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Congenital toxoplasmic retinochoroiditis in the mouse—the use of the peroxidase anti-peroxidase method to demonstrate Toxoplasma antigen
106 TIUNSMX~NS OF THB ROYAL Socmn OF TROPICAL MEDIUNEAND HYGIENE(1985)79, 106-109
Congenital peroxidase
toxoplasmic retinochoroiditis anti-peroxidase method antigen
to
in the mouse-the use of the demonstrate Toxoplasma
J. HAY’*, G. N. DUTTON’ AND J. RALSTON~ ‘Dept of Bioscience and Biotechnology, Biology Division, University of Strathclyde, GeorgeStreet, Glasgow Gl IXW; UK* 2Tennent Institute of Ophthulmology, University of Glasgow, WesternInfirmay, Glasgow GlI 6NT, UK; ‘3Dept. of Pathology, Universi@ of Glasgow, Western Infirmary, Glasgow GII 6NT, UK Abstract
immunocytochemical staining method has been used to demonstrate Toxoplasmaantigen within paraffin-embedded sections of the eyesof mice congenitally infected with Toxopkzsma.Intact Toxoplasmatissue cysts were demonstrated within the retina but in no other ocular structure. No endozoites and no extra-cystic antigens were detected by this technique within anv of the evesexamined. The nossible imolications of thesefindings in relation to the pathogenesisof The peroxidase anti-peroxidase
toxoplasmiE retinochoroiditis
-&e discusskd.
Introduction
A number of hypotheses have been advanced to explain the pathogenesis of the focal necrotizing retinochoroiditis which is the characteristic lesion of ocular toxoplasmosis in man. These hypotheses have been sumfnarized bv DUTTON & HAY (1983). However, experimental investigation of the ocular disease has been limited by the lack of a suitable laboratory model in which the mode of acauisition of infection is analogous to that of man. HAY et al. (1981) have described a murine model of
ocular toxoplasmosis which fulfils this criterion. Toxoplasmaorganisms are transmitted to the developing foetus in utero from a dam which is experimentally infected with the parasite during gestation. Previous histopathological investigations of the eyes of these congenitally infected mice showed a range of severity of diseasefrom mild to severe(LEE et al., 1983; HAY et al., 1984). Although Toxoplasmatissue cysts were frequently seen in the retinae of the mice, no endozoites were observed. As it has been suggested that retinal inftammation results from a hypersensitivity response to toxoplasms released from ruptured tissue cysts (FRBNKEL, 1955) or from mechanical damage
resulting
from
proliferating
organisms
1977), the failure to demonstrate free forms of the parasite within infected eyes may be an important finding. However, it must be noted that in histological preparations of ocular tissue stained by conventional tinctorial methods, endozoites are notoriously difficult to differentiate from disintegrating (O’CONNOR,
nuclei (ZIMMERMAN, 1961).
In order to establish with greater certainty whether free organisms play a part in the pathogenesis of the ocular disease produced in this model, we have incorporated into our experimental regime; the peroxidase anti-peroxidase (PAP) immunocytochemical staining technique (STERNBERGER et al., 1970; STERNBERGER. 1979). This teclmiaue has previouslv
been used by CONL~Y et al. (198i) and ~ONLEY & JENKINS (1981) to demonstrate Toxoplasma antigen (both tissue cysts and endozoites) within the brains of *Present address: School of Pharmacy, Hawthorn Building, $cester Polytechxnc, P.O. Box 143, Lercester LEl 9BH,
man and mouse respectively. The present communication records the methods used and the results obtained using the PAP technique on ocular tissue
from congenitally infected mice.
Materials and Methods Preparation of ocular tissues Eyes were obtained from congenitally infected mice which were the offspring of Strain A albino females which had been injected subcutaneously on the 12th day after mating with a brain emulsion containing 10 tissue cysts of the avirulent Rabbit A strain (BEVERLEY,1959)of Toxoplasma. The cysts had been harvested from the brains of donor mice 17 weeks after infection with Toxoplasma. A comprehensive description of the methodfor the preparation of the congenitally infected mice has been given elsewhere (HUTCHISONet al., 1982).
