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Cuterebra buccata: Immune response in myiasis of domestic rabbits
EXPERIMENTALPARASITOLOGY
34,22-31
Cuterebra
STEVEN Division State
H.
(1973)
buccata: Immune Response of Domestic Rabbits WEISBROTH,
REENIE
WANG,
AND
in Myiasis
SHELDON
of Laboratory Animal Resources, Health Sciences University of New York, Stony Brook, New York (Submitted
for publication,
SCHER Center, 11790
5 June 1972)
WEISBROTH, S. H., WANG, R., AND SCHER, S. 1973. Cuterebra buccata: Immune Response in Myiasis of Domestic Rabbits. Experimental Parasitology 34, 2231. Naturally infected rabbits (Oryctolugus) were used to define further the nature of the immune response in myiasis due to Cuterebra buccata. Third instar larvae were dissected into four fractions; (1) alimentary tract with attached organs, (2) hemolymph, (3) fat body with tracheae, and (4) cuticle with attached muscles. The antigens provoking immune phenomena in naturally infected rabbits were found to reside in the alimentary tract and hemolymph fractions only. All rabbits which were skin tested were found to exhibit delayed hypersensitivity and to have serum precipitins with specificity against these antigens. Passive cutaneous anaphylaxis activity was demonstrated only in sera from rabbits also exhibiting reaginic and/or Arthus-type skin hypersensitivities. With the use of immunoelectrophoresis four separate antigens were demonstrated in alimentary tract fractions. Larval dissections revealed the alimentary tract to be filled with cellular elements of rabbit whole blood. The immunologic findings are discussed in relation to this newly recognized feeding pattern and it is proposed that sensitization of the host occurs as a consequence of exogenous larval secretions injected at the time of feeding. INDEX DESCRIPTORS : Cuterebru buccata; Oryctolagus; Rabbits ; Hypersensitivity immediate ; Hypersensitivity delayed ; Antibody ; Myiasis ; Immunity ; Immunoelectrophoresis ; Immunodiff usion,
In a previous report from this laboratory (Weisbroth et al. 1973) the pathologic consequences of spontaneous cuterebriasis in domestic rabbits (Oryctolagus) were reported. They were found to include localized skin lesions consisting of a cavity with a thin necrotic lining containing the Cuterebra larva, and extending radially from the cavity lining, zones of (1) acute inflammation, (2) granulation tissue with fibrous and vascular elements, and (3) chronic inflammation with macrophages and lymphocytes predominating, but also including plasma cells and eosinophils. Regional lymphadenopathy typical of lymph nodes draining an infectious
Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
process were described as the only other pathologic finding from infected rabbits, These lesions suggested that immune processes might be an active concomitant of Cuterebra myiasis. It was found that of six rabbits carrying third instar Cuterebra buccata larvae some, (1/s) would demonstrate immediate hypersensitivity of the wheal and flare type, most ($&) had Arthus reactivity, and all (GA) had delayed-type hypersensitivity, when skin tested intradermally with whole-larva extracts. The sera of these rabbits all had precipitins as detected by agar-gel immunodiffusion. The purpose of this
report
is to characterize
further
the
Cuterebra
FIG.
nature of antigenicity and also the antibody infected rabbits, MATERIALS
1. Third
buccata
instar larva of C. buccata
in Cztterebra larvae response in naturally
AND
IN
METHODS
Antigens. Cuterebra buccata larvae were collected from naturally infected rabbits and prepared for antigenic analysis by grinding washed whole third stage larvae or their fractions for 30 min in a Ten Broeck mortar at 4 C. using 1: 1 dilutions in sterile distilled water. The grindings were centrifuged using an International model PRJ refrigerated centrifuge at 5 C, at a speed of 4000 rpm for 30 min, and the supernate retained. Supernates were plated on blood agar as a test for absence of bacteria and after proven to be bacterially sterile frozen at -90 C without further treatment for use as stock antigen. Stock antigens were analyzed for
RABBITS
(scale in millimeters).
protein content using bovine serum albumin standards (Lowry et al. 1951) and in all cases diluted with phosphate buffered saline (PBS) at pH 7.4 to contain 2 mg protein/ ml for use. Fractions of whole larvae were prepared by dissecting the larvae (Fig. 1) into four parts : ( 1) hemolymph (collected by hypodermic syringe and needle), (2) alimentary tract with attached organs, (3) fat body with tracheal respiratory system, and (4) cuticle with attached muscles. In practice, the dissection was performed by pinning the larva to a dissection board in a dorsoventral position. A midline incision was made through the cuticle for the whole length of the larva. The hemolymph was collected and the cuticle reflected back and pinned to the board (Fig. 2). The alimentary tract fraction was then removed and weighed leaving
24
WEISBROTH,
FIG. 2. Cuticle fractions.
reflected
to show
WANG,
alimentary
tract
fractions C and D (Fig. 3). The fat body and tracheae (fraction C) were then removed and weighed leaving only the cuticle with its muscles (Fig. 4). The weight relationships of whole larvae to their fractions is summarized in Table I. The preparation of larval fractions follows in general the pro-
Larva
no.
1 2 3 4 5 Mean a Percentage
Weight
of Cuterebra
Whole
larva
Third
Hemolymph
1.65 2.04 1.91 2.20 1.52 1.86 of whole
buccata
0.70 1.05 0.86 1.11 0.64 0.87 weight
(42)” (50) (45) (50) (42) (47)
in parentheses.
AND
SCHER
(B),
fat
body
(C),
and
cuticular
(D)
cedure used in dissection of Hypodernca larvae for similar purposes (Beesley 1970). Skin testing. Skin tests on both infected and noninfected laboratory rabbits were performed by parallel intradermal injections of 0.1 ml of antigen and various controls using tuberculin syringes and 27-gauge needles.
