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Immunoperoxidase staining characteristics of Dirofilaria immitis in the dog
Research in Veterinary Science 1991, 50, 33-37
Immunoperoxidase staining characteristics of Dirofilaria immitis in the dog K. I. TANAKA*, R. B. ATWELL, Department of Companion Animal Medicine and Surgery, University o f Queensland, St Lucia, 4067, Queensland, Australia
The immunoperoxidase staining characteristics of Dirofilaria immitis and pulmonary tissues from infected dogs were studied by using the following sera: anti-fresh D immitis, anti-processed D immitis, anti-dog lgG, anti-dog IgG Fc, anti-dog IgM and antidog C 3. Marked staining was observed using antifresh D immitis serum. Body cavity fluid and cuticle were strongly stained and hypodermis, muscle, lateral cord, testis, vas deferens, ovary, oviduct and uterus were moderately stained. Oesophagus and intestine were mildly stained. Degenerate worms were stained by all antisera. The intact and cut surfaces of microfilariae and eggs and sperm present in filariae were stained, but not their internal contents. Circulating and stored immotile microfilariae did not stain. Excreted eggs, presumed to be unfertilised and, or, degenerate, stained positively, lmmunoperoxidase staining of routinely processed histological samples provides a means of assessing D immitis antigen.
potential antigenic material and of filariae themselves. Materials and methods D immitis antigen and antiserum The procedure performed in this study followed that described in a previous report (Tanaka and Atwel11990). 'Fresh female D immitis antigen', 'fresh male D immitis antigen' and 'processed female D immitis antigen' were prepared. For fresh D immitis antigen preparation, segments of adult male and female worms were ground separately in a pestle and mortar, and were then sonicated in phosphate buffered saline-EDTA at pH 7.2. The resultant solution was sedimented and the supernatant was sterilised by filtration through a 0-45 #m filter. To prepare processed D immitis antigen, whole adult female worms that had been fixed in 10 per cent formalin solution were washed with running tap water for more than 12 hours. Worms were then waxed and dewaxed by routine processing methods for histological tissues (Campbell 1951). They were then prepared as for fresh material. Three groups of two New Zealand rabbits were immunised three times at 14 day intervals by intramuscularly injecting 1 ml of one of three different antigen adjuvant preparations. Suitable rabbit antisera to fresh female D immitis extracts (anti-Fw serum) and to processed female D immitis extracts (anti-Pw serum) prepared forthis study were selected by counterimmunoelectrophoresis. Rabbit antisera to fresh male D immitis extracts were prepared but subsequently not used because of unacceptably lower titres when compared with antisera to fresh female D immitis extracts. Normal rabbit serum was also obtained from one unimmunised rabbit.
IMMUNOPEROXIDASE staining (PAP) is a recently developed technique being used in preference to immunofluorescence staining because of its better sensitivity, specificity and sample preservation (Sternberger et al 1970, Taylor 1978). The immunofluorescent staining characteristics of microfilariae and adult Dirofilaria immitis have been reported (Mantovani and Sulzer 1967, Wong 1974, Abramowsky et al 1981, Hayasaki 1983, Ohara et al 1985). However, these results have not always been of high resolution. The PAP technique has been used for identifying helminths in human tissue sections (Suzuki et al 1981) and to confirm the quality of monoclonal antibody directed against D immitis (Weil et al 1985). In this study, PAP staining techniques using anti-D immitis and anti-immunoglobulin sera were used to locate D immitis 'antigen' in lung tissue, and to investigate the staining characteristics of this
Antisera to dog immunoglobulin and dog complement *Present address: Exploratory Research Laboratories, Chugai Pharmaceuticals Co Ltd, 1-135, Komakado, Gotemba-shi, Shizuoka, Japan, 412
Anti-dog IgG rabbit serum (anti-IgG serum), antidog IgG fragment crystalline goat serum (anti-IgG Fc 33
K. L Tanaka, R. B. Atwell
34
TABLE 1 : Summary of observations of D immitis using PAP staining technology Primary Anti-
Anti-
Anti-
Anti-
Anti-
Anti-
serum
FW
PW
IgG
IgG Fc
IgM
C3
Cuticle
-
+ + +
+ .
