Quantitation of acute phase protein Cx-reactive (CxRP) in rabbits infected with Trypanosoma brucei

Veterinary Parasitology, 5 (1979) 107-115 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

107

QUANTITATION OF ACUTE PHASE PROTEIN Cx-REACTIVE (CxRP) IN R A B B I T S I N F E C T E D W I T H T R Y P A N O S O M A B R UCEI

R.M. C O O K

Department of Zoology and Applied Entomology, Imperial College of Science and Technology, London SW7 2AZ (Great Britain) Present address: Beecham Pharmaceuticals Research Division, Brockham Park, Betchworth, Surrey RH3 7AJ (Great Britain) (Accepted 19 October 1978) ABSTRACT Cook, R.M., 1979. Quantitation of acute phase protein Cx-reactive (CxRP) in rabbits infected with Trypanosoma brucei~ Vet. ParasitoI., 5: 107--115. The concentrations of the acute-phase serum protein CxRP were measured in rabbits chronically infected with Trypanosoma brucei. In typical fulminating infections, CxRP increased during the acute stage of the disease before the appearance of overt symptoms. As tissue involvement increased, the levels of detectable CxRP gradually declined. The levels of CxRP were dependent upon the onset and severity of inflammation, and could not be correlated with blood parasitaemia, nor with the titre of trypanosome agglutinating antibodies. Oral administration of the anti-inflammatory drug indomethecin to infected animals early in the infection delayed both the appearance of pathological lesions and the subsequent rise in CxRP. Similar treatment later in the infection transiently depressed the CxRP titres, but did not affect the subsequent course of the disease.

INTRODUCTION C-reactive p r o t e i n (CRP) is an antigenicaUy c o m p l e x plasma p r o t e i n which has b e e n described in t h e b l o o d o f m a n following i n f e c t i o n w i t h m a n y p a t h o genic organisms, especially w h e n t h e r e is associated i n f l a m m a t i o n , tissue damage o r necrosis (Raffel, 1 9 6 1 ) . It was initially described b y Tillet and Francis ( 1 9 3 0 ) in p a t i e n t s suffering f r o m p n e u m o c o c c a l p n e u m o n i a . T h e somatic Cpolysacchari'd e o f t h e organism f o r m e d a p r e c i p i t a t e with a c u t e phase sera in the p r e s e n c e o f calcium ions. It has s u b s e q u e n t l y b e e n s h o w n t h a t this is a nonspecific r e a c t i o n p r e s e n t in a v a r i e t y o f disease c o n d i t i o n s ( H e d l u n d , 1 9 4 7 ) . CRP appears in t h e sera early in i n f e c t i o n and develops w i t h t h e inflammat o r y r e a c t i o n , persisting o n l y d u r i n g the activity o f t h a t lesion. A l t h o u g h t h e origin and f u n c t i o n o f this ' a c u t e phase p r o t e i n ' is obscure, its p r e s e n c e in t h e sera is indicative o f an i n f l a m m a t o r y response. Its e s t i m a t i o n is t h u s useful in giving an i n d i c a t i o n o f the severity o f illness ( C o o m b s and Gell, 1 9 6 8 ) f o r ex-

108 ample, in rheumatic fever (Anderson and McCarty, 1950). CRP concentrations usually increase before changes in erythrocyte sedimentation rate (ESR) or pyrexia can be detected, and the levels return to normal before ESR (Kindmark, 1972). An acute phase protein, Cx-reactive protein (CxRP) has also been detected in the blood of rabbits during infection. This protein reacts with a pneumococcal carbohydrate (Cx) related to that found in man. CxRP has been demonstrated by immunofluorescent staining in damaged rabbit myocardial tissue, and these deposits are probably a local accumulation of protein derived from the blood stream or from necrotic or inflammatory lesions (Kushner and Kaplan, 1961). Thomasson et al. (1973) examined the levels of CxRP in rabbits infected with Trypanosoma congolense. Increased concentrations were noted early in the infection which rapidly reached a maximum and then slowly declined as tissue necrosis became more severe. In the present study the concentrations of CxRP were examined in rabbits infected with Trypanosoma brucei. The effect of anti-inflammatory drug treatment on the course of infection was also investigated in relation to pathological features and to the titre of CxRP in the serum. MATERIALS AND METHODS

Animals Outbred, male New Zealand white rabbits weighing 2.5--3.0 kg were obtained from Morton Commercial Rabbits, Parsonnage Farm, Essex. Trypanosoma brucei 427 was originally obtained from the Lister Institute of Preventive Medicine, London, and stabilates cryopreserved in liquid nitrogen at Silwood Park (Lumsden and Herbert, 1973). Maintenance was by the syringe passage of infected blood through outbred mice every 2 or 3 days. Details of strain 427 are given by Boreham and Facet (1974).