TABLE
I
Instar
Whole
Alimentary 0.11 0.11 0.13 0.17 0.11 0.13
Larvae tract
(6.0) (5.3) (7.0) (8.0) (7.0) (7.0)
and their
Fractions
Fat
body
0.24 0.25 0.27 0.27 0.22 0.25
(15) (12) (14) (12) (15) (13)
(in g) Cuticle 0.60 0.69 0.75 0.65 0.55 0.64
(36) (31) (34) (30) (36) (34)
Cuterebra
FIG. 3. Alimentary
buccata
IN
25
RABBITS
tract fraction removed to show fat body (C) and cuticular
(D)
fractions.
Antigen-injected and control sites were obImmunoelectrophoresis (IEP). Agar-gel served 15-20 min, 2-4 hr, and 24-48 hr after for immunoelectrophoresis was prepared with injection. Skin reactions when present, were 1.5% Agarose Behringwerke (obtained from measured and described. Typical sites were Lloyd Brothers, Cincinatti, OH) dissolved biopsied for histologic examination. (w/v) in a solution of 1: 5 Michaelis buffer Immunodifusion (IO). Agar-gel im- in distilled water. The agarose solution was munodiffusion (Ouchterlony ) preciptin tests poured while molten on glass slides, and were performed with the use of both com- after cooling central wells of various sizes mercially obtained patterns (Hyland Im- were cored in the solid gel. Cuterebru whole muno-Plate, Pattern C., Hyland Division of larval antigens, or fraction antigens, or unTravenal Labs., Costa Mesa, CA) and also diluted rabbit sera (from natural infections) with those prepared in the laboratory. Imwere placed in the central well for electromunodiffusion preparations made in the phoresis. Electrophoresis was performed laboratory utilized 0.8% Agarose Behring- using a Universal electrophoretic apparatus weke (obtained from Lloyd Brothers, Cin- (obtained from Colab Laboratories, Chicago cinatti, OH) dissolved (w/v) in PBS (pH Heights, IL) utilizing a potential of 50 V 7.4) with 0.5% each of stock sodium azide and operating times of 60 and 90 min, re(0.1 M) and EDTA (0.01 M) as preserva- spectively, for antigens and sera. Immediately troughs were cut in tives, Immunodiffusion preparations were in- after electrophoresis cubated in a moist chamber at room temper- the agarose gel at various distances from the ature, examined daily for precipitin lines and central well. Immunologic differentiation of the electrophoretically separated proteins was discarded after 7 days.
26
WEISBROTH,
FIG.
WANG,
AND
SCRER
4. Fat body removed to show cuticular
achieved by filling the troughs with antisera (in the case of electrophoresed antigens) and vice versa. Preparations were incubated at 37 C for 1-2 hr and then placed in moist chambers in the refrigerator (5 C). IEP preparations were examined daily for 7 days and precipitin lines and their positions recorded. After 7 days the IEP plates were dialysed against physiological saline for 24 hr (to remove nonprecipitated proteins) and stained in a solution of Buffalo Black to visualize individual precipitin lines better. The agar was dried down to a thin film and preserved as a semipermanent record of the experiment. Passive cutaneous anaFlzylaxis (PCA) . Guinea pigs, (between 200 and 250 g), of the Rockefeller Albino Moen-Chase stock were obtained from Brookhaven National Laboratory for use in PCA experiments. These were performed by injecting 0.1 ml
(D)
fraction.
aliquots of appropriate rabbit serum dilutions and controls intradermally over the shaved lateral body wall. Two replicate guinea pigs were used to titrate each rabbit serum. Four hours later the animals were each injected intravenously with a 1 ml mixture containing 0.5 ml of whole larval antigens and 0.5 ml of lo/O Evans Blue dye in distilled water. Skin sites were examined for blue rings of extravasated dye at the end of 30 min. Blue skin lesions typical of PCA reactions were measured in vivo and recorded. These results were compared with remeasurementsof the same site on the underside of pelted skins. Results were such that measurements of lesions in intact skins were used as the routine procedure. RESULTS
Three naturally infected albino (New Zealand White) rabbits were used for intra-
Cuterebra
buccata TABLE
Results
Rabbit number
1330 1343A 1343B
of Intradermal Skin Cuterebra
Whole larva 2
+
+ -t” -+ +
--
3
a Data presented with indication representing (1) 15-20 min, (2) 2-4
1 +++ ---
2
RABBITS
II
Tests in Naturally Infected buccata Larval Antigens
Alimentary tract
1
IN
Fat
3
1
+ +
-------
for reactive (+) hr, and (3) 24-48
dermal skin testing of antigens extracted from whole larvae and larval fractions. Each individual was skin tested on the day that third instar larvae were removed from its skin so that the time lapse following natural exposure to antigens was minimal. In each case 0.1 ml intradermal injections of sterile physiological saline accompanied antigen injections in different sites. As the saline sites were rountinely nonreactive they do not appear in the tables or descriptive results. Similarly, a total of 12 rabbits on different occasions, known not to have been previously exposed to antigens of Cuterebra sp. were skin tested with antigens from whole larvae or larval fractions and since these skin test sites were routinely nonreactive these results do not appear elsewhere. Skin sites were observed for immediate reactions 15-20 min after antigen injection. When present the lesions presented as round or ovoid (but not serpentine) dome-shaped elevated wheals averaging 10-12 x 12-15 mm in diameter. The color varied from pale or colorless to reddish pink. Skin sites similarly were observed 2-4 hr after injection and redescribed again 24 and 48 hr after injection. These lesions were typically smaller (68 X S-12 mm), pinkish, firmer to the touch, and were not dependent on the presence of skin reactivity when observed earlier. The results of skin testing with the various antigens are summarized in Table II. These results are in agreement with those pre-
body
2
Rabbits
using
Cuticle
3
and nonreactive hr observations,
1
2
Hemolymph
3
------(-) skin respectively.
