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Massive increase in splenic germinal centre of chickens experimentally-infected with Trypanosoma brucei brucei
Veterinary Parasitology, 13 (1983) 101--108 Elsevier Science Publishers B.V., Amsterdam - - P r i n t e d in The Netherlands
101
MASSIVE INCREASE IN SPLENIC GERMINAL CENTRES OF CHICKENS EXPERIMENTALLY-INFECTED WITH TR YPANOSOMA BR UCEI BR UCEI
R.A. J OS HUA*
University Department of Bacteriology & Immunology, Western Infirmary, Glasgow, G11 6NT (Gt. Britain)
ABSTRACT Joshua, R.A., 1983. Massive increase in splenic germinal centres of chickens experimentally-infected with Trypanosoma brucei brucei. Vet. Parasitol., 13: 101--108. Domestic chickens infected with Trypanosoma brucei brucei developed a latent parasitaemia which lasted for one year. Six distinct variable antigen types were isolated. Spleens from infected birds were studied histologically at different stages of the infection. Trypanosome infection produced a progressive increase in the number of germinal centres during the early stages of the infection. Peak numbers of germinal centres were reached in the spleen at Day 84 after which the numbers were maintained during an active infection. A tenfold increase in the number of germinal centres was found in trypanosome-infected birds compared to controls. No splenomegaly was observed. Lymphoid cells of the caecal tonsil and the thymus remained apparently normal during the course of infection. Chemotherapeutic termination of infection with berenil resulted in an initial increase in the number of germinal centres. A gradual return to normal level was observed on Day 100 post cure. It is suggested that the development of such a large number of germinal centres reflects the bird's response to the elaboration of a succession of trypanosome variable antigens.
INTRODUCTION
The domestic chicken has long been reported to be among the possible hosts of salivarian trypanosomes (Corson, 1931; Joshua et al., 1978; Zillman and Mehlitz, 1979; Minter-Goedbloed, 1981), though the use of birds for the elucidation of lymphoid changes in trypanosomiasis has had relatively little investigation. The avian immune system possesses a number of features different from that of the mammal, the most prominent being the possession of a lymphoepithelial organ peculiar to birds -- the bursa of Fabricius. Organised lymph *Present address: Nigerian Institute for Trypanosomiasis Research, Vom, Plateau State,
Nigeria.
0304-4017/83/$03.00
© 1983 Elsevier Science Publishers B.V.
102 nodes of the mammalian type are n o t present in the domestic chicken. The peripheral organs are almost wholly represented by the spleen and caecal tonsils. The characteristic features of lymphoid cells are their ability to react to antigens by producing antibodies, to proliferate in response to antigenic stimulation and to retain specific immunological memory. In mammals trypanosome infections result in intense antigenic stimulation as evidenced by extraordinarily high IgM responses (Mattern, 1964), splenomegaly (Assoku, 1975), greatly enlarged germinal centres and massive infiltration of plasma cells (Hudson and Byner, 1973; Moulton and Coleman, 1977) in the spleen. Studies by Wallace et al. (1976) have shown that during active Trypanosoma brucei ssp. infections in chickens many splenic germinal centres are always formed. Thus the absence of numerous germinal centres in a suspected bird can be regarded as evidence against the presence of T. brucei ssp. infection. It appears useful to establish whether an increase in the number of germinal centres invariably occurs in a chronic infection or whether the number of germinal centres fluctuates with fluctuations in parasitaemia. It is also not known if increased numbers of germinal centres are seen following therapy. The present investigation was carried out to provide detailed information about the lymphoid changes that occur in the splenic white pulp of domestic chickens during the course of T. brucei brucei infections. These findings were to be correlated with the observed low parasitaemia in infected birds and the overall constitution of the immune apparatus of Gallus domesticus. MATERIALS AND METHODS Birds Babcock 380 breeds were used. They were not vaccinated against any infectious diseases. Trypanosomes Busoga/60/EATRO/3. These were descendants of a stock of T. brucei rhodesiense. It was known as stabilate WIG 37. Mavubwe/66/EATRO/1125. This stock was originally isolated in Uganda. The sub-species identity of this stock has been established as T. brucei brucei (Le Ray et al., 1977). Lugala/55/EATRO/495. This stock was obtained originally from Glossina pallidipes in Uganda and has been called the Lugala 1 strain. Cloned populations were obtained from a stabilate after it had established an infection in chickens. The sub-species identity of the clone (WITat 1) was confirmed to be T. brucei brucei (Joshua et al., 1978). Cloned organisms were viably preserved as WIG 76.