Method of infection Rabbits were infected with T. brucei 427 by intravenous, intramuscular or subcutaneous injections of between 1.0 × 1 0 7 - 5 . 0 × l 0 s organisms. Trypanosomes were previously separated from heavily infected rat blood by the anion-exchange method of Lanham (1968), washed, and resuspended in phosphate saline glucose buffer, pH 8.0. T. brucei 427 produces a chronic infection in rabbits, usually fatal between 5 and 8 weeks postinfection. Occasional animals may become asymptomatic and aparasitaemic after 4 or 5 weeks. Control rabbits were given a sham, subcutaneous inoculation of 1.0 ml of buffer.

109 Sera Samples of rabbit blood were obtained from the marginal ear vein at intervals throughout infection (Herbert, 1973). Blood was allowed to clot at 37 °C for 4 h. The serum was removed by centrifugation at 1500 × g for 10 min and 300 ~1 aliquots were stored at -20 °C until required. Sera were tested for the presence of 7 globulin and antitrypanosome antibodies b y the Ouchterlony double diffusion method. In addition, the immune sera from three rabbits were examined for agglutinating antibodies according to the m e t h o d of Cunningham and Vickerman (1962). Estimation o f parasitaemia Parasitaemia was assessed b y microscopical examination of wet film preparations of whole blood. Trypanosome counts were made on an improved Neubauer haemocytometer, counting as for erythrocytes. Cx reactive protein Measurement Concentrations of CxRP in the sera of rabbits infected with T. brucei 427 and in drug-treated animals were both qualitatively and quantitatively determined using the Hyland CR slide test kit (Travenol Laboratories Inc.). All sera were heat inactivated at 56 °C for 30 min before testing. Indomethacin treatment Rabbits infected with T. brucei 427 were dosed anti-inflammatory drug, indomethacin BP (Merck, distilled water at a concentration of 5 mg/kg b o d y postinfection. The dosage regimens were followed as shown in Each rabbit was bled at weekly intervals, and at

orally with 1.0 ml of the Sharp and D o h m e Ltd.), in weight, at varying intervals Table I. dally intervals during the

TABLE I Dosage regimens

Rabbit No. Inoculation Indomethacin treatment T. brucei 427 Start (X 108 )

days postinfection Stop

$594 $595 $596 $569 $556

11 18 28 39 57

8.2 8.2 8.2 8.2 3.O

8 15 22 35 51

110 course of treatment, 18 h after each administration. Non-infected control rabbits were similarly treated. RESULTS Levels o f C x R P in infected animals Five rabbits were inoculated subcutaneously with live trypanosomes and bled at regular intervals up to 7 weeks postinfection. All sera samples were initially examined for the presence or absence of CxRP. Those that gave a positive response were again tested to give a Cx-reactive titre. Sera were taken from sham-inoculated control rabbits at five weekly intervals and from rabbits inoculated intramuscularly with disrupted trypanosomes and Freund's Complete Adjuvant (FCA); disrupted trypanosomes alone; and FCA alone. None of the control animals showed any disease s y m p t o m s and no trypanosomes were detected in the blood. The rabbits inoculated with disrupted trypanosomes and FCA gave agglutinating antibody titres in excess of 1/ 5 1 2 0 . Those animals inoculated with FCA developed localized necrotic lesions and an abscess formed at the inoculation site. Acute phase protein was detected in the sera of all the infected rabbits examined and rose during infection (Table II). However, as the severity of the disease increased, so the concentrations of CxRP gradually declined. There was an occasional secondary rise during the final stages of infection. None of the control rabbits showed any increase in CxRP, suggesting t h a t its elevation was a host response towards the tissue damage caused by the trypanosome infection. Although FCA can induce localized inflammation (Dumonde and Glynn, 1962), this was n o t detected in elevated levels of CxRP. All the infected rabbits gave similar reactions to the infection and this appeared to be reflected in the re-

