The peroxidase anti-peroxidase technique for the demonstration of immunoglobulin G in rat livers infected with Fasciola hepatica

J. COMP. PATH. 1981. VOL. 91.

573

THE PEROXIDASE ANTI-PEROXIDASE TECHNIQUE FOR THE DEMONSTRATION OF IMMUNOGLOBULIN IN RAT LIVERS INFECTED WITH FASCIOLA HEPATICA

G

BY

I.J. MAFF,

Central

SINCLAIR Veterinary

andD.A.

Laboratory,

.New Haw,

WASSALL Weybridge,

Surrey,

U.K.

INTRODUCTION

It has been shown consistently that rats develop an appreciable degree of immunity to reinfection with the trematode FuscioZu hepatica (Hayes, Bailer and Mitrovic, 1972, 1973, 1974a; Goose and MacGregor, 1973). That this resistance has an immunological basis has been demonstrated by the passive transfer of immunity from infected to naive rats with either serum or lymphoid cells (Armour and Dargie, 1974; Hayes, Bailer and Mitrovic, 1974b). Histological examination of rats implanted subcutaneously with a live adult fluke, has shown changes characteristic of both cell mediated immunity and antibody production in the prefemoral lymph nodes draining the implantation site (Anderson, Hughes and Harness, 1975). There is evidence that the gut is an important site in the immune reaction of the rat (Rajasekariah and Howell, 1977), although it is not the only one, as Kelley, Campbell and Dineen (1980) have shown that the passage of juvenile flukes through the gut is not essential for the acquisition of resistance. This is borne out in experiments with mice (Harness, Doy and Hughes, 1976) and cattle (Anderson, Berrett and Patterson, 1978; Kendall, Sinclair, Everett and Parfitt, 1978) which implicate the liver as a site at which the immune mechanism operates. This report describes the use of the peroxidase anti-peroxidase (PAP) staining technique as a method of demonstrating IgG in liver sections from rats infected with F. hepaticaand seeksto correlate this with the immunological status of the animals.

MATERIALS

AND

METHODS

Rats. Twenty inbred F344/N + rats, aged 6 weeks were assigned to the following groups: Group 1, 2 rats (uninfected controls) ; Group 2, 9 rats were infected with 6 metacercariae ofF. hepatica (obtained from experimentally infected Lpnaea truncatula) and challenged 48 days later (Day 0) with a further 10 metacercariae; Group 3, 9 rats infected with 10 metacercariae on Day 0. Nec~o~sy. Rats were killed according to the schedule in Table 1. Serum was recovered from blood samples taken immediately after death from the subclavian artery and stored at -20 “C. The macropathology of the livers was recorded, the bile ducts examined for adult flukes and the caudate lobes of the livers removed, 6xed in formalin, embedded in paraffin wax and sectioned.

574

I. J.

SINCLAIR

AND

D.

A. WASSALL

TABLE 1 OUTLINE

Infection

GO@

OF EXPERIMENTS

Rat number

AND

Day killed

1

None

:

0 58

2

6plusmetacercariae 10 metacercariae 48 days later (Day 0)

:

i

2 7 : ::

3

10 metacercariae on day 0

i’3 14 15 16 17 18

SUMMARY

OF RESULTS

Macro-pathology

OF SEROLOGICAL

of liver

Number ofjukes recovered

TESTS

Immunod@ision test

Micro ELBA

Normal Normal Several lesions on caudate lobe Slight lesions on caudate lobe Lesions on part of caudate lobe Several lesions on caudate lobe Lesions on part of caudate lobe Small lesions on caudate lobe Puffyappearancetocaudatelobe Puffyappearancetocaudatelobe Normal Normal Normal Normal Normal Normal Lesions on caudate lobe Lesions on caudate lobe Lesions on caudate lobe Lesions on caudate lobe