1 +++ ---
sites
in groups
2
3
+ + of three
viously reported from this laboratory (Weisbroth et al. 1973), in that naturally infected rabbits routinely exhibit delayed hypersensitivity, and occasionally, reaginic and/or Arthus hypersensitivity. More significantly however it appears that the antigens responsible for eliciting these reactions originate in the larval hemolymph. They do not appear to be present in the cuticle with its attached muscles or in the fat body or respiratory system. The latter (cuticle and fat body fractions) are substantial elements of the larval bulk together accounting for approximately 50% of the total weight (Table I). The relationships between larval fractions were further analyzed by agar-gel immunodiffusion. These results are summarized in Fig. 5 which is a representative photograph of results using sera from six separate C. buccata-infected rabbits with naturally acquired antibodies. Analysis by immunodiffusion confirmed the results of skin testing with regard to the anatomic distribution of larval antigens. Antigens are present in higher concentration in the alimentary tract fraction than either whole larval extracts or the hemolymph fraction. Typically titered end points for alimentary tract extracts approximated 1:64 when using the same undiluted antisera. Sera from naturally infected rabbits typically had diluted end points varying from 1:4 to 1: 16 when using stock alimentary tract antigens, but dilution end points of 1:2 or 1:4 when using stock whole
2s
WEISBROTH,
FIG.
rabbit larval lymph,
5. Immunodiffusion in agar gel serum (center well) against estract, (2) alimentary tract, (4) fat body, and (5) cuticle of
WANG,
of immune (1) whole(3) hemoC. buccata.
larva or hemolymph antigens. We have concluded from these experiments that the most likely anatomic location of antigens operative in natural immunization are related to the alimentary tract and its associated organs. That this is likely is suggested as well by
FIG.
larva.
AND
SCHER
the manner of larval feeding, not previously established with certainty in lagomorph or rodent hosts. The larvae are apparently active blood feeders and the lumen of the alimentary tract of all dissected larvae was observed to be filled with the cellular elements of rabbit whole blood (Fig. 6). Considering that Cuterebra larvae undergo a 200-fold increase in weight (from 10 mg first instar to approximately 200 mg third instar larvae) in 25-30 days, it may be concluded that they consume considerable quantities of rabbit blood in the course of this development. Antibodies detected in vitro by immunodiffusion analysis were demonstrated in V&JO as well by heterologous PCA technique in guinea pig skin. Four sera were available for PCA analysis. These results are summarized in Table III. PCA activity (end point dilutions of 1: 128 and 1: 1024 in sera from rabbits 1164 and 1330, respectively) was observed to be present only in sera from rabbits also exhibiting skin hypersensitivity of the immediate or Arthus types. The significance of this finding is difficult to assess
6. Photomicrograph of Giemsa-stained smear Note rabbit erythrocytes (A) and leukocytes
of alimentary (B). X 1000.
tract
contents
of C. buccata
Cuterebra
buccata TABLE
Results Rabbit number
of Sereral
Immunologic
Assays”
Immediate or Arthus hypersensitivities
1164 1330 1343A 1343B
III Infected
Delayed hypersensitivity
for activity
Cuterebra
buccata PC‘4 activity<
+ + + +
+ + -
absent.
The variability detected in immune responses may in part be related to genetically determined individual capability, but also may be related to degree, if any, of previous exposure. While all of the immune properties detected in these rabbits were a consequence of natural immunization by parasitic larvae, it is unknown if this is of value to the host in the prevention or abortion of future parasitic invasions. The interpretation that it is may derive support from epidemiological surveys that report an inverse relationship between age of host and degree of Cuterebra sp. infestation in wild (Sylvilagus sp.) rabbit populations (Boisvenue 1955). The rabbits used in this report were of unknown background with regard to previous exposure.
DISCUSSION
In understanding the immunologic events in natural rabbit (and rodent) cuterebriasis it is necessary to bear in mind that the parasitic period is comparatively short. The total association between parasite and host approximates 25-30 days (Boisvenue 1955). For this reason phenomena characteristic of early immune responses (delayed hypersensitivity) were an expected finding. Benjamini and his co-workers have characterized the sequence of immune consequences in fleabite hypersensitivity and have concluded that delayed hypersensitivity is the earliest detectable type of skin reactivity in timed infections (Benjamini et al. 1961).
with
Serum precipitins
+ + + +
because of the possibility that the apparent failure to react (in PCA) of sera known to possess precipitins may be an artifact of low antigen concentration in whole larval extracts. Alimentary tract antigens were not available to evaluate this possibility. These antisera were used also to visualize electrophoretically separated antigens of the alimentary tract fraction. Although only one or two distinct antigen-antibody systems were resolvable by agar-gel immunodiffusion, at least four such systems were identifiable by IEP technique (Table IV). It is assumed that all four of these antigens are important in the development of the immune response since the sera used to resolve them were derived from naturally infected rabbits.
29
RABBITS
in Rabbits
+* + -
a \Vhole larval antigens used for assay. * (+) Appears as indication for activity (-) c Of rabbit serum in guinea pig test system.
IN
TABLE
IV
Precipitin Lines in Immunod$usion (Ouchterlony) and Immunoelectrophoresis (IEP) of Larval Alimentary Tract Antigens of C. buccata and Naturally Immunized Oryctolagus Rabbits Rabbit no.
1164 1130 1343A 1343B
No. of precipitin lines in immunodiffusion 2 1 1 2
No. of precipitin lines in IEF (+)
C--J
Total
1 0 0 1
3 2 2 2
4 2 2 3
0 (+) and (-) represent indication for precipitin lines on the cathodal and anodal sides of center well, respectively.