TABLE II Titres o f Cx-reactive p r o t e i n in D a y s post- R a b b i t infection $722

no. $723

the sera of rabbits infected with T. brucei 4 2 7

$724

$725

$447

0

0

0

0

0

0

8 15 21 28 36 53

0 128 256 128 64 256

0 16 256 32 32 0

0 128 128 128 64 128

0 64 256 2 0 0

64 128 128 64 32 --

111 corded titres of CxRP. The results of one representative animal are described in detail. Rabbit $447 showed typical symptons with low blood parasitaemia present throughout infection and the development of lesions around the head and forelimbs. Recorded levels of CxRP rose after 5 days of infection and rapidly reached a maximum titre by day 15. CxRP could be detected before any external symptoms were evident, with peak values between days 14 and 28. The first signs of oedema and erythema occurred on day 19 and thereafter became more severe. Parasites were first recorded on day 24, by which time exudative dermatitis had developed. By the fourth week, small skin lesions had formed, the eyes were swollen and purulent rhinitis was established. The animal lost weight rapidly after day 35. As the overall tissue necrosis became more progressive, so the levels of detectable CxRP slowly declined. Two rabbits ($448 and $449) were infected by the intramuscular and intravenous routes, respectively. Rabbit $448 developed an asymptomatic reaction to the infection. Parasitaemia remained low, first recorded 33 days postinfection. The animal was aparasitaemic by the fifth week of infection as determined by rodent subinoculations. No external disease symptoms were evident, and no CxRP was detectable at any stage. Rabbit $449 had an uncharacteristically high blood parasitaemia throughout infection. Parasites were first detected on day 3 and the maximum recorded parasitaemia was on day 17 (2.0 X l 0 s/ml.) The animal died 54 days after infection. There was minimal tissue involvement and only a slight facial oedema by day 15. CxRP was first detected at a very low titre (1:8) by day 10 and remained at this level up to day 47. Rabbits $447, $448 and $449 gave similar agglutinating antibody profiles, rising to peak titres of 1/5120 within 7 days of infection.

Effect of indomethacin treatment Since anti-inflammatory drug treatment might be of use in controlling the symptoms of trypanosomiasis (Goodwin, 1974), the effects of indomethacin (5 mg/kg) were monitored. The results for the individual infected rabbits are given in Fig. 1. In all cases the disease ran a typical course with regular waves of parasitaemia and associated tissue involvement. Rabbit $594 showed no symptoms until day 15. The initial oedema gradually increasing in severity with heavy involvement of the eyes and nostrils. Concentrations of CxRP remained low until day 36 when a titre of 1:32 was recorded. A similarresponse was obtained with rabbit $595. A slightoedema was present by day 15, but was markedly reduced following drug treatment. C x R P levelsagain remained depressed until 36 days afterinfection. Rabbit $596 showed heavy facialoedema and inflammation by day 22. During and for fivedays afterthe cessationof drug treatment the symptoms be-

112 J

128

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S594

J

J

J

S595

32

I--

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J

128

J

$596

J

J

S569

@

I,a, Iz: 0

8

0

O

10

2O

3O

4~

20

Days post infection

•

30

20

sb

Days post infection

3;

n

¢

10

20

3O

4O

50

60

Days post infection Fig. 1. T i t r e s o f C x - r e a c t i v e p r o t e i n in r a b b i t s i n f e c t e d w i t h T. bruceL E f f e c t o f i n d o m e t h a c in t r e a t m e n t during i n f e c t i o n . Oral a d m i n i s t r a t i o n o f i n d o m e t h a c i n (5 m g / k g ) inclusive days for e a c h r a b b i t .

came less severe. By day 36 t h e y had reverted and were very heavy 43 days after infection. CxRP levels initially rose but fell dramatically during treatment. Upon cessation, the values increased, reaching a m i x i m u m titre of 1:64. Rabbit $569 showed typical s y m p t o m s after 22 days of infection, which remained static during drug treatment. The necrotic lesions became more involved, the animal dying on day 43. Concentrations of CxRP slowly increased to reach a peak titre of 1:128 and then fell as tissue involvement became more