Control eyes were obtained from uninfected littermates of the congenitally infected mice as well as uninfected mice which were offspring of dams uninfected with Toxoplasma. All ocular tissues examined were derived from mice which were between 14 and 20 weeks of age when killed. Mice were anaesthetixedby carbon dioxide inhalation and fixed by whole body perfusion with 10% buffered fortnalin after whole body perfusion with heparinized saline (both solutions prewarmed to 37°C). After enucleation, the eyes were post-fixed in 10% buffered forrnalin containing 5% glacial acetic acid (v/v) before recessing into parafBn wax. Toxoplasma infection was coIll rtned by a positive dye test (!&BIN-FELDMAN,1948) performed upon terminal sera and the presence of ToxoplartM tissue cysts within histological preparations of brain tissue. Preparation of antiserum Two NZW rabbits (supplied by Hacking & Churchill) of approximately 2.5 kg body weight and seronegative for Toxophma antibodies by the direct agglutination test (DESMONTS& REMMGTON, 1980) were used to raise anti-Tox&zsma antiserum. The rabbits were injected intravenously with a suspension of saline-washed forrnalinkilled endozoites of the RH strain of Tox&zsma on four consecutive weeks (week 1, 1 x 103; week 2, 75 x 10’; week 3, 1 x 106;week 4, 1 x 106).The parasites had been harvested from the peritoneal exudates of mice using the method of TRYON et al. (1978). The rabbits were then challenged on two further occasions (again on consecutive weeks) with live endozoites of the RH Strain (week 5. 1 x lb61 week 6, 1 x 106). 14 days after the final challenge; the rabbns were anaesthetixedand exsanguinated. Blood was
J.
HAY
et
incubated for one hour at 37°C to produce serum which was then centrifuged to remove cells and debris. The sera from both rabbits had a Sabin-Feldman dye teat of 1:1250. The IgG fraction of the pooled sera was isolated using a single batch adsorption with DE-52 cellulose (Whatman) employing the method of Reif (1969). The IgG fraction had an IgG-IFA titre of 1:16000. Protocol for PAP Staining (i) sectionsof ocular tissue (5 p thick) were dewaxed using xylene and hydrated through graded alcohols; (ii) endogenous peroxidaae activity was blocked by immersing sections for 20 min in a solution of 05% hydrogen peroxide in methanol (one part 30% H202 to 59 parts methanol); (iii) sections were washed in two changes (10 min each) of T&buffered saline (TBS) at pH 7.6. Slides were dried except for the area of the section, and placed in a moist chamber; (iv) non-specific binding was inhibited by Poxa#usme antibody free normal swine serum (Nordic), (diluted US) in TBS at room temperature for 20 min; (v) eections were rimed in TBS for one to two min and drted aa in (iii); (vi) sections were then incubated at room temperature for 30 min with the firat layer? comprising the rabbit antiTom@asma immunoglobuhn diluted with TBS containing l/25 normal swine aermn (predetermined optimum staining dilution 1:200); (vii) sections were treated aa in (iii); (viii) sectionswere incubated at room temperature for 30 mm with the second layer comprising Tom@asmaantibody-free swine anti-rabbit IgG (Dako) diluted to 11100with TBS containing l/25 normal swine serum; (ix) sections were treated aa in (iii); (x) sections were incubated at room temperature for 30 min with the third layer comprising Toxophma antibodyfree rabbit PAP complex (Dako) diluted to l/200 with TBS containing l/25 normal swine serum; (xi) sections were treated as in (iii); (xii) sections were incubated with 50 ml of diaminobazidine (DAB)* for 5 to 10 min, the degree of staining being controlkd microscopically. DAB (100 mg, Sigma Isopac) was dissolved in a diluent of pH 7-6 (17 ml 0.2 M Tris buffer, 19 ml O-1M HCI, 19 ml distilled water). Fii, 1.0 ml aliquots were prepared and stored at -20°C. For eachincubation 1.0 ml DAB stock solution was made up to 10 ml with 0.2 M Tris-HCl buffer (PH 7-6), adding 0.1 ml 1% fresh H202 immediately before use: (xiii) sections *DAB is a rnxsible carcinogen and should be used with caution. -
Table I-Controls
107
al.
were treated as in (iii); (xiv) nuclei were lightly counterstained using Harris’s haematoxyiin, dehydrated, cleared and mounted in D.P.X. (R. A. Lamb).