63 9

;; 37 58

6 9 :z 37 58

Serum examination. Rat sera were tested for antibodies to F. hepatica by the double immunodiffusion test (Ouchterlony and Nilsson, 1973) and a modification of the microplate ELISA described by Voller, Bidwell, Huldt and Engvall (1974). A soluble antigen of adult F. hepatica was prepared by subjecting flukes (obtained from experimentally infected sheep) to snap freezing in liquid nitrogen and then ultrasonification in phosphate buffered saline pH 7.2 (PBS). The sonicate was centrifuged at 40 000 g for 60 min at 4 “C and the supernatant recovered. The protein concentration was determined by spectrophotometry at 280 nm, with albumin standards. This antigen was used at a concentration of 20 mg per ml of PBS in the double immunodiffusion test. A concentration of 10 pg per ml of O-1 M carbonate buffer (pH 9.6) was used to coat micro-ELISA plates (M129A, Dynatech Ltd). The indirect microELISA test was carried out in the micro-ELISA plates as follows: (1) O-1 ml of antigen per well, incubated at 37 “C for 30 min; (2) plate washed 3 times with PBS containing O-5 per cent Tween 20,5 min per wash. (3) 0.1 ml of rat serum per well (in doubling dilutions from 1 in lo), incubated at 37 “C with rotation for 30 min; (4) washed (as stage 2); Ltd) per (5) 0.1 ml of 1 in 900 rabbit-anti rat IgG (Nordic Immunochemicals well, incubated at 37 “C with rotation for 30 min; (6) washed (as stage 2) 5 min per wash; (7) O-1 ml of 1 in 1500 goat anti-rabbit IgG conjugated to horseradish peroxidase (Nordic Ltd) per well, incubated at 37 “C with rotation for 30 min; washed (as stage 2) 5 min per wash; [ij O-1 ml of substrate (5 aminosalicylic acid) per well, rotated at room temperature for 1 h; (10) O-025 ml 1N NaOH per well added to stop reaction. The presence of rat antibody to F. hepatica was detected by the production of a brown colour in the substrate which was quantified with a Titertek Multiskan (Flow Laboratories Ltd).

Fasciola

THEPAPTECHNIQUEIN

INFECTION

575

Liver sections. Five-pm sections of the caudate lobe of the liver were de-waxed, brought to water, and stained for IgG. Three immunohistochemical stains were used. (1) Immunofluorescence by the method of Sainte-Marie (1962). (2) An indirect immunoperoxidase technique by the method of Petts and Roitt (1971). (3) A peroxidase anti-peroxidase (PAP) method adapted from the method of Sternberger, Hardy, Cuculis and Meyer (1970). The most informative results were obtained with the PAP method which was performed as follows: (1) expose sections to 1 in 5 horse serum in PBS pH 7.2 for 5 min; (2) tip off horse serum and add 1 in 200 rabbit anti-rat IgG (Miles Biochemicals) in PBS for 30 min ; (3) wash in 1 in 100 horse serum in PBS for 20 min; (4) add 1 in 40 goat anti-rabbit IgG (Miles) for 30 min; (5) wash (as stage 3) for 20 min; (6) add 1 in 40 rabbit PAP complex (Miles) for 30 min; (7) wash (as stage 3) for 20 min; (8) add freshly prepared substrate (50 mg diaminobenzidine in 100 ml PBS and 0.33 ml of 3 per cent w/v hydrogen peroxide) for 5 min; (9) rinse in PBS and wash in running tap water for 10 min; ( 10) counterstain in Ehrlich’s haematoxylin for 5 min; (11) blue in tap water, dehydrate, clear and mount in DPX. RESULTS

Table 1 records the macropathology of the rat livers, the number of adult flukes recovered and a summary of the results of the serological tests.

Gross Liver Pathology The livers from rats given one infection (Group 3) were normal in appearance from Day 0 to Day 9. After Day 9 the livers showed lesions, although only 2 rats (killed on Days 37 and 58) harboured flukes. The lesions were restricted to the caudate lobe of the livers, both in this group and in the rats of Group 2 which received 2 infections. In Group 2 the damage to this lobe decreased over the period of the experiment and the last rat killed had a liver which was normal in appearance, although two flukes were found in its bile duct.

Serum Antibody T&es by the micro-ELBA, expressed as the optical density of the substrate. The results in Group 2 show a sharp fall in antibody on Days 2 and 3, followed by a peak on Day 9. In Group 3 the antibody increased from zero on Day 2 to a titre similar to that found in Group 2 on Day 15. Thereafter both groups show antibody titres which remained relatively constant for the remainder of the experiment. Precipitating antibodies were detected by immunodiffusion in all rats which had been infected for 23 days or more (Table I). Fig.

