%
381
317
140
41
Days after challenge
Worms recovered
E R F m
5
[ C. r/
P
470
WONG,
GUEST
AND
LAVOIPIERRE
The aorta, the carotids, the posterior vena cava, and the jugulars were exposed by blunt dissection. They were clamped and removed with the heart and lungs and transferred to a pan. After transfer the clamps were removed and the blood collected in the pan for later examination. The heart and lungs were examined thoroughly with the aid of a dissecting microscope. Blood vesselswere followed into the lungs, opened, and examined. Larger veins of the liver were also dissected open and searched. Gross lesions or nodules were removed for histopathological examination. In group B, euthanization was based on times at which the fourth larval stage could be expected to be found under the skin, and the fifth stage in the heart (Orihel 1961). Dog 678A died before it was due t’o be euthanized, but is included in Table II. Dogs in group C were euthanized only at times (140 to 480 days postchallenge infection) when challenge larvae had had sufficient time to migrate to the heart and become sexually mature. An exception was made for Dog 688, in which a search was made for worms on the 41st day after the challenge infection was administered. Recovery of Larvae and Adult Worms The procedure used in groups B and C In order to determine the fate of the in- was somewhat different to that in group A. oculated larvae, all 19 dogs were eutha- After euthanization with sodium pentobarnized at appropriate intervals of time fol- bital, the hair was clipped and a depilatory lowing the last inoculation of L3. was used to remove the remaining hair. The dogs in group A (the control group), After being rinsed, the skin was removed with one exception, were killed and ex- in sections, and each section was placed in amined well ,after the infection had become physiol’ogical saline. An inspection was patent, as evidenced by the appearance of made of the viscera. Evidence of host remicrofilariae in the blood stream. Followaction to the presence of larvae, as well as ing euthanization with sodium pentobarbideveloping worms was sought. Gross letal, the skin was removed and its under- sions or nodules were preserved. The heart surface examined for lesions and/or larvae, and lungs were removed for more detailed and the viscera were inspected for gross examination, and portions of the right lesions. Only the heart and lungs were re- atrium and ventricle, the pulmonary artery, moved for closer study using the following and the lobes of the lungs were taken for method. subsequent histological study. The chest was opened and the sternum The carcass, with the exception of the removed so that the heart could be seen. head, feet, and tail, was dismembered and
animals received an initial dose of 200 larvae. Subsequently, 2 of the animals were given an additional dose of 140 and 196 larvae, respectively, 19 to 21 days after the first inoculation. The third group (C) consisted of 8 animals (3 males and 5 females). The purpose of this experiment was to investigate the effect of a single challenge dose of nonirradiated infective larvae (normal larvae ) , in dogs which had been exposed previously to varying doses of irradiated L3. The irradiated larvae were administered to each animal in the dosages shown in Table III. The total number of larvae given varied from 200, as in the case of Dog 47, to as many as 2401 in Dog 31. The regimen under which the LB were administered varied from a single inoculum of 200 worms in Dog 47 to 4 separate inocula of 200, 200, 1000, and 1000, and 200, 201, 1000, and 1000 in Dogs 24 and 31, respectively. The single challenge dose of nonirradiated larvae varied from 200 to 250 LB, the doses being injected 57 to 190 days after the final inoculation of irradiated larvae (see Table III).
D. immitis placed in saline. After incubation at 37°C for approximately 3 hr, the skin portions, the heart, the lungs, and the saline in which these had been soaked were examined with a ‘dissecting microscope. After being inspected, various portions ‘of the skin and the carcass were placed separately in fresh saline and incubated overnight. The tissues were discarded the following morning and the saline allowed to stand for sedimentation. Excess fluid was then decanted, and the sediment concentrated by centrifugation before it was examined for the presence of larvae. Larvae recovered were examined microscopically and then fixed for detailed morphological study.