Results and Discussion The results obtained from the various absorption controls (shown in Table I) demonstrate that the PAP staining method, as described in the previous section, is specific for both the endoxoite and cystozoite forms of Toxoplasmu. Ten serial sections were taken from each of 38 eyes of mice congenitally infected with Toxoplasmu. In 24 eyes, isolated and intact Toxo&sma tissue cysts of
varying diameter were selectively stained (Fig. l,A, B). These were found within the retina but iu no other ocular structure. No endozoites were.detected in any of the sections examined, thus confirming the findings of previous studies in which tinctorial staining methods only were used (LEE et al., 1983; HAY et al., 1984).
In contradistinction, FRBNRBL (1955), who studied in hamsters which were experimentally infected with Toxoplasma as adults, identified endozoites within the retina. Frenkel described $is form of the parasite only in eyesin which feeyctmn of m&vrdual cells was accompanied.by m8ammatory reactton. In the present mvestrgaocular toxoplasmosis
tion, 12 eyes presented @rimsl histopathological damage with little or no m8ammstory reaction and little disturbance to the retinal architecture (Fig.
l,A). However, as stated above, no endozoites were identilk-d
even in this form of the disease. antibody levels were found within the aqueous of patients with
On the basis that raised anti-Toxoplama
acute toxoplasmic retinochoroiditis it has been suggested that immune-complex deposition in combinstion with vasculitis may may be associated with the ocular disease (SAARI et al., 1981). Antigen-antibody complexes have been reported within the glomeruli of mice infected with Toxoplasma (HULDT, 1971). In the
used for the peroxidase anti-peroxidase method of stainiig ToxopZama Result
Control 1.
Normal rabbit serum (Nordic) used as primary antiserum on known Toxopkmu infected brain.
No staining
2.
Rabbit anti-mouse IgG (Nordic) used as primary antiserum on known Toxoplasma infected brain.
3. 4.
Omission of primary antiserum. Omission of primary and secondary antisera. Incubation with diaminobenzidine only. Incubation of uninfected eyes with anti-Toxoplasma
No staiuiug of cysts, staining of IgG where present No staiuing No staiuing No staining No staining
5. 6.
primary 7.
IgG as
antiserum.
Incubation of known Toxoplasma infected brain with anti-
ToxopLasma cysts stain
Toxo#usma IgC. 8. 9.
Incubation of a ‘cytospin’ preparation of formalin hilled Toxoplasma endozoites with anti-Toxoplasma IgG. Pre-absorption of a~~ti-Toxoplas~~~~IgG with formalin killed Toxoplasma endozoites, followed by incubation with Toxoplasma infected brain.
Toxoplamra organisms stain No staining
108
CONGENITAL
TOXOPLASMIC
RETINOCHOROIDITIS
IN
MICE
Fig. 1. Toxoplasmatissue cysts within murine retina, stained by the PAP technique (haematoxylin counterstain). A. Cyst within bipolar cell layer: note that the outer retina and pigment epithelium are normal in appearance(X 344). B. Two small cysts within disorganized retina (X 1376).