30
WEISBROTH,
WANG, ANII SCIIER
During the classical investigation of Cordylobia myiasis by Blacklock and his colleagues (Blacklock and Gordon 1927; Blacklock et al. 1930) it was shown that hemocoelefluid (hemolymph) and excreta of third instar larvae contained antigens with specificity for precipitins in the sera of infected hosts. Larval cuticle, salivary gland, gut, and whole-larval extracts either did not contain these antigens or they were present in too low a concentration to be detected with the available technology. The latter interpretation is suggestedin the case of their inability to demonstrate the presence of antigens in gut. It is difficult to account for an antigen in larval excreta not also present in the gut (and its contents) except as a statement of concentration. It may be that the antigens were concentrated in larval feces. Similarly, there is difficulty in accounting for the presenceof an antigen in hemolymph not also present in whole larvae as other than an effect of concentration. Immunization of the host in the course of natural Cuterebra infection appears to occur as a consequenceof the feeding process. The cuticle of migrating third instar Cuterebra larvae was shown to be immunologically inert and similar in this regard to the cuticle of Hypodema (Beesley 1970) and Cordylobia (Blacklock et al. 1930) larvae. The orientation of the larva within its subcutaneouscavity is such that the anal end is exposed through a fistula (“air hole”) in the host skin to the outside. Excretion of waste products thus is directed with other exudates from the cavity to the surface of the skin. For this reason it is unlikely that immunization occurs as an incidental process consequent to the excretion of antigens in larval feces.A more likely explanation would entail the local release of exogeneous secretions that enhance or facilitate the feeding process because these antigens would be injected or absorbed directly into the host’s vascular system. It is known that Cuterebra larvae induce clinical findings (e.g., lowered hematocrit, lowered hemoglobin, splenomeg-
sly, extramedullary hematopoiesis) consistent with blood loss anemia in small wild rodents (Childs and Cosgrove 1966; Clough 1965; Dunaway et al. 1967 ; Mckinney and Christian 1970; Payne and Cosgrore 1966; Sealander 1961) and further (on the basis of this report) were shown to be active blood feeders in rabbits. In this regard it is known that the naturally operative antigens of other blood-feeding dipterans (e.g., mosquitoes) are in fact related to the feeding processand anatomically located in organs (e.g., salivary gland) discharging exogenous secretions into the alimentary tract. A suggested approach to the more accurate anatomic localization of antigens would involve examination of immunofluorescent preparations of larval cross sections and work is being directed to this end. It is unknown at this time if the antigens present in third instar larvae are present also (in whole or part) in larvae of the first and second instars. Similar uncertainty exists with regard to other questions of larval development. Do the first instar larvae feed during migration to the subcutaneous final localization? Do they release exoantigens that facilitate migration? Are these exoantigens not present in later stages and thus undetectable by systems evaluating rabbit sera with antigens extracted from third instar larvae? These are illustrative of the many unresolved questions that prohibit a complete understanding of host-parasite interactions in cuterebriasis to be drawn at present. A sufficiently broad outline has now been established however as to regard the interface between host and parasite to represent a variation of the pattern typical for the blood feeding Diptera. The life cycle is such that these events occur during the larval (parasitic) stages rather than in the adult (freeliving) phase. ACKNOWLEDGMENTS The authors gratefully acknowledge the advice and technical assistance of their colleague in the Department of Pathology, Dr. Frederick Miller.
Cuterebra
buccata IN RABBITS
REFERENCES W. N. 1970. Observations on the biology of the ox warble, Hypoderma (Diptera: Oestridae). IV. Some serological reactions against the parasite in natural and artificial hosts. Ann& of Tropical Medicine and Parasitology 64, 277-281. BENJAMINI, E., ‘FEINFOLD, B. E., AND KARMAN, L. 1961. Skin reactivity in guinea pigs sensitized to flea bites. The sequence of reactions. ProBEESLEY,
ceedings Biology
of
the
Society
for
Experimental
and Medicine 108, 700-702. BLACKLOCK, D. B., AND GORWN, R. M. 1927. The experimental production of immunity against metazoan parasites and an investigation of its nature. Alvnals of Tropical Medicine and parasitology 21, 181-224. BLACKLOCK, D. B., GORDON, R. M., AND FINE, J. 1930. Metazoan immunity: A report of recent investigations. Annals of Tropical Medicine and
Parasitology
24, 5-54.
R. S. 1955. “Studies on the life history and ecology of Cuter&a spp. occuring in Michigan cottontails with systematic studies on cuterebrine larvae from other animals.” Ph.D. thesis, School for Advanced Graduate Studies, Michigan State University. CHILDS, H. E., JR., AND COSGROVE, G. E. 1966. A study of pathological conditions in wild rodents BOISVENE,
31
in radioactive areas. American Midland Naturalist 76, 306-324. CLOUGH, G. C. 1965. Physiological effect of botfly parasitism on meadow voles. Ecology 46, 344346. DUNAWAY, P. B., PAYNE, J. A., LEWIS, L. L., AND STORY, J. D. 1967. Incidence and effects of Cuterebra in Peromyscus. Journal of Mammalogy 48, 38-51. LOWRY, 0. H., ROSENBROUGH, N. J., FARR,L., AND RANDALL, R. J. 1951. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265-275. MCKINNEY, T. D., AND CHRISTIAN, J. J. 1970. Incidence and effects of botfly parasitism in the Eastern chipmunk. Journal of Wildlife Disease 6, 140-143. PAYNE, J. A., AND COSGROVE, G. E. 1966. Tissue changes following Cuterelm infestation in rodents. Americau Midland Natwalist 75, 205-213. SEALANDER, J. A. 1961. Hematological values in deer mice in relation to botfly infection. Journal of Mammalogy
42,
57-60.
S. H., WANG, R., AND SCHER, S. 1973. Immune and pathologic consequences of spontaneous Cuterebrcn myiasis in domestic rabbits (Oryctolagus cunicubs) . Laboratory Animal Science 23 : 241-247.