1 shows

Immunohistochemical

the

titre

of serum

antibody

detected

Stains

Only the PAP method, of the three immunohistochemical stains employed on the liver sections, produced satisfactory results. The immunofluorescent

576

I. J.

SINCLAIR

AND

D.

A.

23 Day

Fig.

WASSALL

n

0

37

I 58

of experiment

1. Micro-ELISA results expressed as the mean optical density the day of the experiment. ( x ) Group 2; ( 0) Group 3.

of the colour

reaction

in relation

to

technique produced areas of stain around the damaged portions of the liver but was less sensitive than the PAP technique for rat IgG. The indirect immunoperoxidase method did not produce satisfactory negative controls. PAP detected differences in the concentration of IgG between the livers in the 2 groups ofinfected rats. In Group 3 the staining indicated that IgG was associated with pathological changes caused by the fluke infection. Stain was observed around the necrotic tissue and granulomas produced by the burrowing activities of the flukes and from Day 15 the degree of coloration increased in intensity. The liver sections from Group 2 not only showed IgG associated with the internal pathological changes similar to those found in Group 3, but also demonstrated an increase in IgG concentration at the liver surface on Days 3 and 6. The peak of intensity was reached on Day 6 when the surface staining included the first 4 layers of cells beneath the liver surface (Fig. 2). This surface staining was less on Day 9 and had almost disappeared by Day 15.

DISCUSSION

Although the normal mammalian liver appears to play no part in the synthesis of gamma-globulin (Miller and Bale, 1954)) hepatic immunoglobulin synthesis has been demonstrated in calves infected with F. hepaticu (Flagstad and Eriksen, 1974). Our preliminary observations with the PAP technique lend support to this observation. The stain did not detect IgG in the control livers, but did show it in livers of infected rats. In previously infected rats the liver capsule stained heavily for IgG between the third and ninth day after administration of a second infection. This would suggest that the capsule is an important site at which the host’s protective mechanisms operate. The maximal production of

THEPAPTECHNIQUEIN

Fig. 2. Caudate liver lobe surface and 3 (b); 6 (c). Note the increasing

underlying intensity

Fasciola INFECTION

cell layers of Group 2 rats, killed on days 0 of staining on days 3 and 6. PAP x 100.

577

(a);

IgG at the liver surface occurred about the sixth day of the challenge infection and might have been stimulated by antigens released from the migrating juvenile flukes after they had passed through the gut wall. If, however, the stimulus was supplied by the entry of the flukes into the liver, this would reduce the time taken in migration from the gut to a maximum of 4 to 5 days, when allowance is made for the production of the IgG. This is even shorter than the

578

I.

J.

SINCLAIR

AND

D.

A.

WASSALL

8 days allowed by Lang (1967) for migration of flukes in mice, and further work is necessary to clarify the position. The decrease in humoral antibody on Days 2 and 3 in rats given a second infection suggests that the juvenile flukes removed specific antibody from the circulation or produced a transitory inhibition of antibody production. An analogous depression of antibody titre has been observed in cattle when a challenge infection of metacercariae was given (Kendall et al., 1978). Examination of the infected livers revealed that F, hepatica showed a marked preference for the caudate lobe in rats. A similar preference has been noted by Anderson et al. (1978) in cattle. They found that when the livers were examined 45 weeks after the cattle had received an infection of 1000 metacercariae, the ventral lobe showed almost complete atrophy. The immune responses of the laboratory rat to fluke have attracted considerable attention as a model system which bears some relationship to the behaviour of cattle to the same parasite. The demonstration of IgG in the liver capsule by the PAP staining technique raises the question of the relative importance of the gut and the liver as barriers to reinfection. SUMMARY

Rats were given either one or two infections of F. hepatica and were killed between 0 and 58 days after the final infection. Antibody to F. hepatica was demonstrated in the serum by the Enzyme-linked Immunosorbent Assay (ELISA) and the Ouchterlony double-diffusion precipitin test in all rats which received two infections, but only ELISA detected antibody in rats before the 15th day of a single infection. Three to nine days after a second infection, the area round the liver capsule was shown by peroxidase anti-peroxidase staining (PAP) to contain an increased concentration of IgG. It is suggested that this IgG may play a role in the immune mechanism of the rat. ACKNOWLEDGMENT

The authors thank Mr A. D. Cookson for his able technical assistance, particularly with the histochemical techniques. REFERENCES

Anderson,

P. H., Berrett, S., and Patterson, D. S. P. (1978). Resistance to Fusciola hepatica in cattle, II. Biochemical and morphological observations. Journal of Comparative Pathology, 88, 245-25 1.