present study, perivascular lymphocyte and mononuclear cell infiltration was a characteristic feature of affected eyes and has been reported in our previous investigations (LEE et al., 1983; HAY et al., 1984). However, no PAP staining, indicative of Toxoplasma antigens was detected in relation to the retinal vessels. The absence of PAP stainine outwith tissue cvsts also appears to discount th; possibility that- an antigenic toxin released by the parasite is responsible for the retinal damage. A diffuse fluorescence around cysts and in the region of perivascular cuffing has been reported by WERNER et d. (1981) who used fluorescent antibody labelling on cryostat sections of brain from mice experimentally infected with Toxoidama as adults. These authors concluded from their *&lings that Toxoplasma was producing an ‘exotoxin’. However. FRENKEL & PIEKARSK! (1978) have drawn attention to the drawbacks of the fluorescent antibody staining method when appropriate absorption controls are not included in the experimental regime. It is also possible that the use of frozen sections causes aberrant staining since Toxodusma antigens, at least in the endozoite form of the parasite, appear to be mainlv orotein in nature (HANDMAN et al.. 1980)and water-soluble (JOHNSON‘et al., 1983). REID kt al. (1984) have also reported immunostaining of diffusable antigens after tissue autolysis. Thus the ‘exotoxin’ described by WERNER et al. (198 1) may simply be due to antigenic diffusion within unfixed tissue. CARVER & GOLDMAN(1959) and CONLEY& JENKINS (1981) using immunofluorescence and immunoperoxidase methods respectively on paraffin sections of
mouse brain did not report such diffuse staining outwith tissue cysts. In conclusion, the failure to demonstrate specific staining of extra-cystic antigen or endozoites within any of the eyesexamined, lends further support to the hypothesis that in this murine model at least, the ocular inflammatory response occurs as an indirect consequence of Toxoplasma infection. Acknowledgements We thank Professor W. R. Lee for valuable comments, Dr. H. Williams for carrying out the dye tests and Professor W. H. Stimson and his staff for technical support. G. N. Dutton was in receipt of a Wellcome Trust Fellowship and this research was supported in part by the W. H. Ross Foundation (Scotland) for the Study of Prevention of Blindness. References Beverlev, J. K. A. (1959). Connenital transmission of toxoplasmosis through successi;e generations of mice. Nature, 183, 1348-1349. Carver, R. K. & Goldman, M. (1959). Staining Toxoplasma gomfii with fluorescein-labelled antibody. AmricanJournal of Clinical Patholoev. 32. 159-164. Conley, g. K. & Jenkins,, g ‘A. (1981). Immunohistological study of the anatormc relationship of Toxopkasma antigens to the inflammatory response in the brains of mice chronically infected with Toxoplasma gondii. Infection and Immunity, 31, 1184-1192. Conley, F. K., Jenkins, K. A. & Remington, J. S. (1981). Toxoplasma go&i infection of the central nervous system. Use of the peroxidase-antiperoxidase method to demonstrate Toxoplasma in formalin fixed, paraffin embedded tissue sections. Human Pathology, 12, 690698.
J. HAY
Desmonts, G. & Remington, J. S. (1980). Direct agglutination test for diagnosis of Toxoplasma infection. Method for increasing sensitivity and specificity. ~oumal of Chical
Microbiology,
11, 562-568.
Dutton, G. N. & Hay, J. (1983). Toxoplasmic retinochoroiditis--current concepts in pathogenesis. T~wuacrions of the3~o~halmalogtcaogreal Soames of the Umted Ktngdom, 103,
Frenkel, J.* K. (1955). Ocular lesions in hamsters with chronic Toxohsma and Bemoitia infection. Am&can Joumal of O$thalnw&y, 39, 203-225. Frenhel, J. K. & Pie.karshi, G. (1978). The demonstration of Toxopb. and other organisms by imnumofluoresceny;&a pufall. Journal of Infectwus Dwases, 138, Hand&n, E., Goding, J. W. & Remington, J. S. (1980). Detection and “;IBcterixation of membrane antigens of ~‘.xkasnla
go&.
Journal of I wmuawk,
124, 2578-
L,O>.
Hay, J., Hutchison, W. M., Lee, W. R. & Siim, J. C. (1981). Cataract in mice congenitally infected with Toxoplanna gondii. Annals of Tropic& Medicine and Panlsitol5gy
75, 455-457.
Hay,.!., Lee, W. R., Dutton, G. N., Hutch&t, y. M., & S-, J. C. (1984). Congeniud toxoplasmic retmochoroidms in a mouse model. Annals of Tmpical Medicine and 78, 109-116. HUE= ‘%? ht., Hay, J., Lee, W. R. & Siim, J. C. (1982).-A study of cat&kin m.urine covtal toxoplas~~0;;. Annals of Troptcal Medscme and
amsdogy,
76,
- . Huldt, G. (1971). Studies on experimental toxoplasmosis. fk%50f
- - -- - .
the New
York Acadeny
of Saences, 177,
Johnson, A. M., Haynes, W. D., Leppard, P. J., McDomild, P. J. & Neoh, S. H. (1983). Ultrastructural and biochemi&l SNdkS on the imukobistochcmisay of Toxop&sma godi
et
al.