Hypodermis
-
+ +
+ -
Body
-
+ + +
+ +
-
-
-
-
+ +
+
-
-
-
-
-
Common
organs
cavity
Muscle Lateral
cord
.
.
. -
-
+ +
+
-
+
.
Intestine
-
+
-
-
-
Gut
-
+ +
-
+ +
+ +
+ +
Ovary
-
+ +
+
-
-
-
Oviduct
-
+ +
+
-
-
-
Uterus
-
+ +
+
-
-
-
Shell
-
+ +
+
+ -
-
Surface
-
+ +
.
Internal
.
Cut
-
+ -
-
-
-
+ -
-
contents adult
.
.
.
worm
Intrauterine
eggs
surface
Intrauterine
.
.
.
.
.
+
.
.
-
. -
mf
Surface
-
Internal
.
Cut
-
-
Oesophagus
Female
surface
+ +
+
.
-
.
.
.
.
-
+
-
Testis
-
+ +
+ .
.
.
.
Vas
-
+ +
+ .
.
.
.
Male
adult
-
worm
deferens
Intratesticular
sperm
Surface
-
Internal
.
Cut
-
+
-
-
+ ++
++
surface
Degenerate Whole Fresh
+
-
.
-
.
.
+ -
-
-
+ -
-
+ +
+ +
+ +
.
.
worm
body
motile
++
mf
Surface
.
.
.
.
NP
NP
Internal
.
.
.
.
NP
NP
Stored
immotile
mf
Surface
-
-
NP
-
NP
NP
NP
Internal
-
-
NP
-
NP
NP
NP
Surface
-
+ +
-
-
NP
NP
Internal
-
-
-
NP
NP
Cut
-
+ +
-
-
NP
NP
Surface
-
+ +
-
-
NP
NP
Internal
-
-
-
NP
NP
Circulating
live mf
Cut
in pulmonary
vessel
surface
Egg in pulmonary
-
serum
Normal
UI
vessel
UI
surface
Negative
FW Fresh
+ + stain,
female
+ -
Faint
D immitis,
stain
(implies
PW Processed
doubtful), female
+
Mild
D irnmitis,
serum), anti-dog IgM goat serum (anti-IgM serum) and anti-dog third complement goat serum (anti-C3 serum) were obtained commercially as primary sera for the PAP stain. Tissue sampling Whole worm. To prepare processed whole worm material, five fresh adult male and five fresh adult
stain, NP N o t
+ +
NP
Moderate
performed,
stain,
NP + + +
Ul Unidentified,
Strong mf
stain
Microfilariae
female D immitis were fixed in buffered I0 per cent formalin solution as soon as possible after removal from the pulmonary arteries. Samples were prepared in 4 ~tm sections. Whole microfilariae. Heparinised canine blood containing motile microfilariae was added to an equal volume of sterile distilled water. The prepared haemolysate was smeared, dried on glass slides and then
Immunoperoxidase staining o f Dirofilaria immitis
35
Adult D immitis stained by using anti-fresh D imrnitis serum
! !i~ii ~ ii FIG 1: Male, cross section of apical part. Cuticle, hypodermis, muscle, oesophagus and testis are stained, internal tissue of sperm is not stained, x 1 1 5
FIG 2: Male, cross section of rnidbody. Cuticle, hypodermis, body cavity, intestine, gut contents are stained. Cuticle and body cavity are stained the strongest, x 1 15
FIG 3: Female, longitudinal section of midbody. Cuticle showed multilayering. Cut surface and uncut surface of microfilariae are moderately stained. Stained unfertilised eggs are also observed. x115
FIG 4: Female, longitudinal section of midbody. Cuticle, body cavity are stained. Internal tissue of egg is not stained but egg surfaces are stained. × 1 15
fixed with 100 per cent ethanol for 10 minutes. The remainder of the prepared blood was preserved at 4°C for 10 days until microfilariae had become immotile. Slides were then prepared as for live microfilariae using these immotile microfilariae.