103
Initiation o f trypanosome infection in chickens Cockerels in three groups of six were each inoculated subcutaneously with trypanosomes derived from each of the three stocks. Each bird received 107.8 organisms from one stock only. The course of the infection was monitored by inoculation of chicken blood into mice.
Diagnosis by mouse inoculation Two ml of heparinized blood was collected from the brachial vein of each bird. One ml of the blood was inoculated into each of two mice. Parasitaemia in the inoculated mice was monitored for t w e n t y days by examination of blood from the tail vein.
Infectivity titration o f infected chicken blood in mice This was carried out essentially as described by Lumsden et al. (1963) except t h a t a 1-ml volume of each dilution was inoculated into each of six mice. Blood diluent was prepared as described by Lumsden et al. (1973).
Isolation o f clones (WITat) Infected blood from a chicken was inoculated intraperitoneally into each of two mice. From a series of mice infected in this manner, clones were obtained as described by Joshua et al. (1981). These clones are called WITat (Western Infirmary Trypanozoon antigen type).
Histological investigation Chickens were killed on appropriate days, in groups of two/three, by cervical dislocation. A section of spleen was fixed in Bouin--Holland fixative, processed and cast in paraffin wax. Embedded tissues were sectioned (5-p) and m o u n t e d on slides. Tissues were stained for 6 min in 0.5% eosin rinsed in water, stained for 1 min in 0.5% toluidine blue, rinsed in water and finally dehydrated in alcohol. Stained tissues were cleared in xylene, m o u n t e d in Canada balsam and examined microscopically using a X 10 objective and X 10 eye piece.
Chemotherapeutic treatment Berenil (Diminazene aceturate, Hoechst, U.K.) was used for treatment of t r y p a n o s o m e infections in chickens at 7 mg kg-1 . The drug (230 mg) was dissolved in 10 ml of distilled water and 1 ml was given per kg of body weight.
104 RESULTS
Infectivity o f trypanosomes to chickens Organisms derived from the stock, Lugala/55/EATRO/495 produced a persistent infection which lasted for over one year. Derivatives of Mavubwe/ 6 6 / E A T R O / l 1 2 5 produced a transient infection that cleared up spontaneously on Day 5. Organisms derived from Busoga/60/EATRO/3 failed to infect the chickens. No infected birds showed any evidence of ill-health. Histological examination revealed a massive increase in splenic germinal centres.
Antigen variants isolated Six variable antigen types (WITat 2--7) were isolated from two chickens infected with a cloned population of T. brucei brucei (WITat 1).
Parasitaemia in birds Initially all T. brucei brucei infections in chickens were confirmed by direct blood inoculation into mice. Attempts at diagnosis by direct blood microscopy were totally unsuccessful. Blood inoculation into mice gives little indication of the number of trypanosomes present in the original host. The practicality of the infectivity titration m e t h o d was tested in each of four fowls that had been infected with T. brucei brucei at different times, i.e., 35, 8 3 , 2 2 0 and 247 days previously. The number of infective trypanosomes in all the birds was found to be extremely low (~ 100 m1-1 of blood).