109
toxoplasmic retinochoroiditis. Acta Ophthalmologica, 61, 818-830. O’Connor, G. R. (1977). Protozoan dkases of the uvea. Zntemational Ophrhaldogy Clinics, 17, 163-176. Reid, W. A., Thorn son, W. D. & Kay, J. (1984). Immuu+aining oP**sable antigens after autolysis. N of the Pathological Society of Great Britain and Ireland. 148th Meeting. Bkmiqham: K. P. Reprographics. Zwmarimal Journal of Pathology, 142, A5. Reif, A. E. (1%9). Batch preparation of rabbit y-G globulin with DEAE-cellulose. Zmmtawchemistiy, 6, 723-731. Saari, K. M., Turunen, H., Leinikhi, P. G., Krause, U., Pohjanpelto, P. J., Parviainen, M. & Ame, E. (1981). Enxyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmic chorioretinitis. Acra Ophthabwlagica, 59, 800-809. Sabin~A. B. & Feldman, H. A. (1948). Dyes as microchemtcal indicators of a new itmmmity phenomenon affecting a protozoan parasite (Tox&a.wna). Science, 108, Stem-&, L. A. (1979). kraanoqrcc~ (2nd edit.). New York: John Wiley and Sons Inc. Sternberger, L. A., Hardy, P. H. Jr., Cucculis, J. J. & Meyer, H. G. (1970). The unlabelled antibody-enxyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradnh peroxidase-antihorseradishpemxidase) and its use in identification of spirochetes.Journal of Histochendy 6 cytochemisL?y, 18, 315-333.
Tryon, J. C., Weidner, E., Larson, A. D. & Hart, L. T. (1978). A rapid method for isohuing purified Toxoplasmu tachyxoites from peritoneal exudate and cell culture. Journal of Parakdgy, 64, 1127-1129. Werner, H., Masihi, K. N. & Set&, U. (1981). Latent Toxoplurma-infection was a possible rislt factor for CNS
antigens using monaclond antibodies.
Histochetnisty, 77, 209-215. Lee, W. R., Hay, J., Hutchison, W. M., Dutton, G. N. & Siim, J. C. (1983). A murine model of congenital
Accepted fm publication
10th June,
1984.
Congenital peroxidase
toxoplasmic retinochoroiditis anti-peroxidase method antigen
to
in the mouse-the use of the demonstrate Toxoplasma
J. HAY’*, G. N. DUTTON’ AND J. RALSTON~ ‘Dept of Bioscience and Biotechnology, Biology Division, University of Strathclyde, GeorgeStreet, Glasgow Gl IXW; UK* 2Tennent Institute of Ophthulmology, University of Glasgow, WesternInfirmay, Glasgow GlI 6NT, UK; ‘3Dept. of Pathology, Universi@ of Glasgow, Western Infirmary, Glasgow GII 6NT, UK Abstract
immunocytochemical staining method has been used to demonstrate Toxoplasmaantigen within paraffin-embedded sections of the eyesof mice congenitally infected with Toxopkzsma.Intact Toxoplasmatissue cysts were demonstrated within the retina but in no other ocular structure. No endozoites and no extra-cystic antigens were detected by this technique within anv of the evesexamined. The nossible imolications of thesefindings in relation to the pathogenesisof The peroxidase anti-peroxidase
toxoplasmiE retinochoroiditis
-&e discusskd.
Introduction
A number of hypotheses have been advanced to explain the pathogenesis of the focal necrotizing retinochoroiditis which is the characteristic lesion of ocular toxoplasmosis in man. These hypotheses have been sumfnarized bv DUTTON & HAY (1983). However, experimental investigation of the ocular disease has been limited by the lack of a suitable laboratory model in which the mode of acauisition of infection is analogous to that of man. HAY et al. (1981) have described a murine model of
ocular toxoplasmosis which fulfils this criterion. Toxoplasmaorganisms are transmitted to the developing foetus in utero from a dam which is experimentally infected with the parasite during gestation. Previous histopathological investigations of the eyes of these congenitally infected mice showed a range of severity of diseasefrom mild to severe(LEE et al., 1983; HAY et al., 1984). Although Toxoplasmatissue cysts were frequently seen in the retinae of the mice, no endozoites were observed. As it has been suggested that retinal inftammation results from a hypersensitivity response to toxoplasms released from ruptured tissue cysts (FRBNKEL, 1955) or from mechanical damage
resulting
from
proliferating
organisms
1977), the failure to demonstrate free forms of the parasite within infected eyes may be an important finding. However, it must be noted that in histological preparations of ocular tissue stained by conventional tinctorial methods, endozoites are notoriously difficult to differentiate from disintegrating (O’CONNOR,
nuclei (ZIMMERMAN, 1961).