WEISBROTH,
34,22-31
Cuterebra
STEVEN Division State
H.
(1973)
buccata: Immune Response of Domestic Rabbits WEISBROTH,
REENIE
WANG,
AND
in Myiasis
SHELDON
of Laboratory Animal Resources, Health Sciences University of New York, Stony Brook, New York (Submitted
for publication,
SCHER Center, 11790
5 June 1972)
WEISBROTH, S. H., WANG, R., AND SCHER, S. 1973. Cuterebra buccata: Immune Response in Myiasis of Domestic Rabbits. Experimental Parasitology 34, 2231. Naturally infected rabbits (Oryctolugus) were used to define further the nature of the immune response in myiasis due to Cuterebra buccata. Third instar larvae were dissected into four fractions; (1) alimentary tract with attached organs, (2) hemolymph, (3) fat body with tracheae, and (4) cuticle with attached muscles. The antigens provoking immune phenomena in naturally infected rabbits were found to reside in the alimentary tract and hemolymph fractions only. All rabbits which were skin tested were found to exhibit delayed hypersensitivity and to have serum precipitins with specificity against these antigens. Passive cutaneous anaphylaxis activity was demonstrated only in sera from rabbits also exhibiting reaginic and/or Arthus-type skin hypersensitivities. With the use of immunoelectrophoresis four separate antigens were demonstrated in alimentary tract fractions. Larval dissections revealed the alimentary tract to be filled with cellular elements of rabbit whole blood. The immunologic findings are discussed in relation to this newly recognized feeding pattern and it is proposed that sensitization of the host occurs as a consequence of exogenous larval secretions injected at the time of feeding. INDEX DESCRIPTORS : Cuterebru buccata; Oryctolagus; Rabbits ; Hypersensitivity immediate ; Hypersensitivity delayed ; Antibody ; Myiasis ; Immunity ; Immunoelectrophoresis ; Immunodiff usion,
In a previous report from this laboratory (Weisbroth et al. 1973) the pathologic consequences of spontaneous cuterebriasis in domestic rabbits (Oryctolagus) were reported. They were found to include localized skin lesions consisting of a cavity with a thin necrotic lining containing the Cuterebra larva, and extending radially from the cavity lining, zones of (1) acute inflammation, (2) granulation tissue with fibrous and vascular elements, and (3) chronic inflammation with macrophages and lymphocytes predominating, but also including plasma cells and eosinophils. Regional lymphadenopathy typical of lymph nodes draining an infectious
Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
process were described as the only other pathologic finding from infected rabbits, These lesions suggested that immune processes might be an active concomitant of Cuterebra myiasis. It was found that of six rabbits carrying third instar Cuterebra buccata larvae some, (1/s) would demonstrate immediate hypersensitivity of the wheal and flare type, most ($&) had Arthus reactivity, and all (GA) had delayed-type hypersensitivity, when skin tested intradermally with whole-larva extracts. The sera of these rabbits all had precipitins as detected by agar-gel immunodiffusion. The purpose of this
report
is to characterize
further
the
Cuterebra
FIG.
nature of antigenicity and also the antibody infected rabbits, MATERIALS
1. Third
buccata
instar larva of C. buccata
in Cztterebra larvae response in naturally
AND
IN
METHODS
Antigens. Cuterebra buccata larvae were collected from naturally infected rabbits and prepared for antigenic analysis by grinding washed whole third stage larvae or their fractions for 30 min in a Ten Broeck mortar at 4 C. using 1: 1 dilutions in sterile distilled water. The grindings were centrifuged using an International model PRJ refrigerated centrifuge at 5 C, at a speed of 4000 rpm for 30 min, and the supernate retained. Supernates were plated on blood agar as a test for absence of bacteria and after proven to be bacterially sterile frozen at -90 C without further treatment for use as stock antigen. Stock antigens were analyzed for
RABBITS
(scale in millimeters).
protein content using bovine serum albumin standards (Lowry et al. 1951) and in all cases diluted with phosphate buffered saline (PBS) at pH 7.4 to contain 2 mg protein/ ml for use. Fractions of whole larvae were prepared by dissecting the larvae (Fig. 1) into four parts : ( 1) hemolymph (collected by hypodermic syringe and needle), (2) alimentary tract with attached organs, (3) fat body with tracheal respiratory system, and (4) cuticle with attached muscles. In practice, the dissection was performed by pinning the larva to a dissection board in a dorsoventral position. A midline incision was made through the cuticle for the whole length of the larva. The hemolymph was collected and the cuticle reflected back and pinned to the board (Fig. 2). The alimentary tract fraction was then removed and weighed leaving
24
WEISBROTH,
FIG. 2. Cuticle fractions.
reflected
to show
WANG,
alimentary
tract
fractions C and D (Fig. 3). The fat body and tracheae (fraction C) were then removed and weighed leaving only the cuticle with its muscles (Fig. 4). The weight relationships of whole larvae to their fractions is summarized in Table I. The preparation of larval fractions follows in general the pro-
Larva
no.
1 2 3 4 5 Mean a Percentage
Weight
of Cuterebra
Whole
larva
Third
Hemolymph
1.65 2.04 1.91 2.20 1.52 1.86 of whole
buccata
0.70 1.05 0.86 1.11 0.64 0.87 weight
(42)” (50) (45) (50) (42) (47)
in parentheses.
AND
SCHER
(B),
fat
body
(C),
and
cuticular
(D)
cedure used in dissection of Hypodernca larvae for similar purposes (Beesley 1970). Skin testing. Skin tests on both infected and noninfected laboratory rabbits were performed by parallel intradermal injections of 0.1 ml of antigen and various controls using tuberculin syringes and 27-gauge needles.