Germinal centre development during the course o f an infection Trypanosoma brucei brucei infection, monitored by blood inoculation into mice, was found to persist in the birds t h r o u g h o u t the duration of the investigation. A progressive increase in the number of germinal centres was noticeable in the spleen sections as the infection persisted (Table I). Germinal centres increased as much as five fold by Day 84 post infection and this level was sustained thereafter w i t h o u t apparent increase in n u m b e r of trypanosomes. Gross normal appearance o f spleen during the course o f infection One of the c o m m o n e s t post mortem findings was the inability to distinguish grossly between the spleens from infected and control chickens. Despite the striking increase in the number of germinal centres in the spleens
105 TABLE I Mean
number of germinal centres per meridional section of spleen from groups of 2--3
birds Days post Germinal centres infection in spleen sections (Arithmetic mean) 7 14 21 62 84 180 225 290 310 392
37 64 72 126 202 171 177 162 233 178
o f infected birds, this organ did n o t reveal any abnormal increase in size over those o f c o n t r o l birds. The ratio o f the spleen to the live body weight of each bird was calculated. This is referred to as splenic index (S.I.) S.I. = Weight o f spleen X 1 0 0 / T o t a l live b o d y weight The splenic index of infected birds (mean = 0.145) showed no apparent increase as c o m p a r e d with the ratio of this organ in the control (mean = 0.150). DISCUSSION These experiments have shown t hat a stock of T. brucei brucei is able t o p r o d u c e a persistent infection lasting for one year in domestic chickens. It is obvious also t h a t splenic germinal centres respond to stimulation by t r y p a n o s o m e antigen during the course of an active infection. A striking feature of T. brucei brucei infected chickens, is the great increase in the n u m b e r of splenic germinal centres. Quantitatively, the n u m b e r of germinal centres present in the spleens of t r y p a n o s o m e - i n f e c t e d birds was 15--20 times greater than the n u m b e r found in the spleens of u n i n f e c t e d birds. R ecen t studies (Wallace, 1976; Herbert et al., 1981) have shown that the d e v e l o p m e n t o f large numbers of splenic germinal centres reflects the birds' i m m u n e response to the elaboration of a succession of variable antigen types. The n u m b e r o f germinal centres increased progressively in the present investigation during the first 84 days. Different variable antigen types were isolated f r o m the infected chickens within this period. This suggests t h a t germinal centres are f o r m e d in response to emerging t r y p a n o s o m e antigens.
106
There are two types of trypanosome antigens, i.e., the c o m m o n and the variable. If germinal centres are produced to the c o m m o n antigen, increase at a persistent rate would have been observed in the spleen sections during the course of the infection. It seems unlikely that the germinal centres are formed in response to the c o m m o n antigens since an exponential increase in number was not found in infections over one year. It could therefore be said that the periodic emergence of new surface coat (variable) antigen stimulates new germinal centres and that resolution later occurs in older ones. Despite the increased number of germinal centres seen, the gross architecture of the spleens of infected fowls remained undisrupted. The other lymphoid organs (thymus, bursa, bone marrow and caecaltonsils) were grossly and histologically indistinguishable from those of uninfected birds. It could be argued that the compact structure of the chicken spleen did not readily lead to splenomegaly, but this is not so for enlargement of the avian spleen is c o m m o n l y found in diseases such as visceral leucosis, erysipelas (Erysipelothrix insidiosa) and ornithosis. In the latter, spleen increases of up to 40 times the normal size have been recorded in pet birds (Blackmore, 1968). Also domestic chickens have no organized lymph nodes. The spleen would therefore be expected to suffer the whole disruptive effect of a trypanosome infection and be even more markedly affected than that of mammals where an