In order to establish with greater certainty whether free organisms play a part in the pathogenesis of the ocular disease produced in this model, we have incorporated into our experimental regime; the peroxidase anti-peroxidase (PAP) immunocytochemical staining technique (STERNBERGER et al., 1970; STERNBERGER. 1979). This teclmiaue has previouslv
been used by CONL~Y et al. (198i) and ~ONLEY & JENKINS (1981) to demonstrate Toxoplasma antigen (both tissue cysts and endozoites) within the brains of *Present address: School of Pharmacy, Hawthorn Building, $cester Polytechxnc, P.O. Box 143, Lercester LEl 9BH,
man and mouse respectively. The present communication records the methods used and the results obtained using the PAP technique on ocular tissue
from congenitally infected mice.
Materials and Methods Preparation of ocular tissues Eyes were obtained from congenitally infected mice which were the offspring of Strain A albino females which had been injected subcutaneously on the 12th day after mating with a brain emulsion containing 10 tissue cysts of the avirulent Rabbit A strain (BEVERLEY,1959)of Toxoplasma. The cysts had been harvested from the brains of donor mice 17 weeks after infection with Toxoplasma. A comprehensive description of the methodfor the preparation of the congenitally infected mice has been given elsewhere (HUTCHISONet al., 1982).
Control eyes were obtained from uninfected littermates of the congenitally infected mice as well as uninfected mice which were offspring of dams uninfected with Toxoplasma. All ocular tissues examined were derived from mice which were between 14 and 20 weeks of age when killed. Mice were anaesthetixedby carbon dioxide inhalation and fixed by whole body perfusion with 10% buffered fortnalin after whole body perfusion with heparinized saline (both solutions prewarmed to 37°C). After enucleation, the eyes were post-fixed in 10% buffered forrnalin containing 5% glacial acetic acid (v/v) before recessing into parafBn wax. Toxoplasma infection was coIll rtned by a positive dye test (!&BIN-FELDMAN,1948) performed upon terminal sera and the presence of ToxoplartM tissue cysts within histological preparations of brain tissue. Preparation of antiserum Two NZW rabbits (supplied by Hacking & Churchill) of approximately 2.5 kg body weight and seronegative for Toxophma antibodies by the direct agglutination test (DESMONTS& REMMGTON, 1980) were used to raise anti-Tox&zsma antiserum. The rabbits were injected intravenously with a suspension of saline-washed forrnalinkilled endozoites of the RH strain of Tox&zsma on four consecutive weeks (week 1, 1 x 103; week 2, 75 x 10’; week 3, 1 x 106;week 4, 1 x 106).The parasites had been harvested from the peritoneal exudates of mice using the method of TRYON et al. (1978). The rabbits were then challenged on two further occasions (again on consecutive weeks) with live endozoites of the RH Strain (week 5. 1 x lb61 week 6, 1 x 106). 14 days after the final challenge; the rabbns were anaesthetixedand exsanguinated. Blood was
J.