IN BEAGLES
471
Irradiated L3 of D. immitis in Beagles
The fate of the irradiated L3 in the dogs was studied by necropsy examination of 6 euthanized dogs at varying intervals p.i., corresponding to developmental times of the postinfective stages as given by Orihel (1961) for normal worms. All the worms which were recovered were found during the soaking of subcutaneous tissues taken from the same side of the body as that into which the Ls had been inoculated. With few exceptions, the helminths recovered were entire and living. The rate of recoveries of inoculated worms ,decreased as the intervals between inoculation and examination increased Blood Collection (Table II). The greatest number of worms Midafternoon samples of venous blood (20) was recovered from Dog 678A. This were collected routinely from each dog at dog had received 200 and 196 irradiated fortnightly intervals. One milliliter portions L3, 36 and 15 days respectively prior to were oxylated for examination fcor micro- euthanization. Included in this number are filariae by Knott’s technique; the remainder portions of 2 worms. One of these was found in the dorsal lumbar fascia, the other provided sera for use in IFA tests. in the dorsal thoracic fascia. Living worms IFA Tests for L3 Antibodies varied in length from 1.4 to 6.1 mm. Five IFA tests were done according to the worms, all 4th stage larvae ( L4), were remethod of W’ong and Guest ( 1969), using covered from Dog 21 into which a single infective larvae of D. immitis as the anti- inoculum of 200 irradiated LB had been gen, Sera from dogs in groups B and C introduced 66 days prior to euthanization. were tested in double blind series over a The length ‘of these worms, one of which was dead, ranged from 5.2 to 8.7 mm. The period of one year. last recovery of living worms (2) was from Dog 25, which had been inoculated 87 and RFXJLTS 66 days pre-euthanization, with 200 and 140 Normal (Nonirradiated) L3 of D. immitis irradiated LB in the respective inocula. in Beagles These worms were also L* and measured The results of experimental infections 6.1 and 6.5 mm in length. Compared to using normal L3 in 5 beagles are summa- normal Lq (Orihel 1961), they appeared rized in Table I. With the exception of less developed. No worms were recovered Dog 1, which was killed 136 days postinoc- from Dogs 29, 28, and 26, although the ulation ( p.i. ) during the prepatent period, skins and carcasses as well as the hearts infections in all dogs resulted in the pro- and lungs were examined thoroughly. duction of microfilaremia. Prepatent periods These dogs had each been inoculated once detected ranged from 184 to 210 days. Per- with 260 irradiated L3 at 88, 137, and 425 centage recovery of adult worms ranged days respectively, prior to euthanization. from 22% in Dog 5, killed 651 days p.i., The maximum longevity of the irradiated to 54% in Dog 2, killed 266 days p.i. larvae was thus found to be about 66 days.
472 Challenge
WONG,
GUEST
AND
Infections
Eight dogs which had received various regimens of irradiated larvae were each given a single challenge dose of approximately the same number of Ls ( Table III ) . Depending on the individual dog, the challenge was delivered at approximately 2, 2, 3, 3, 4, 4, 5, and 6 months respectively after the final immunizing inoculum. Adult worms ranging in number from a single individual (a female) in the case of Dog 47 to 34 worms (males and females ) in the case ‘of Dog 730D were recovered from 5 of the 8 d’ogs challenged. These represented from 0.5 to 17% recovery. Microfilaremia was detected in 2 of the 5 dogs within the normal prepatent period. Of the 3 remaining dogs, 1 (Dog 48) was sacrificed during prepatency, 1 (Dog 47) had only a single unfertilized female worm as previously noted, and 1 (Dog 730D) did not have any circulating microfilariae although 34 mature worms were recovered from it. No worms were found in 3 dogs: 24, 36, and 688. Dogs 24 and 36, which had received 200 irradiated Ls over a period of 1 year and 3 months, and which were challenged 3 months after the final inoculations, were sacrificed 480 days later. Dog 688 received 2 inoculations totaling 415 irradiated Ls; it was challenged 4 months later and sacrificed 41 days postchallenge. IFA Antibodies
against L3
With the exception of 1 dog (678A), which was necropsied 36 days after the first inoculation of irradiated La, all dogs which had received irradiated worms developed antibodies to Ls detectable by the IFA method. The titers were generally low, with the highest being 1: 128. Antibodies became detectable in the unchallenged dogs generally about 3 weeks after the first inoculation, and titers peaked around 4 months postinoculation. Increased titers were not consistently seen following subsequent inoculations (boosters) of irradiated L3, but increased titers were observed in
LAVOIPIEFtRE
5 of 8 dogs which had received multiple injections of irradiated Ls and/or challenge inocula. Peak titers in challenged dogs usually occurred after challenge inoculation. As a whole, higher titers were observed in dogs with a lower percentage of worm recoveries and vice versa. DISCUSSION
Although a small number of dogs was used in this experiment, the results obtained from this preliminary study appear to provide useful guidelines for future investigations in the utilization of irradiated larvae ,as live vaccine against D. immitis infection. Since no worm was ever found in the heart ‘or lung of the dogs which were given irradiated larvae only, it is reasonable to conclude that the infective larva of D. immitis when irradiated at 20 krads loses its ability to establish patent infection in its host. It is noteworthy that the last day on which irradiated live worms were recoverable was 66 days pi. As no live worms were found 87 days or later, it would seem reasonable to assume that the longevity of irradiated larvae of D. immitis, when exposed to 20 krads, is somewhere between 2 and 3 months. According to Orihel ( 1!361), the normal L4 ‘of D. immitis ,develops for about 70 days before moulting to the 5th stage, at which point it begins its migration to the heart. Our experiments indicate that irradiated worms died bedore moulting to the 5th stage. Whether this failure of irradiated D. immitb to become established in the host is due solely to factors inherent in the irradiated worm, or is due in part also to adverse host response, can only be speculative at present. That host factors may also play a part in limiting development of the worms is shown by the finding that normal D. immitis in an abnormal host (the macaque monkey) meets with a similar fate as irradiated larvae in the dog ( Wong, unpublished data).