TABLE
I
Instar
Whole
Alimentary 0.11 0.11 0.13 0.17 0.11 0.13
Larvae tract
(6.0) (5.3) (7.0) (8.0) (7.0) (7.0)
and their
Fractions
Fat
body
0.24 0.25 0.27 0.27 0.22 0.25
(15) (12) (14) (12) (15) (13)
(in g) Cuticle 0.60 0.69 0.75 0.65 0.55 0.64
(36) (31) (34) (30) (36) (34)
Cuterebra
FIG. 3. Alimentary
buccata
IN
25
RABBITS
tract fraction removed to show fat body (C) and cuticular
(D)
fractions.
Antigen-injected and control sites were obImmunoelectrophoresis (IEP). Agar-gel served 15-20 min, 2-4 hr, and 24-48 hr after for immunoelectrophoresis was prepared with injection. Skin reactions when present, were 1.5% Agarose Behringwerke (obtained from measured and described. Typical sites were Lloyd Brothers, Cincinatti, OH) dissolved biopsied for histologic examination. (w/v) in a solution of 1: 5 Michaelis buffer Immunodifusion (IO). Agar-gel im- in distilled water. The agarose solution was munodiffusion (Ouchterlony ) preciptin tests poured while molten on glass slides, and were performed with the use of both com- after cooling central wells of various sizes mercially obtained patterns (Hyland Im- were cored in the solid gel. Cuterebru whole muno-Plate, Pattern C., Hyland Division of larval antigens, or fraction antigens, or unTravenal Labs., Costa Mesa, CA) and also diluted rabbit sera (from natural infections) with those prepared in the laboratory. Imwere placed in the central well for electromunodiffusion preparations made in the phoresis. Electrophoresis was performed laboratory utilized 0.8% Agarose Behring- using a Universal electrophoretic apparatus weke (obtained from Lloyd Brothers, Cin- (obtained from Colab Laboratories, Chicago cinatti, OH) dissolved (w/v) in PBS (pH Heights, IL) utilizing a potential of 50 V 7.4) with 0.5% each of stock sodium azide and operating times of 60 and 90 min, re(0.1 M) and EDTA (0.01 M) as preserva- spectively, for antigens and sera. Immediately troughs were cut in tives, Immunodiffusion preparations were in- after electrophoresis cubated in a moist chamber at room temper- the agarose gel at various distances from the ature, examined daily for precipitin lines and central well. Immunologic differentiation of the electrophoretically separated proteins was discarded after 7 days.
26
WEISBROTH,
FIG.
WANG,
AND
SCRER
4. Fat body removed to show cuticular
achieved by filling the troughs with antisera (in the case of electrophoresed antigens) and vice versa. Preparations were incubated at 37 C for 1-2 hr and then placed in moist chambers in the refrigerator (5 C). IEP preparations were examined daily for 7 days and precipitin lines and their positions recorded. After 7 days the IEP plates were dialysed against physiological saline for 24 hr (to remove nonprecipitated proteins) and stained in a solution of Buffalo Black to visualize individual precipitin lines better. The agar was dried down to a thin film and preserved as a semipermanent record of the experiment. Passive cutaneous anaFlzylaxis (PCA) . Guinea pigs, (between 200 and 250 g), of the Rockefeller Albino Moen-Chase stock were obtained from Brookhaven National Laboratory for use in PCA experiments. These were performed by injecting 0.1 ml
(D)
fraction.
aliquots of appropriate rabbit serum dilutions and controls intradermally over the shaved lateral body wall. Two replicate guinea pigs were used to titrate each rabbit serum. Four hours later the animals were each injected intravenously with a 1 ml mixture containing 0.5 ml of whole larval antigens and 0.5 ml of lo/O Evans Blue dye in distilled water. Skin sites were examined for blue rings of extravasated dye at the end of 30 min. Blue skin lesions typical of PCA reactions were measured in vivo and recorded. These results were compared with remeasurementsof the same site on the underside of pelted skins. Results were such that measurements of lesions in intact skins were used as the routine procedure. RESULTS
Three naturally infected albino (New Zealand White) rabbits were used for intra-
Cuterebra
buccata TABLE
Results
Rabbit number
1330 1343A 1343B
of Intradermal Skin Cuterebra
Whole larva 2
+
+ -t” -+ +
--
3
a Data presented with indication representing (1) 15-20 min, (2) 2-4
1 +++ ---
2
RABBITS
II
Tests in Naturally Infected buccata Larval Antigens
Alimentary tract
1
IN
Fat
3
1
+ +
-------
for reactive (+) hr, and (3) 24-48
dermal skin testing of antigens extracted from whole larvae and larval fractions. Each individual was skin tested on the day that third instar larvae were removed from its skin so that the time lapse following natural exposure to antigens was minimal. In each case 0.1 ml intradermal injections of sterile physiological saline accompanied antigen injections in different sites. As the saline sites were rountinely nonreactive they do not appear in the tables or descriptive results. Similarly, a total of 12 rabbits on different occasions, known not to have been previously exposed to antigens of Cuterebra sp. were skin tested with antigens from whole larvae or larval fractions and since these skin test sites were routinely nonreactive these results do not appear elsewhere. Skin sites were observed for immediate reactions 15-20 min after antigen injection. When present the lesions presented as round or ovoid (but not serpentine) dome-shaped elevated wheals averaging 10-12 x 12-15 mm in diameter. The color varied from pale or colorless to reddish pink. Skin sites similarly were observed 2-4 hr after injection and redescribed again 24 and 48 hr after injection. These lesions were typically smaller (68 X S-12 mm), pinkish, firmer to the touch, and were not dependent on the presence of skin reactivity when observed earlier. The results of skin testing with the various antigens are summarized in Table II. These results are in agreement with those pre-
body
2
Rabbits
using
Cuticle
3
and nonreactive hr observations,
1
2
Hemolymph
3
------(-) skin respectively.