HAY
et
incubated for one hour at 37°C to produce serum which was then centrifuged to remove cells and debris. The sera from both rabbits had a Sabin-Feldman dye teat of 1:1250. The IgG fraction of the pooled sera was isolated using a single batch adsorption with DE-52 cellulose (Whatman) employing the method of Reif (1969). The IgG fraction had an IgG-IFA titre of 1:16000. Protocol for PAP Staining (i) sectionsof ocular tissue (5 p thick) were dewaxed using xylene and hydrated through graded alcohols; (ii) endogenous peroxidaae activity was blocked by immersing sections for 20 min in a solution of 05% hydrogen peroxide in methanol (one part 30% H202 to 59 parts methanol); (iii) sections were washed in two changes (10 min each) of T&buffered saline (TBS) at pH 7.6. Slides were dried except for the area of the section, and placed in a moist chamber; (iv) non-specific binding was inhibited by Poxa#usme antibody free normal swine serum (Nordic), (diluted US) in TBS at room temperature for 20 min; (v) eections were rimed in TBS for one to two min and drted aa in (iii); (vi) sections were then incubated at room temperature for 30 min with the firat layer? comprising the rabbit antiTom@asma immunoglobuhn diluted with TBS containing l/25 normal swine aermn (predetermined optimum staining dilution 1:200); (vii) sections were treated aa in (iii); (viii) sectionswere incubated at room temperature for 30 mm with the second layer comprising Tom@asmaantibody-free swine anti-rabbit IgG (Dako) diluted to 11100with TBS containing l/25 normal swine serum; (ix) sections were treated aa in (iii); (x) sections were incubated at room temperature for 30 min with the third layer comprising Toxophma antibodyfree rabbit PAP complex (Dako) diluted to l/200 with TBS containing l/25 normal swine serum; (xi) sections were treated as in (iii); (xii) sections were incubated with 50 ml of diaminobazidine (DAB)* for 5 to 10 min, the degree of staining being controlkd microscopically. DAB (100 mg, Sigma Isopac) was dissolved in a diluent of pH 7-6 (17 ml 0.2 M Tris buffer, 19 ml O-1M HCI, 19 ml distilled water). Fii, 1.0 ml aliquots were prepared and stored at -20°C. For eachincubation 1.0 ml DAB stock solution was made up to 10 ml with 0.2 M Tris-HCl buffer (PH 7-6), adding 0.1 ml 1% fresh H202 immediately before use: (xiii) sections *DAB is a rnxsible carcinogen and should be used with caution. -
Table I-Controls
107
al.
were treated as in (iii); (xiv) nuclei were lightly counterstained using Harris’s haematoxyiin, dehydrated, cleared and mounted in D.P.X. (R. A. Lamb).
Results and Discussion The results obtained from the various absorption controls (shown in Table I) demonstrate that the PAP staining method, as described in the previous section, is specific for both the endoxoite and cystozoite forms of Toxoplasmu. Ten serial sections were taken from each of 38 eyes of mice congenitally infected with Toxoplasmu. In 24 eyes, isolated and intact Toxo&sma tissue cysts of
varying diameter were selectively stained (Fig. l,A, B). These were found within the retina but iu no other ocular structure. No endozoites were.detected in any of the sections examined, thus confirming the findings of previous studies in which tinctorial staining methods only were used (LEE et al., 1983; HAY et al., 1984).
In contradistinction, FRBNRBL (1955), who studied in hamsters which were experimentally infected with Toxoplasma as adults, identified endozoites within the retina. Frenkel described $is form of the parasite only in eyesin which feeyctmn of m&vrdual cells was accompanied.by m8ammatory reactton. In the present mvestrgaocular toxoplasmosis
tion, 12 eyes presented @rimsl histopathological damage with little or no m8ammstory reaction and little disturbance to the retinal architecture (Fig.
l,A). However, as stated above, no endozoites were identilk-d
even in this form of the disease. antibody levels were found within the aqueous of patients with
On the basis that raised anti-Toxoplama
acute toxoplasmic retinochoroiditis it has been suggested that immune-complex deposition in combinstion with vasculitis may may be associated with the ocular disease (SAARI et al., 1981). Antigen-antibody complexes have been reported within the glomeruli of mice infected with Toxoplasma (HULDT, 1971). In the
used for the peroxidase anti-peroxidase method of stainiig ToxopZama Result
Control 1.
Normal rabbit serum (Nordic) used as primary antiserum on known Toxopkmu infected brain.
No staining
2.
Rabbit anti-mouse IgG (Nordic) used as primary antiserum on known Toxoplasma infected brain.
3. 4.