Anderson, J. C., Hughes, D. L., and Harness, E. (1975). The immune responseof rats to subcutaneousimplantation with Fasciola hepatica. British Veterinary Journal, 131, 509-518. Armour, J., and Dargie, J. E. (1974). Immunity to Fusciola hepatica in the rat: successfultransfer of immunity by lymphoid cells and by serum. Experimental Parasitolo~, 35, 3% l-388. Flagstad, T., and Eriksen, L. (1974). Hepatic immunoglobulin synthesisin Fasciola hepatica infected calves. Research in Veterinary Science, 17, 59-63. Goose,J., and MacGregor, M. (1973). Natural acquired immunity to Faxiola hepatica in the rat. British Veterinary Journal, 129, xlix.

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~APTECHNIQUEIN

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INFECTION

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Harness, E., Doy, T. C., and Hughes, D. L. (1976). Host-parasite relationships of Fasciola hepatica in the mouse during the early stages of infection. Parasitology, 173, xxv-xxvi. Hayes, T. J., Bailer, J., and Mitrovic, M. (1972). Immunity in rats to superinfection with Fasciola hepatica. Journal of Parasitology, 58, 1103-l 105. Hayes, T. J., Bailer, J., and Mitrovic, M. ( 1973). Immunity to Fasciola hepatica in rats : the effect of two different levels of primary exposure on superinfection. Journal of Parasitology, 59, 810-812. Hayes, T. J., Bailer, J., and Mitrovic, M. (1974a). Acquired immunity and age resistance in rats with chronic fascioliasis. Journal of Parasitology, 68, 247-250. Hayes, T. J., Bailer, J., and Mitrovic, M. (1974b). Serum transfer of immunity to Fasciola hepatica in rats. Journal of Parasitology, 68, 722-723. Kelley, J. D., Campbell, N. J., and Dineen, J. K. (1980). The role of the gut in acquired resistance to Fasciola hepatica in the rat. Veterinary Parositolopy, 6,359-367. Kendall, S. B., Sinclair, I. J., Everett? G., and Parfitt, J. W. (1978). Resistance to Fasciola hepatica in cattle, I. Parasrtological and serological observations. Journal of Comparative Pathology, 88, 115-122. Lang, B. Z. (1967). Host-parasite relationships of Fasciola hepatica in the white mouse II. Studies on acquired immunity. Journal of Parasitology, 53, 21-30. Miller, L. L., and Bale, W. F. (1954). Synthesis of all plasma protein fractions except gamma globulins by the liver, Journal of Experimental Medicine, 99, 125-l 3 1. Ouchterlony, O., and Nilsson, L. A. (1973). I mmunodiffusion and immuno-electrophoresis. In Handbook of Experimental Immunology, D. M. Weir Ed., Blackwell Scientific Publications, Oxford. Petts, V., and Roitt, I. M. (1971). Peroxidase conjugates for demonstration of tissue antibodies: evaluation of the technique. Clinical and Experimental Immunology, 9, 407-418. Rajasekariah, G. R., and Howell, M. J. (1977). The fate of Fasciola hepatica metacercariae following challenge infection of immune rats. Journal of Helminthology,

51, 289-294. Sainte-Marie, G. (1962). A p araffin embedding technique for studies employing fluorescence. Journal of Histochemistry and Cytochemistry, 10, 250-256. Sternberger, L. A., Hardy, Jr. P. H., Cuculis, J. J., and Meyer, H. G. (1970). The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antiperoxidase) and its use in identification of spirochetes. Journal of Histochemistry and Cytochemistry, 18, 3 15-333. Voller, A., Bidwell, D. E., Huldt, G., and Engvall, E. (1974). A microplate method of ELISA and its application to malaria. Bulletin of the World Health Organization, 51,209-22 1. [Received for publication,

October 6th, 19801