1 +++ ---
sites
in groups
2
3
+ + of three
viously reported from this laboratory (Weisbroth et al. 1973), in that naturally infected rabbits routinely exhibit delayed hypersensitivity, and occasionally, reaginic and/or Arthus hypersensitivity. More significantly however it appears that the antigens responsible for eliciting these reactions originate in the larval hemolymph. They do not appear to be present in the cuticle with its attached muscles or in the fat body or respiratory system. The latter (cuticle and fat body fractions) are substantial elements of the larval bulk together accounting for approximately 50% of the total weight (Table I). The relationships between larval fractions were further analyzed by agar-gel immunodiffusion. These results are summarized in Fig. 5 which is a representative photograph of results using sera from six separate C. buccata-infected rabbits with naturally acquired antibodies. Analysis by immunodiffusion confirmed the results of skin testing with regard to the anatomic distribution of larval antigens. Antigens are present in higher concentration in the alimentary tract fraction than either whole larval extracts or the hemolymph fraction. Typically titered end points for alimentary tract extracts approximated 1:64 when using the same undiluted antisera. Sera from naturally infected rabbits typically had diluted end points varying from 1:4 to 1: 16 when using stock alimentary tract antigens, but dilution end points of 1:2 or 1:4 when using stock whole
2s
WEISBROTH,
FIG.
rabbit larval lymph,
5. Immunodiffusion in agar gel serum (center well) against estract, (2) alimentary tract, (4) fat body, and (5) cuticle of
WANG,
of immune (1) whole(3) hemoC. buccata.
larva or hemolymph antigens. We have concluded from these experiments that the most likely anatomic location of antigens operative in natural immunization are related to the alimentary tract and its associated organs. That this is likely is suggested as well by
FIG.
larva.
AND
SCHER
the manner of larval feeding, not previously established with certainty in lagomorph or rodent hosts. The larvae are apparently active blood feeders and the lumen of the alimentary tract of all dissected larvae was observed to be filled with the cellular elements of rabbit whole blood (Fig. 6). Considering that Cuterebra larvae undergo a 200-fold increase in weight (from 10 mg first instar to approximately 200 mg third instar larvae) in 25-30 days, it may be concluded that they consume considerable quantities of rabbit blood in the course of this development. Antibodies detected in vitro by immunodiffusion analysis were demonstrated in V&JO as well by heterologous PCA technique in guinea pig skin. Four sera were available for PCA analysis. These results are summarized in Table III. PCA activity (end point dilutions of 1: 128 and 1: 1024 in sera from rabbits 1164 and 1330, respectively) was observed to be present only in sera from rabbits also exhibiting skin hypersensitivity of the immediate or Arthus types. The significance of this finding is difficult to assess
6. Photomicrograph of Giemsa-stained smear Note rabbit erythrocytes (A) and leukocytes
of alimentary (B). X 1000.
tract
contents
of C. buccata
Cuterebra
buccata TABLE
Results Rabbit number
of Sereral
Immunologic
Assays”
Immediate or Arthus hypersensitivities
1164 1330 1343A 1343B
III Infected
Delayed hypersensitivity
for activity
Cuterebra
buccata PC‘4 activity<
+ + + +
+ + -
absent.
The variability detected in immune responses may in part be related to genetically determined individual capability, but also may be related to degree, if any, of previous exposure. While all of the immune properties detected in these rabbits were a consequence of natural immunization by parasitic larvae, it is unknown if this is of value to the host in the prevention or abortion of future parasitic invasions. The interpretation that it is may derive support from epidemiological surveys that report an inverse relationship between age of host and degree of Cuterebra sp. infestation in wild (Sylvilagus sp.) rabbit populations (Boisvenue 1955). The rabbits used in this report were of unknown background with regard to previous exposure.
DISCUSSION
In understanding the immunologic events in natural rabbit (and rodent) cuterebriasis it is necessary to bear in mind that the parasitic period is comparatively short. The total association between parasite and host approximates 25-30 days (Boisvenue 1955). For this reason phenomena characteristic of early immune responses (delayed hypersensitivity) were an expected finding. Benjamini and his co-workers have characterized the sequence of immune consequences in fleabite hypersensitivity and have concluded that delayed hypersensitivity is the earliest detectable type of skin reactivity in timed infections (Benjamini et al. 1961).
with
Serum precipitins
+ + + +
because of the possibility that the apparent failure to react (in PCA) of sera known to possess precipitins may be an artifact of low antigen concentration in whole larval extracts. Alimentary tract antigens were not available to evaluate this possibility. These antisera were used also to visualize electrophoretically separated antigens of the alimentary tract fraction. Although only one or two distinct antigen-antibody systems were resolvable by agar-gel immunodiffusion, at least four such systems were identifiable by IEP technique (Table IV). It is assumed that all four of these antigens are important in the development of the immune response since the sera used to resolve them were derived from naturally infected rabbits.
29
RABBITS
in Rabbits
+* + -
a \Vhole larval antigens used for assay. * (+) Appears as indication for activity (-) c Of rabbit serum in guinea pig test system.
IN
TABLE
IV
Precipitin Lines in Immunod$usion (Ouchterlony) and Immunoelectrophoresis (IEP) of Larval Alimentary Tract Antigens of C. buccata and Naturally Immunized Oryctolagus Rabbits Rabbit no.
1164 1130 1343A 1343B
No. of precipitin lines in immunodiffusion 2 1 1 2
No. of precipitin lines in IEF (+)
C--J
Total
1 0 0 1
3 2 2 2
4 2 2 3
0 (+) and (-) represent indication for precipitin lines on the cathodal and anodal sides of center well, respectively.