described previously by Tanaka and Atwell (1990). Tissue sections were exposed to hydrogen peroxide solution to block endogenous peroxidase activity and then were exposed to normal pig serum to inhibit any non-specific reaction. The following were used as primary sere: anti-Fw serum, anti-Pw serum, normal rabbit serum (control serum), anti-dog IgG serum, anti-dog C 3 serum, anti-dog IgM serum or anti-dog IgG Fc serum. As secondary sere, anti-rabbit IgG goat serum or anti-goat IgG rabbit serum (heavy and light chains specific) were used. As pAP-complex serum, PAPcomplex rabbit or goat serum was used. A 3,3diaminobenzidine solution was used as chromogen.
Infected canine lung. Canine lung tissue from five naturally infected dogs was fixed with buffered 10 per cent formalin solution at the time of necropsy. Tissues were prepared in 4 #m sections. Immunoperoxidase staining The PAP stain performed followed the method
36
K. L Tanaka, R. B. A twell Pulmonary tissues (naturally infected) stained by using anti-fresh D irnmitis serum
FIG 5: Stained microfilariae are obvious in pulmonary tissue. × 225
FIG 6: Stained degenerated pulmonary vessel, x 480
The preparations were counterstained with Mayer's haematoxylin.
and anti-IgM sera. No stain was evident on the slides when anti-C3 serum was used. Strong staining of dead and degenerate worms in :he pulmonary artery was observed in sections stained by anti-FW, anti-Pw, anti-IgG and anti-C3 sera. The surface of live microfilariae in peripheral blood and of stored immotile microfilariae did not stain, but the microfilariae in the pulmonary tissue were clearly stained (Fig 5). Positive staining was also observed on the surface of intrauterine eggs (Fig 4) and 'free' eggs in the pulmonary tissue (Fig 6).
Results
The PAP staining characteristics of D immitis are summarised in Table 1. In the D immitis worm sections stained with anti-Fw serum, the strongest staining was of the body cavity, particularly body cavity fluid, followed by the staining of the cuticle (Figs 1 to 4), which is clearly shown to be multilayered (Figs 3 and 4). The hypodermis, muscle and lateral cord of male and female worms were moderately stained (Figs 1 to 4). The slides stained using anti-dog IgG sera, anti-dog IgM sera and anti-dog C a sera showed a negative reaction as did all sections stained using control sera. Mild staining was observed in the oesophagus and intestinal tract (Figs 1 and 2). Sections of testes produced a moderately positive stain whereas sperm did not stain, except for the outer surface and the cut surface which was mildly stained (Figs 1 and 2). Both uterus and oviduct were moderately stained. The surface and cut surface of intrauterine microfilariae, eggs, sperm and egg shell, and intratesticular sperm were moderately stained. In contrast, internal structures of liberated microfilariae, eggs and sperm did not stain. Organs common to male and female showed similar stain intensity (Figs l to 4). The intensity of staining using anti-Pw serum was definitely inferior to that for anti-Fw serum. The body cavity fluid stained faintly and the gut contents were moderately stained using anti-IgG, anti-IgG Fc
egg obstructing a peripheral
Discussion
It appears that this anti-rw serum used here has a high sensitivity toward D immitis, adult male and female body components as well as microfilariae, egg and sperm. Weil et al (1985), using monoclonal antibody as primary serum, observed a similar distribution of stain intensities for each organ. However, immotile microfilariae prepared in this study were not positive. This may have been due to the difference in microfilarial preparation between damaged (Weft et al 1985) and immotile microfilariae. Results using the direct immunofluorescence test (Abramowsky et al 1981) were also similar to the present observations. However, microfilariae did not show positive immunostaining. Ohara et al (1985), using indirect immunofluorescence and antisera produced by IgE and by IgG-inducing antigen, reported that allergen was detected in the body cavity fluid and excretory canal of filariae. The IgG-