113 marked. Drug treatment between days 35 and 39 resulted in a rapid decline in CxRP which could n o t be detected from day 38 onwards. The concentration of CxRP in the sera of rabbit $566 again rose during infection and reached peak values b y day 36 -- the first day any external symptoms were evident. Thereafter the CxRP titres slowly declined. Drug treatment, although n o t affecting the disease symptoms, caused a secondary increase in CxRP which reached a final titre of 1:64. Sham-inoculated control rabbits were similarly treated with indomethacin. Acute phase protein was detected in only one animal, 24 h after the injection of buffer, when a titre of 1: 8 was recorded. This may have represented a local inflammatory reaction to the initial inoculation or to the bleeding regime. DISCUSSION With typical fulminating infections of T. brucei in rabbits, serum concentrations of CxRP showed an initial rise, reached a peak and declined slowly as the tissue s y m p t o m s became more severe. The increase in CxRP appeared to be a response to the inflammatory s y m p t o m s produced during infection, and in most instances gave a good indication of the pending inflammation. Increased CxRP titres first became apparent during the acute stage of the disease and rapidly reached a peak, often before any external s y m p t o m s were visible. They subsequently declined as tissue necrosis became more severe. A secondary elevation was occasionally recorded in some of the infected animals during terminal parasitaemia. This somewhat uncharacteristic premature disappearance of CxRP in infectious disease is in agreement with the findings of Thomasson et al. (1973) using T. congolense-infected rabbits. Treatment with the anti-inflammatory drug indomethacin early on in the course of infection retarded both the subsequent development of external s y m p t o m s and the initial rise in serum CxRP. If treatment was effected early enough, the levels remained low for several weeks after dosage was stopped, and only rose when a severe inflammatory reaction was visible. If treatment was carried o u t during the initial rise in CxRP levels, they were rapidly depressed, b u t immediately u p o n cessation the levels increased to relatively high titres, even before external s y m p t o m s became evident. Drug treatment after the initial CxRP peak caused a rapid fall in activity which thereafter remained low. Treatment during the terminal part of the infection caused an increase in CxRP levels, which may have been a result of local inflammation or gastric bleeding. Indomethacin treatment itself had no effect on blood parasitaemia and appeared n o t to alter markedly the subsequent course of the disease. Antiinflammatory treatment may, however, be effective in conjunction with trypanocidal agents. Certainly the steroid prednisolone appears to be a useful adjunct in the treatment of certain aspects of T. rhodesiense infection (Foulkes, 1975). Enhanced CxRP titres did n o t appear to be of value in the early diagnosis of infection, although they may represent a good index of tissue response to

114 injury. The recorded levels could n o t be correlated with parasitaemia, nor to the production of trypanosome agglutinating antibody. It seems, therefore, that the levels of CxRP are d e p e n d e n t upon the course of infection and reflect both the severity and the longevity of the various temporal disease s y m p t o m s (Dutton, 1955). Ree (1971) detected raised levels of CRP in Gambian Africans with Plasmodium falciparum malaria. No correlation was noted with the level of any immunoglobulin, although the intensity of parasitaemia could be regarded as a determinant of the presence or absence of CRP in the blood. This was n o t found in the present studies, T. brucei being primarily a tissue parasite, although profitable investigations might be made using T. vivax or T. congolense infections~ Alterations in serum CRP do n o t appear to have been investigated in human trypanosome infections, b u t if present, its value in effective diagnosis of specific infection would be limited. It is possible, however, that initial screening of susceptible sleeping sickness patients using a parasite-specific serolgicai technique would be useful in presumptive diagnosis. The association between elevated titres of acute phase serum protein and tissue damage does suggest, however, that monitoring these levels may be of use in assessing the pathological consequences of trypanosome infections. This m e t h o d may represent a useful alternative to some of the haematological techniques currently used to determine clinical involvement (Urquhart, 1978). In 1969, Ganrot and Kindmark, investigating the phagocytosis of Gaffkya tetragena b y isolated human neutrophils in vitro, found that the addition of pure human CRP could considerably enhance the phagocytic activity of the cells, and they further suggested that CRP might be of importance in the cellular defence against bacteria in vivo. As indicated b y Thomasson et al (1973), the increased levels of CRP may have similar significance in early host defence mechanisms against trypanosome infections. It has been demonstrated that immune sera from rabbits chronically infected with T. brucei are capable of promoting the specific attachment and subsequent ingestion of considerable numbers of trypanosomes by rabbit peritoneal macrophages in vitro (Cook, 1977). If CRP molecules are able to bind to the surface structures of encapsulated bacteria thus rendering them liable to phagocytosis, perhaps an analogous opsonic mechanism m a y occur in trypanosome infections. ACKNOWLEDGEMENTS I should like to thank Dr P.F.L. Boreham for his advice and helpful criticism and Mr M.G. Parry for technical assistance. The indomethacin was a kind gift from Merck, Sharp and D o h m e Ltd. The w o r k was carried o u t whilst in receipt of an MRC studentship for training in research methods.