increase of over ten times has been recorded (Assoku, 1975). This assumption was not borne out in this investigation. Hanna (1965) and White et al. (1970) showed that germinal centres appear in the white pulp of the spleen following antigenic stimulation. They are formed by the progressive agglutinative growth of antigen-bearing dendritic cells and B-cells (bursa derived lymphocytes). The increased number of germinal centres in spleen of birds infected with T. brucei brucei therefore represents a massive segregation of B-lymphocytes to the germinal centres of the spleen during the chronic trypanosome infection. A probable cause of the increased germinal centre development in the spleen of infected chickens is the persistence of internal non-variant trypanosome antigens in the bird during the chronic infection. In addition, new antigens emerging on the coat of the trypanosome could cause repeated burst of antigenic stimulation. Lack of a persisting infection is analogous to the absence of antigenic stimulus in a situation such as is found in germ-free animals (Silverstein and Lukes, 1962). The gradual return to normal level of germinal centres following the cure of the infections confirms that the numerous germinal centres are due to a persisting trypanosome infection. The normal ability of domestic chickens to control T. brucei brucei infection may, therefore, be due to their ability to elaborate numerous germinal centres. If the immune apparatus of mammals could be made to react as that
107
of birds, it might be possible for trypanosomiasis to be self-limiting in mammals. CONCLUSION
The immune response of chickens is intimately involved in the control of parasitaemia. The astonishing increase in splenic germinal centre numbers caused by T. brucei brucei infections in chickens is probably immunologically induced. These organelles probably arise in response to the sequential presentation to them of numerous trypanosome variant antigens. ACKNOWLEDGEMENT
The author is grateful to Dr. W.J. Herbert and Prof. R.G. White for advice and guidance. I am indebted to the Governing Board and the Director of Nigerian Institute for Trypanosomiasis Research for providing financial support for the work. REFERENCES Assoku, R.K.G., 1975. Immunological studies of the mechanism of anaemia in experimental Trypanosoma evansi infections in rats. Int. J. Parasitol., 5: 137--145. Blackmore, D.K., 1968. Some observations on Ornithosis. (ii). The disease in cage and aviary birds. In: O. Graham-Jones (Editor), Some Diseases of Animals Communicable to Man in Britain. Pergamon, Oxford, p. 151. Corson, J.F., 1931. Direct infection of native fowls with Trypanosoma rhodesiense. J. Trop. Med. Hyg., 34: 109. Hanna, M.G., 1965. Germinal centre changes and plasma cell reaction during the primary immune response. Int. Arch. Allergy Appl. Immunol., 26: 230--251. Herbert, W.J., Joshua, R.A. and White, R.G., 1981. Vaccination of domestic chickens against a stock of Trypanosoma brucei. Trans. R. Soc. Trop. Med. Hyg., 75: 149. Hudson, K.M. and Byner, C., 1973. Changes in the lymphoid architecture of trypanosome infected mice. Trans. R. Soc. Trop. Med. Hyg., 67 : 265. Joshua, R.A., Herbert, W.J. and White, R.G., 1978. Acquisition by Trypanosoma brucei brucei of potential infectivity for man by passage through birds. Lancet, 1: 724--725. Joshua, R.A., Kayit, Y.S. and Sule, R.A., 1981. A simple technique for isolating pure populations of African trypanosomes from blood. Scientific Proceedings Fifth Annual Conference of the Nigerian Society for Parasitology, Calabar, pp. 30--31. Le Ray, D., Barry, J.D., Easton, C. and Vickerman, K., 1978. First tsetse fly transmission of the AnTat serodeme of Trypanosoma brucei. Ann. Soc. Belge Med. Trop., 57: 369--381. Lumsden, W.H.R., Cunningham, M.P., Webber, W.A.P. and Van Hoeve, K., 1963. A method for the measurement of the infectivity of trypanosome suspensions. Exp. Parasitol., 14: 269--279. Lumsden, W.H.R., Herbert, W.J. and McNeillage, G.J.C., 1973. Techniques with Trypanosomes. Churchill Livingstone, Edinburgh, pp. 29--38. Mattern, P., 1964. Techniques and epidemiological significance of human African trypanosomiasis by means of demonstration of B~ macroglobulin in the blood and cerebrospinal fluid. Ann. Inst. Pasteur, 107: 415.