Immunoperoxidase staining of Dirofilaria immitis inducing antigen was found in the subcuticular area with a weaker reaction in the muscle layer. In the present study, no such associated anti-IgG sera staining was observed. This difference could be due to the different original antigenic substances; Ohara's anti-IgG serum being prepared from specific IgGinducing D immitis antigen. The surface of apparently degenerate eggs in the lung stained positively. The same degree of staining was seen for both intrauterine eggs and excreted eggs. This was in contrast to the positive staining of intrauterine microfilariae and the negative staining of circulating microfilariae. These results could be explained by the degree of antigenic stimulus and thus immunological reaction once perhaps damaged or degenerate eggs are lodged in the peripheral pulmonary vasculature (Kihara 1987). Several authors using circulating microfilariae of other filarial species as an antigen for the immunofluorescence test, have reported poor staining of intact circulating microfilariae (Lucasse 1962, Sadun 1963, Mantovani and Sulzer 1967, Santos 1976). This may have been caused by exposure to some unknown inhibitory substance(s) coating microfilariae once in circulation, particularly when these results were compared to those for intrauterine microfilariae. A similar coating mechanism which would help to avoid immunological recognition and reaction by the host to microfilariae could also exist for D immitis. When comparing the sensitivity of the PAP stain and the immunofluorescence stain, extremely high resolution was observed with the PAP stain. Sternberger (1979) reported that the PAP stain was 100 to 1000 times more sensitive than the immunofluorescence test. Positively stained substances that are coloured light brown are easily distinguished from other tissues even if they are fragmented and scattered. This observation would suggest that routinely processed histological samples could be assessed using the PAP technique and anti-Fw sera. This could be of benefit to the understanding of pathophysiology in the dog and also assist in the identification of pulmonary parasitic debris in people. Acknowledgements The authors appreciate the support of Dr J. Opdebeeck for her immunological assistance, Dr W. Kelly and Dr R. Sutton for histopathological interpretations and Mr J. Massey and laboratory staff for assistance with PAP staining.
37
References ABRAMOWSKY, C. R., POWERS, K. G., AIKAWA, M. & SWINEHART, G. (1981) Dirofilaria immitis. 5. Immunopathology of filarial nephropathy in dogs. American Journal of Pathology 104, 1-12 CAMPBELL, W. N. (1951) Methods for the microscopic examination of tissues. In: Approved Laboratory Technic. 5th edn. Eds J. A. Kolmer et al. New York, Appleton-Century-Crofts. pp 1115-1144 HAYASAKI, M.' (1983) Antigenicity of microfilarial and adult Dirofilaria immitis in indirect fluorescent test. Japanese Journal of Veterinary Science 45, 113-115 KIHARA, S. (1987) Effect of intravenous injection with degenerated eggs o f the canine heartworm on dogs. Journal of the Japanese Veterinary Medical Association 40, 260-267 LUCASSE, C. (1962) Fluorescent antibody test for onchocerciasis. Zeitschrift fiir Tropenmedizin Parasitologie 13, 404-408 MANTOVANI, A. & SULZER, J. S. (1967) Indirect fluorescent antibody technique for diagnosis of canine filariasis. American Journal of Veterinary Research 28, 351-354 OHARA, H., IKEDA, T., TANI, S. & FUJITA, K. (1985) Determination of localization of allergen and IgG inducing antigen in Dirofilaria immitis by immunofluorescent antibody method. Japanese Journal of Experimental Medicine 55, 167-175 SADUN, E. H. (1963) Seminar on immunity to parasitic helminths. VII. Fluorescent antibody technique for helminth infections. Experimental Parasitology 13, 72-82 SANTOS, L. G. D. (1976) Diagnosis of Wuchereria bancrofti filariasis by immunofluorescence using microfilariae as antigen. Annals of Tropical Medicine and Parasitology 70, 219-225 STERNBERGER, L. A., HARDY Jr, P. H., CUCULIS, J. J. & MEYER, H. G. (1970) The unlabelled antibody enzyme method of immunobistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. Journal of Histochemistry and Cytochemistry 18, 313-333 STERNBERGER, L. A. (1979) The unlabelled antibody peroxidaseantiperoxidase (PAP) method. In: Immunocytochemistry. 