D. immitis The effect produced by multi-inoculations ,of irradiated larvae remains an open question. It is not cIear whether the worms in the booster doses had an equal chance to develop, ‘or were eliminated by the already immunized/sensitized host. Judging by the results of the IFA tests as well as resulting worm burdens after challenge inocula, there did not appear to be a very obvious advantage in administering multiinoculations of irradiated larvae. The results of the tests did show, however, that the highest titers ‘of anti-L3 antibodies appeared after the death of all immunizing larvae. The role played by the humoral antibodies to LI as detected by the IFA technique is not clear at present. Although there appears to be some evidence of correlation between LB antibody levels and the degree of resistance achieved in dogs which received inocula of irradiated L3, the levels as a whole were rather low. Similar or even higher levels of antibodies to L3 have been noted in infected dogs which were found to harbor many ad& worms. Therefore, it remains a matter for conjecture as to whether these humoral antibodies indicate the presence of functional antibodies leading to immunity. By far the best results of protective immunity appeared in “vaccinated” dogs which were challenged at least 3 months after receiving irradiated larval vaccines. The least effect was seen in 2 dogs challenged only 57 ,days after final inoculation. In one of these dogs, the worm recovery rate of 17% was only slightly lower than those in nonvaccinated controls (22 to 54% ). It thus appears that the timing of the challenge exposure after all the immunizing larvae were dead was significant. Numbers of immunizing larvae alone did not seem important, as seen in Dog 31, but large numbers of larvae combined with proper timing gave the best results, as seen in Dogs 24 and 36. Based on these data, we are repeating this part of the experi-
473
IN BEAGLES
ment using larger numbers and other breeds of ,dogs. In conclusion, our study suggests that the use of live vaccine for protection against D. immitis is possible. However, commercial production of such a vaccine has many inherent problems, such as the high cost of maintaining populations of live worms in experimental dogs and large colonies of vector mosquitoes. Much still remains to be learned of the mechanism of protective immunity to D. immitis and the nature of the functional antigens. Before vaccine production becomes a practical proposition, the isolation, characterization, concentration, preservation, and standardization of these antigens, be they exsheathing fluid, tissue migratory enzymes, or metabolic products, must be undertaken. ACKNOWLEDGMENTS We take pleasure in acknowledging technical assistance ‘received from S. Mullin, D. Evans, K. Brown, and I. Sakaizi; all were veterinary students at the time of this study. Appreciation is due to Professor E. Maxie, Department of Pomology, School of Agricultural and Environmental Sciences, University of California at Davis, for excellent services rendered in irradiation of the infective larvae used in this experiment. This investigation was supported by GRS grant 69-23, University of California at Davis, and by the U.S.-Japan Cooperative Medical Science Program, ‘administered by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, grants R22 AI09441 and A109741, and partially by grant RRO-0169 from the National Institutes of Health.
REFERENCES Dow,
C., JARRETT, W. F. H., JENNINGS, F. W., MCINTYRE, W. I. M., AND MULLIGAN, W. 1959. Studies on immunity to Uncinaria stenocephala infection in the dog-double vaccination with irradiated larvae. Journal of the Veterinary Medical Association 135, 407-411. EPHRUSSI, B., AND BEADLE, G. W. 1936. A technique of transplantation for Drosophiliu. American Naturalist 70, 21?+225. Hsii, S. Y., AND Hsti, H. F. 1961. New approach ,to immunization against Schistosoma japonicum. Science 133, 766.
474
WONG,
GUEST
AND
JARRETT, W. F. H., JENNINGS, F. W., MCINTYRE, W. I. M., MULLIGAN, W., SHARP, N. C. C., AND URQUHART, G. M. 1959. Immunological studies on Dictyocaulus viviparus infection in calves: Double vaccination with irradiated larvae. American Journul of Veterinary Research 20, 522-526. KNOTT, J. 1939. Method for making microfilarial surveys on day blood. Transactions of the Royal Society of Tropical Medicine and Hygiene 33, 191-196. MILLER, T. A. 1964. Effect of X-irradiation upon the infective larvae of Ancylostoma caninum and the immunogenic effect in dogs of a single infection with 40 kr-irradiated larvae. Journal of Parasitology 50, 735-742. ORIHEL, T. C. 1961. Morphology of the larval stages of DiToflaTia immitis in the dog. JOUTnal of Parasitology 47, 251-262.
LAVOIPIERRE
POYNTER, D., JONES, B. V., NELSON, A. M. Il., PEACOCK, R., ROBINSON, J., SILVERMAN, P. H., AND TERRY, R. J. 1960. Recent experiences with vaccination. Veterinary RKOTCZ 72, 10781086. RADKE, M. G., AND SADUN, E. H. 1963. Resistance produced in mice by exposure to irradiated Schistosoma munsoni cercariae. Experimental Parasitology 13, 134-142. WONC, M. M., FREDERICKS, H. J., AND RAMACHANDRAN, C. P. 1969. Studies on immunization against Brugia mulayi infection in the rhesus monkey. Bulletin of the World Health Organization 40, 493-501. WONC, M. M., AND GUEST, M. F. 1969. Filarial a#ntibodies and eosinophilia in human subjects in an endemic area. Transactions of the Royal Society of Tropical Medicine and Hygiene 63, 796-800.