Omission of primary antiserum. Omission of primary and secondary antisera. Incubation with diaminobenzidine only. Incubation of uninfected eyes with anti-Toxoplasma
No staiuiug of cysts, staining of IgG where present No staiuing No staiuing No staining No staining
5. 6.
primary 7.
IgG as
antiserum.
Incubation of known Toxoplasma infected brain with anti-
ToxopLasma cysts stain
Toxo#usma IgC. 8. 9.
Incubation of a ‘cytospin’ preparation of formalin hilled Toxoplasma endozoites with anti-Toxoplasma IgG. Pre-absorption of a~~ti-Toxoplas~~~~IgG with formalin killed Toxoplasma endozoites, followed by incubation with Toxoplasma infected brain.
Toxoplamra organisms stain No staining
108
CONGENITAL
TOXOPLASMIC
RETINOCHOROIDITIS
IN
MICE
Fig. 1. Toxoplasmatissue cysts within murine retina, stained by the PAP technique (haematoxylin counterstain). A. Cyst within bipolar cell layer: note that the outer retina and pigment epithelium are normal in appearance(X 344). B. Two small cysts within disorganized retina (X 1376).
present study, perivascular lymphocyte and mononuclear cell infiltration was a characteristic feature of affected eyes and has been reported in our previous investigations (LEE et al., 1983; HAY et al., 1984). However, no PAP staining, indicative of Toxoplasma antigens was detected in relation to the retinal vessels. The absence of PAP stainine outwith tissue cvsts also appears to discount th; possibility that- an antigenic toxin released by the parasite is responsible for the retinal damage. A diffuse fluorescence around cysts and in the region of perivascular cuffing has been reported by WERNER et d. (1981) who used fluorescent antibody labelling on cryostat sections of brain from mice experimentally infected with Toxoidama as adults. These authors concluded from their *&lings that Toxoplasma was producing an ‘exotoxin’. However. FRENKEL & PIEKARSK! (1978) have drawn attention to the drawbacks of the fluorescent antibody staining method when appropriate absorption controls are not included in the experimental regime. It is also possible that the use of frozen sections causes aberrant staining since Toxodusma antigens, at least in the endozoite form of the parasite, appear to be mainlv orotein in nature (HANDMAN et al.. 1980)and water-soluble (JOHNSON‘et al., 1983). REID kt al. (1984) have also reported immunostaining of diffusable antigens after tissue autolysis. Thus the ‘exotoxin’ described by WERNER et al. (198 1) may simply be due to antigenic diffusion within unfixed tissue. CARVER & GOLDMAN(1959) and CONLEY& JENKINS (1981) using immunofluorescence and immunoperoxidase methods respectively on paraffin sections of
mouse brain did not report such diffuse staining outwith tissue cysts. In conclusion, the failure to demonstrate specific staining of extra-cystic antigen or endozoites within any of the eyesexamined, lends further support to the hypothesis that in this murine model at least, the ocular inflammatory response occurs as an indirect consequence of Toxoplasma infection. Acknowledgements We thank Professor W. R. Lee for valuable comments, Dr. H. Williams for carrying out the dye tests and Professor W. H. Stimson and his staff for technical support. G. N. Dutton was in receipt of a Wellcome Trust Fellowship and this research was supported in part by the W. H. Ross Foundation (Scotland) for the Study of Prevention of Blindness. References Beverlev, J. K. A. (1959). Connenital transmission of toxoplasmosis through successi;e generations of mice. Nature, 183, 1348-1349. Carver, R. K. & Goldman, M. (1959). Staining Toxoplasma gomfii with fluorescein-labelled antibody. AmricanJournal of Clinical Patholoev. 32. 159-164. Conley, g. K. & Jenkins,, g ‘A. (1981). Immunohistological study of the anatormc relationship of Toxopkasma antigens to the inflammatory response in the brains of mice chronically infected with Toxoplasma gondii. Infection and Immunity, 31, 1184-1192. Conley, F. K., Jenkins, K. A. & Remington, J. S. (1981). Toxoplasma go&i infection of the central nervous system. Use of the peroxidase-antiperoxidase method to demonstrate Toxoplasma in formalin fixed, paraffin embedded tissue sections. Human Pathology, 12, 690698.
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Accepted fm publication
10th June,
1984.