2nd edn. New York, John Wiley. pp 104-169 SUZUKI, T., AOKI, N. & YAMASHITA, T. (1981) A trial of identification of tissue parasites by using unlabelled anti.body enzyme method. 2. Helminths. Japanese Journal of Parasitology 30, 517-526 TANAKA, K. & ATWELL, R. B. (1990) The optimal condition for peroxidase-anti-peroxidase staining of lung' tissues in canine dirofilariasis. Japanese Journal of Veterinary Science 52, 447-451 TAYLOR, C. R. (1978) Immunoperoxidase techniques. Practical and theoretical aspects. Archives of Pathology and Laboratory Medicine 102, 113- 121 WELL, G. J., MALANE, M. S., POWER, K. G. & BLAIR, L. S. (1985) Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs. Journal of Immunology 134, 1185-1191 WONG, M. M. (1974) Experimental dirofilariasis in macaques: susceptibility and host responses to Dirofilaria immitis, the dog heartworm. Transactions of the Royal Society of Tropical Medicine and Hygiene 68,479-490
Received November 13, 1989 Accepted June 20, 1990
Immunoperoxidase staining characteristics of Dirofilaria immitis in the dog K. I. TANAKA*, R. B. ATWELL, Department of Companion Animal Medicine and Surgery, University o f Queensland, St Lucia, 4067, Queensland, Australia
The immunoperoxidase staining characteristics of Dirofilaria immitis and pulmonary tissues from infected dogs were studied by using the following sera: anti-fresh D immitis, anti-processed D immitis, anti-dog lgG, anti-dog IgG Fc, anti-dog IgM and antidog C 3. Marked staining was observed using antifresh D immitis serum. Body cavity fluid and cuticle were strongly stained and hypodermis, muscle, lateral cord, testis, vas deferens, ovary, oviduct and uterus were moderately stained. Oesophagus and intestine were mildly stained. Degenerate worms were stained by all antisera. The intact and cut surfaces of microfilariae and eggs and sperm present in filariae were stained, but not their internal contents. Circulating and stored immotile microfilariae did not stain. Excreted eggs, presumed to be unfertilised and, or, degenerate, stained positively, lmmunoperoxidase staining of routinely processed histological samples provides a means of assessing D immitis antigen.
potential antigenic material and of filariae themselves. Materials and methods D immitis antigen and antiserum The procedure performed in this study followed that described in a previous report (Tanaka and Atwel11990). 'Fresh female D immitis antigen', 'fresh male D immitis antigen' and 'processed female D immitis antigen' were prepared. For fresh D immitis antigen preparation, segments of adult male and female worms were ground separately in a pestle and mortar, and were then sonicated in phosphate buffered saline-EDTA at pH 7.2. The resultant solution was sedimented and the supernatant was sterilised by filtration through a 0-45 #m filter. To prepare processed D immitis antigen, whole adult female worms that had been fixed in 10 per cent formalin solution were washed with running tap water for more than 12 hours. Worms were then waxed and dewaxed by routine processing methods for histological tissues (Campbell 1951). They were then prepared as for fresh material. Three groups of two New Zealand rabbits were immunised three times at 14 day intervals by intramuscularly injecting 1 ml of one of three different antigen adjuvant preparations. Suitable rabbit antisera to fresh female D immitis extracts (anti-Fw serum) and to processed female D immitis extracts (anti-Pw serum) prepared forthis study were selected by counterimmunoelectrophoresis. Rabbit antisera to fresh male D immitis extracts were prepared but subsequently not used because of unacceptably lower titres when compared with antisera to fresh female D immitis extracts. Normal rabbit serum was also obtained from one unimmunised rabbit.