108 Minter-Goedbloed, E., 1981. The susceptibility of chickens to Trypanosoma brucei species. Trans R. Soc. Trop. Med. Hyg., 75: 345--349. Moulton, J.E. and Coleman, J.L., 1977. Immunosuppression in deer mice with experimentally induced trypanosomiasis. Am. J. Vet. Res., 38: 573--579. Silverstein, A.M. and Lukes, R.J., 1962. Foetal response to antigenic stimulus (i) plasma-cellular and lymphoid reactions in the human foetus to intrauterine injection. Lab. Invest., 11 : 918--932. Wallace, L.E., 1976. Structure and function of dendritic cells in the lymphoid tissue of the domestic fowl. Ph.D. Thesis, University of Glasgow, U.K., pp. 42--71. Wallace, L.E., White, R.G. and Herbert, W.J., 1976. Massive segregation of B-cells in the germinal centres of the spleen of trypanosome-infected birds. Trans. R. Soc. Trop. Med. Hyg., 70: 279. White, R.G., French, V.I. and Stark, J.M., 1970. A study of the localization of a protein antigen in the spleen and its relation to the formation of germinal centres. Med. Microbiol., 3: 65--83. Zillmann, U. and Mehlitz, D., 1979. Occurrence of Trypanozoon in domestic chickens in the Ivory Coast. Tropenmed. Parasitol., 30: 244--248.
101
MASSIVE INCREASE IN SPLENIC GERMINAL CENTRES OF CHICKENS EXPERIMENTALLY-INFECTED WITH TR YPANOSOMA BR UCEI BR UCEI
R.A. J OS HUA*
University Department of Bacteriology & Immunology, Western Infirmary, Glasgow, G11 6NT (Gt. Britain)
ABSTRACT Joshua, R.A., 1983. Massive increase in splenic germinal centres of chickens experimentally-infected with Trypanosoma brucei brucei. Vet. Parasitol., 13: 101--108. Domestic chickens infected with Trypanosoma brucei brucei developed a latent parasitaemia which lasted for one year. Six distinct variable antigen types were isolated. Spleens from infected birds were studied histologically at different stages of the infection. Trypanosome infection produced a progressive increase in the number of germinal centres during the early stages of the infection. Peak numbers of germinal centres were reached in the spleen at Day 84 after which the numbers were maintained during an active infection. A tenfold increase in the number of germinal centres was found in trypanosome-infected birds compared to controls. No splenomegaly was observed. Lymphoid cells of the caecal tonsil and the thymus remained apparently normal during the course of infection. Chemotherapeutic termination of infection with berenil resulted in an initial increase in the number of germinal centres. A gradual return to normal level was observed on Day 100 post cure. It is suggested that the development of such a large number of germinal centres reflects the bird's response to the elaboration of a succession of trypanosome variable antigens.
INTRODUCTION
The domestic chicken has long been reported to be among the possible hosts of salivarian trypanosomes (Corson, 1931; Joshua et al., 1978; Zillman and Mehlitz, 1979; Minter-Goedbloed, 1981), though the use of birds for the elucidation of lymphoid changes in trypanosomiasis has had relatively little investigation. The avian immune system possesses a number of features different from that of the mammal, the most prominent being the possession of a lymphoepithelial organ peculiar to birds -- the bursa of Fabricius. Organised lymph *Present address: Nigerian Institute for Trypanosomiasis Research, Vom, Plateau State,
Nigeria.
0304-4017/83/$03.00
© 1983 Elsevier Science Publishers B.V.