115 REFERENCES Anderson, H.C. and McCarty, M., 1950. Determination of C-reactive protein in the blood as a measure of the activity of the disease process in acute rheumatic fever. Am. J. Med., 8: 445--455. Boreham, P.F.L. and Facet, C.A., 1974. Fibrinogen and fibrinogen/fibrin degradation products in experimental African trypanosomiasis. Int. J. Parasitol., 4: 143--151. Cook, R.M., 1977. Studies of Phagocytic Activity in Experimental African Trypanosomiasis. Ph.D. Thesis, University of London, 412 pp. Coombs, R.R.A. and Gell, P.G.H., 1968. Diagnostic and analytic in vitro methods. In: P.G. H. Gell and R.R.A. Coombs (Editors), Clinical Aspects of Immunology. Blackwell Scientific Publications, Oxford, Edinburgh, pp. 3--55. Cunningham, M.P. and Vickerman, K., 1962. Antigenic analysis in the Trypanosoma brucei group using the agglutination reaction. Trans. R. Soc. Trop. Med. Hyg., 56: 48--59. Dumonde, D.C. and Glynn, L.E., 1962. Production of arthritis in rabbits by an immunological reaction to fibrin. Br. J. Exp. Pathol., 43: 373--383. Dutton, A.A.C., 1955. The influence of the route of injection on lethal infections in mice. Br. J. Exp. Pathol., 36: 128--136. Foulkes, J.R., 1975. An evaluation of prednisolone as a routine adjunct to the treatment of T. rhodesiense. J. Trop. Med. Hyg., 78: 72--74. Ganrot, P.O. and Kindmark, C.O., 1969. C-reactive protein -- a phagocytosis-promoting factor. Scand. J. Lab. Clin. Invest., 24: 215--219. Goodwin, L.G., 1974. The African scene: mechanisms of pathogenesis in trypanosomiasis. In: G.E.W. Wolstenholme and J. Knight (Editors), Trypanosomiasis and Leishmaniasis with Special Reference to Chagas' Disease. Ciba Foundation Symposium 20. Elsevier, Amsterdam, pp. 107--119. Hedlund, P., 1947. The appearance of acute uhase protein in various diseases. Acta Med. Scand. Suppl. 196: 579--601. Herbert, W.J., 1973. Laboratory animal techniques for immunology. In: D.M. Weir (Editor), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, pp. A3.1--A3.27. Kindmark, C.O., 1972. The concentration of C-reactive protein in sera from healthy individuals. Scand. J. Clin. Lab. Invest., 29: 407--411. Kushner, I. and Kaplan, M.H., 1961. Studies of acute phase protein. 1. An immunohistochemical method for the localization of Cx-reactive protein in rabbits. Association with necrosis in local inflammatory lesions. J. Exp. Med., 114: 961--973. Lanham, S.M., 1968. Separation of trypanosomes from the blood of infected rats and mice by anion exchangers. Nature, 218: 1273--1274. Lumsden, W.H.R. and Herbert, W.J., 1973. Techniques with Trypanosomes. ChurchillLivingstone, Edinburgh, 120 pp. Raffel, S., 1961. Immunity, 2nd Edition. Appleton Century Crofts, New York, N.Y., 470 pp. Ree, G.H., 1971. C-reactive protein in Gambian Africans with special reference to P. falciparum malaria. Trans. R. Soc. Trop. Med. Hyg., 65: 754--580. Thomasson, D.L., Mansfield, J.M., Doyle, R.J. and Wallace, J.H., 1973. C-reactive protein levels in experimental African trypanosomiasis. J. Parasitol., 59: 738--739. Tillet, W.S. and Francis, T., 1930. Serological reactions in pneumonia with a non-protein somatic fraction of pneumococcus. J. Exp. Med., 52: 561--571. Urquhart, G.M., 1978. Discussion group on pathology. Trans. R. Soc. Trop. Med. Hyg., 72: 118--119.