IMMUNOPEROXIDASE staining (PAP) is a recently developed technique being used in preference to immunofluorescence staining because of its better sensitivity, specificity and sample preservation (Sternberger et al 1970, Taylor 1978). The immunofluorescent staining characteristics of microfilariae and adult Dirofilaria immitis have been reported (Mantovani and Sulzer 1967, Wong 1974, Abramowsky et al 1981, Hayasaki 1983, Ohara et al 1985). However, these results have not always been of high resolution. The PAP technique has been used for identifying helminths in human tissue sections (Suzuki et al 1981) and to confirm the quality of monoclonal antibody directed against D immitis (Weil et al 1985). In this study, PAP staining techniques using anti-D immitis and anti-immunoglobulin sera were used to locate D immitis 'antigen' in lung tissue, and to investigate the staining characteristics of this
Antisera to dog immunoglobulin and dog complement *Present address: Exploratory Research Laboratories, Chugai Pharmaceuticals Co Ltd, 1-135, Komakado, Gotemba-shi, Shizuoka, Japan, 412
Anti-dog IgG rabbit serum (anti-IgG serum), antidog IgG fragment crystalline goat serum (anti-IgG Fc 33
K. L Tanaka, R. B. Atwell
34
TABLE 1 : Summary of observations of D immitis using PAP staining technology Primary Anti-
Anti-
Anti-
Anti-
Anti-
Anti-
serum
FW
PW
IgG
IgG Fc
IgM
C3
Cuticle
-
+ + +
+ .
Hypodermis
-
+ +
+ -
Body
-
+ + +
+ +
-
-
-
-
+ +
+
-
-
-
-
-
Common
organs
cavity
Muscle Lateral
cord
.
.
. -
-
+ +
+
-
+
.
Intestine
-
+
-
-
-
Gut
-
+ +
-
+ +
+ +
+ +
Ovary
-
+ +
+
-
-
-
Oviduct
-
+ +
+
-
-
-
Uterus
-
+ +
+
-
-
-
Shell
-
+ +
+
+ -
-
Surface
-
+ +
.
Internal
.
Cut
-
+ -
-
-
-
+ -
-
contents adult
.
.
.
worm
Intrauterine
eggs
surface
Intrauterine
.
.
.
.
.
+
.
.
-
. -
mf
Surface
-
Internal
.
Cut
-
-
Oesophagus
Female
surface
+ +
+
.
-
.
.
.
.
-
+
-
Testis
-
+ +
+ .
.
.
.
Vas
-
+ +
+ .
.
.
.
Male
adult
-
worm
deferens
Intratesticular
sperm
Surface
-
Internal
.
Cut
-
+
-
-
+ ++
++
surface
Degenerate Whole Fresh
+
-
.
-
.
.
+ -
-
-
+ -
-
+ +
+ +
+ +
.
.
worm
body
motile
++
mf
Surface
.
.
.
.
NP
NP
Internal
.
.
.
.