30
WEISBROTH,
WANG, ANII SCIIER
During the classical investigation of Cordylobia myiasis by Blacklock and his colleagues (Blacklock and Gordon 1927; Blacklock et al. 1930) it was shown that hemocoelefluid (hemolymph) and excreta of third instar larvae contained antigens with specificity for precipitins in the sera of infected hosts. Larval cuticle, salivary gland, gut, and whole-larval extracts either did not contain these antigens or they were present in too low a concentration to be detected with the available technology. The latter interpretation is suggestedin the case of their inability to demonstrate the presence of antigens in gut. It is difficult to account for an antigen in larval excreta not also present in the gut (and its contents) except as a statement of concentration. It may be that the antigens were concentrated in larval feces. Similarly, there is difficulty in accounting for the presenceof an antigen in hemolymph not also present in whole larvae as other than an effect of concentration. Immunization of the host in the course of natural Cuterebra infection appears to occur as a consequenceof the feeding process. The cuticle of migrating third instar Cuterebra larvae was shown to be immunologically inert and similar in this regard to the cuticle of Hypodema (Beesley 1970) and Cordylobia (Blacklock et al. 1930) larvae. The orientation of the larva within its subcutaneouscavity is such that the anal end is exposed through a fistula (“air hole”) in the host skin to the outside. Excretion of waste products thus is directed with other exudates from the cavity to the surface of the skin. For this reason it is unlikely that immunization occurs as an incidental process consequent to the excretion of antigens in larval feces.A more likely explanation would entail the local release of exogeneous secretions that enhance or facilitate the feeding process because these antigens would be injected or absorbed directly into the host’s vascular system. It is known that Cuterebra larvae induce clinical findings (e.g., lowered hematocrit, lowered hemoglobin, splenomeg-
sly, extramedullary hematopoiesis) consistent with blood loss anemia in small wild rodents (Childs and Cosgrove 1966; Clough 1965; Dunaway et al. 1967 ; Mckinney and Christian 1970; Payne and Cosgrore 1966; Sealander 1961) and further (on the basis of this report) were shown to be active blood feeders in rabbits. In this regard it is known that the naturally operative antigens of other blood-feeding dipterans (e.g., mosquitoes) are in fact related to the feeding processand anatomically located in organs (e.g., salivary gland) discharging exogenous secretions into the alimentary tract. A suggested approach to the more accurate anatomic localization of antigens would involve examination of immunofluorescent preparations of larval cross sections and work is being directed to this end. It is unknown at this time if the antigens present in third instar larvae are present also (in whole or part) in larvae of the first and second instars. Similar uncertainty exists with regard to other questions of larval development. Do the first instar larvae feed during migration to the subcutaneous final localization? Do they release exoantigens that facilitate migration? Are these exoantigens not present in later stages and thus undetectable by systems evaluating rabbit sera with antigens extracted from third instar larvae? These are illustrative of the many unresolved questions that prohibit a complete understanding of host-parasite interactions in cuterebriasis to be drawn at present. A sufficiently broad outline has now been established however as to regard the interface between host and parasite to represent a variation of the pattern typical for the blood feeding Diptera. The life cycle is such that these events occur during the larval (parasitic) stages rather than in the adult (freeliving) phase. ACKNOWLEDGMENTS The authors gratefully acknowledge the advice and technical assistance of their colleague in the Department of Pathology, Dr. Frederick Miller.
Cuterebra
buccata IN RABBITS
REFERENCES W. N. 1970. Observations on the biology of the ox warble, Hypoderma (Diptera: Oestridae). IV. Some serological reactions against the parasite in natural and artificial hosts. Ann& of Tropical Medicine and Parasitology 64, 277-281. BENJAMINI, E., ‘FEINFOLD, B. E., AND KARMAN, L. 1961. Skin reactivity in guinea pigs sensitized to flea bites. The sequence of reactions. ProBEESLEY,
ceedings Biology
of
the
Society
for
Experimental
and Medicine 108, 700-702. BLACKLOCK, D. B., AND GORWN, R. M. 1927. The experimental production of immunity against metazoan parasites and an investigation of its nature. Alvnals of Tropical Medicine and parasitology 21, 181-224. BLACKLOCK, D. B., GORDON, R. M., AND FINE, J. 1930. Metazoan immunity: A report of recent investigations. Annals of Tropical Medicine and
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24, 5-54.
R. S. 1955. “Studies on the life history and ecology of Cuter&a spp. occuring in Michigan cottontails with systematic studies on cuterebrine larvae from other animals.” Ph.D. thesis, School for Advanced Graduate Studies, Michigan State University. CHILDS, H. E., JR., AND COSGROVE, G. E. 1966. A study of pathological conditions in wild rodents BOISVENE,
31
in radioactive areas. American Midland Naturalist 76, 306-324. CLOUGH, G. C. 1965. Physiological effect of botfly parasitism on meadow voles. Ecology 46, 344346. DUNAWAY, P. B., PAYNE, J. A., LEWIS, L. L., AND STORY, J. D. 1967. Incidence and effects of Cuterebra in Peromyscus. Journal of Mammalogy 48, 38-51. LOWRY, 0. H., ROSENBROUGH, N. J., FARR,L., AND RANDALL, R. J. 1951. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265-275. MCKINNEY, T. D., AND CHRISTIAN, J. J. 1970. Incidence and effects of botfly parasitism in the Eastern chipmunk. Journal of Wildlife Disease 6, 140-143. PAYNE, J. A., AND COSGROVE, G. E. 1966. Tissue changes following Cuterelm infestation in rodents. Americau Midland Natwalist 75, 205-213. SEALANDER, J. A. 1961. Hematological values in deer mice in relation to botfly infection. Journal of Mammalogy
42,
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S. H., WANG, R., AND SCHER, S. 1973. Immune and pathologic consequences of spontaneous Cuterebrcn myiasis in domestic rabbits (Oryctolagus cunicubs) . Laboratory Animal Science 23 : 241-247.
WEISBROTH,