NP
NP
Stored
immotile
mf
Surface
-
-
NP
-
NP
NP
NP
Internal
-
-
NP
-
NP
NP
NP
Surface
-
+ +
-
-
NP
NP
Internal
-
-
-
NP
NP
Cut
-
+ +
-
-
NP
NP
Surface
-
+ +
-
-
NP
NP
Internal
-
-
-
NP
NP
Circulating
live mf
Cut
in pulmonary
vessel
surface
Egg in pulmonary
-
serum
Normal
UI
vessel
UI
surface
Negative
FW Fresh
+ + stain,
female
+ -
Faint
D immitis,
stain
(implies
PW Processed
doubtful), female
+
Mild
D irnmitis,
serum), anti-dog IgM goat serum (anti-IgM serum) and anti-dog third complement goat serum (anti-C3 serum) were obtained commercially as primary sera for the PAP stain. Tissue sampling Whole worm. To prepare processed whole worm material, five fresh adult male and five fresh adult
stain, NP N o t
+ +
NP
Moderate
performed,
stain,
NP + + +
Ul Unidentified,
Strong mf
stain
Microfilariae
female D immitis were fixed in buffered I0 per cent formalin solution as soon as possible after removal from the pulmonary arteries. Samples were prepared in 4 ~tm sections. Whole microfilariae. Heparinised canine blood containing motile microfilariae was added to an equal volume of sterile distilled water. The prepared haemolysate was smeared, dried on glass slides and then
Immunoperoxidase staining o f Dirofilaria immitis
35
Adult D immitis stained by using anti-fresh D imrnitis serum
! !i~ii ~ ii FIG 1: Male, cross section of apical part. Cuticle, hypodermis, muscle, oesophagus and testis are stained, internal tissue of sperm is not stained, x 1 1 5
FIG 2: Male, cross section of rnidbody. Cuticle, hypodermis, body cavity, intestine, gut contents are stained. Cuticle and body cavity are stained the strongest, x 1 15
FIG 3: Female, longitudinal section of midbody. Cuticle showed multilayering. Cut surface and uncut surface of microfilariae are moderately stained. Stained unfertilised eggs are also observed. x115
FIG 4: Female, longitudinal section of midbody. Cuticle, body cavity are stained. Internal tissue of egg is not stained but egg surfaces are stained. × 1 15
fixed with 100 per cent ethanol for 10 minutes. The remainder of the prepared blood was preserved at 4°C for 10 days until microfilariae had become immotile. Slides were then prepared as for live microfilariae using these immotile microfilariae.
described previously by Tanaka and Atwell (1990). Tissue sections were exposed to hydrogen peroxide solution to block endogenous peroxidase activity and then were exposed to normal pig serum to inhibit any non-specific reaction. The following were used as primary sere: anti-Fw serum, anti-Pw serum, normal rabbit serum (control serum), anti-dog IgG serum, anti-dog C 3 serum, anti-dog IgM serum or anti-dog IgG Fc serum. As secondary sere, anti-rabbit IgG goat serum or anti-goat IgG rabbit serum (heavy and light chains specific) were used. As pAP-complex serum, PAPcomplex rabbit or goat serum was used. A 3,3diaminobenzidine solution was used as chromogen.
Infected canine lung. Canine lung tissue from five naturally infected dogs was fixed with buffered 10 per cent formalin solution at the time of necropsy. Tissues were prepared in 4 #m sections. Immunoperoxidase staining The PAP stain performed followed the method
36
K. L Tanaka, R. B. A twell Pulmonary tissues (naturally infected) stained by using anti-fresh D irnmitis serum
FIG 5: Stained microfilariae are obvious in pulmonary tissue. × 225
FIG 6: Stained degenerated pulmonary vessel, x 480
The preparations were counterstained with Mayer's haematoxylin.
and anti-IgM sera. No stain was evident on the slides when anti-C3 serum was used. Strong staining of dead and degenerate worms in :he pulmonary artery was observed in sections stained by anti-FW, anti-Pw, anti-IgG and anti-C3 sera. The surface of live microfilariae in peripheral blood and of stored immotile microfilariae did not stain, but the microfilariae in the pulmonary tissue were clearly stained (Fig 5). Positive staining was also observed on the surface of intrauterine eggs (Fig 4) and 'free' eggs in the pulmonary tissue (Fig 6).
Results
The PAP staining characteristics of D immitis are summarised in Table 1. In the D immitis worm sections stained with anti-Fw serum, the strongest staining was of the body cavity, particularly body cavity fluid, followed by the staining of the cuticle (Figs 1 to 4), which is clearly shown to be multilayered (Figs 3 and 4). The hypodermis, muscle and lateral cord of male and female worms were moderately stained (Figs 1 to 4). The slides stained using anti-dog IgG sera, anti-dog IgM sera and anti-dog C a sera showed a negative reaction as did all sections stained using control sera. Mild staining was observed in the oesophagus and intestinal tract (Figs 1 and 2). Sections of testes produced a moderately positive stain whereas sperm did not stain, except for the outer surface and the cut surface which was mildly stained (Figs 1 and 2). Both uterus and oviduct were moderately stained. The surface and cut surface of intrauterine microfilariae, eggs, sperm and egg shell, and intratesticular sperm were moderately stained. In contrast, internal structures of liberated microfilariae, eggs and sperm did not stain. Organs common to male and female showed similar stain intensity (Figs l to 4). The intensity of staining using anti-Pw serum was definitely inferior to that for anti-Fw serum. The body cavity fluid stained faintly and the gut contents were moderately stained using anti-IgG, anti-IgG Fc
egg obstructing a peripheral
Discussion
It appears that this anti-rw serum used here has a high sensitivity toward D immitis, adult male and female body components as well as microfilariae, egg and sperm. Weil et al (1985), using monoclonal antibody as primary serum, observed a similar distribution of stain intensities for each organ. However, immotile microfilariae prepared in this study were not positive. This may have been due to the difference in microfilarial preparation between damaged (Weft et al 1985) and immotile microfilariae. Results using the direct immunofluorescence test (Abramowsky et al 1981) were also similar to the present observations. However, microfilariae did not show positive immunostaining. Ohara et al (1985), using indirect immunofluorescence and antisera produced by IgE and by IgG-inducing antigen, reported that allergen was detected in the body cavity fluid and excretory canal of filariae. The IgG-
Immunoperoxidase staining of Dirofilaria immitis inducing antigen was found in the subcuticular area with a weaker reaction in the muscle layer. In the present study, no such associated anti-IgG sera staining was observed. This difference could be due to the different original antigenic substances; Ohara's anti-IgG serum being prepared from specific IgGinducing D immitis antigen. The surface of apparently degenerate eggs in the lung stained positively. The same degree of staining was seen for both intrauterine eggs and excreted eggs. This was in contrast to the positive staining of intrauterine microfilariae and the negative staining of circulating microfilariae. These results could be explained by the degree of antigenic stimulus and thus immunological reaction once perhaps damaged or degenerate eggs are lodged in the peripheral pulmonary vasculature (Kihara 1987). Several authors using circulating microfilariae of other filarial species as an antigen for the immunofluorescence test, have reported poor staining of intact circulating microfilariae (Lucasse 1962, Sadun 1963, Mantovani and Sulzer 1967, Santos 1976). This may have been caused by exposure to some unknown inhibitory substance(s) coating microfilariae once in circulation, particularly when these results were compared to those for intrauterine microfilariae. A similar coating mechanism which would help to avoid immunological recognition and reaction by the host to microfilariae could also exist for D immitis. When comparing the sensitivity of the PAP stain and the immunofluorescence stain, extremely high resolution was observed with the PAP stain. Sternberger (1979) reported that the PAP stain was 100 to 1000 times more sensitive than the immunofluorescence test. Positively stained substances that are coloured light brown are easily distinguished from other tissues even if they are fragmented and scattered. This observation would suggest that routinely processed histological samples could be assessed using the PAP technique and anti-Fw sera. This could be of benefit to the understanding of pathophysiology in the dog and also assist in the identification of pulmonary parasitic debris in people. Acknowledgements The authors appreciate the support of Dr J. Opdebeeck for her immunological assistance, Dr W. Kelly and Dr R. Sutton for histopathological interpretations and Mr J. Massey and laboratory staff for assistance with PAP staining.
37
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Received November 13, 1989 Accepted June 20, 1990