102 nodes of the mammalian type are n o t present in the domestic chicken. The peripheral organs are almost wholly represented by the spleen and caecal tonsils. The characteristic features of lymphoid cells are their ability to react to antigens by producing antibodies, to proliferate in response to antigenic stimulation and to retain specific immunological memory. In mammals trypanosome infections result in intense antigenic stimulation as evidenced by extraordinarily high IgM responses (Mattern, 1964), splenomegaly (Assoku, 1975), greatly enlarged germinal centres and massive infiltration of plasma cells (Hudson and Byner, 1973; Moulton and Coleman, 1977) in the spleen. Studies by Wallace et al. (1976) have shown that during active Trypanosoma brucei ssp. infections in chickens many splenic germinal centres are always formed. Thus the absence of numerous germinal centres in a suspected bird can be regarded as evidence against the presence of T. brucei ssp. infection. It appears useful to establish whether an increase in the number of germinal centres invariably occurs in a chronic infection or whether the number of germinal centres fluctuates with fluctuations in parasitaemia. It is also not known if increased numbers of germinal centres are seen following therapy. The present investigation was carried out to provide detailed information about the lymphoid changes that occur in the splenic white pulp of domestic chickens during the course of T. brucei brucei infections. These findings were to be correlated with the observed low parasitaemia in infected birds and the overall constitution of the immune apparatus of Gallus domesticus. MATERIALS AND METHODS Birds Babcock 380 breeds were used. They were not vaccinated against any infectious diseases. Trypanosomes Busoga/60/EATRO/3. These were descendants of a stock of T. brucei rhodesiense. It was known as stabilate WIG 37. Mavubwe/66/EATRO/1125. This stock was originally isolated in Uganda. The sub-species identity of this stock has been established as T. brucei brucei (Le Ray et al., 1977). Lugala/55/EATRO/495. This stock was obtained originally from Glossina pallidipes in Uganda and has been called the Lugala 1 strain. Cloned populations were obtained from a stabilate after it had established an infection in chickens. The sub-species identity of the clone (WITat 1) was confirmed to be T. brucei brucei (Joshua et al., 1978). Cloned organisms were viably preserved as WIG 76.
103
Initiation o f trypanosome infection in chickens Cockerels in three groups of six were each inoculated subcutaneously with trypanosomes derived from each of the three stocks. Each bird received 107.8 organisms from one stock only. The course of the infection was monitored by inoculation of chicken blood into mice.
Diagnosis by mouse inoculation Two ml of heparinized blood was collected from the brachial vein of each bird. One ml of the blood was inoculated into each of two mice. Parasitaemia in the inoculated mice was monitored for t w e n t y days by examination of blood from the tail vein.
Infectivity titration o f infected chicken blood in mice This was carried out essentially as described by Lumsden et al. (1963) except t h a t a 1-ml volume of each dilution was inoculated into each of six mice. Blood diluent was prepared as described by Lumsden et al. (1973).
Isolation o f clones (WITat) Infected blood from a chicken was inoculated intraperitoneally into each of two mice. From a series of mice infected in this manner, clones were obtained as described by Joshua et al. (1981). These clones are called WITat (Western Infirmary Trypanozoon antigen type).
Histological investigation Chickens were killed on appropriate days, in groups of two/three, by cervical dislocation. A section of spleen was fixed in Bouin--Holland fixative, processed and cast in paraffin wax. Embedded tissues were sectioned (5-p) and m o u n t e d on slides. Tissues were stained for 6 min in 0.5% eosin rinsed in water, stained for 1 min in 0.5% toluidine blue, rinsed in water and finally dehydrated in alcohol. Stained tissues were cleared in xylene, m o u n t e d in Canada balsam and examined microscopically using a X 10 objective and X 10 eye piece.
Chemotherapeutic treatment Berenil (Diminazene aceturate, Hoechst, U.K.) was used for treatment of t r y p a n o s o m e infections in chickens at 7 mg kg-1 . The drug (230 mg) was dissolved in 10 ml of distilled water and 1 ml was given per kg of body weight.
104 RESULTS
Infectivity o f trypanosomes to chickens Organisms derived from the stock, Lugala/55/EATRO/495 produced a persistent infection which lasted for over one year. Derivatives of Mavubwe/ 6 6 / E A T R O / l 1 2 5 produced a transient infection that cleared up spontaneously on Day 5. Organisms derived from Busoga/60/EATRO/3 failed to infect the chickens. No infected birds showed any evidence of ill-health. Histological examination revealed a massive increase in splenic germinal centres.
Antigen variants isolated Six variable antigen types (WITat 2--7) were isolated from two chickens infected with a cloned population of T. brucei brucei (WITat 1).
Parasitaemia in birds Initially all T. brucei brucei infections in chickens were confirmed by direct blood inoculation into mice. Attempts at diagnosis by direct blood microscopy were totally unsuccessful. Blood inoculation into mice gives little indication of the number of trypanosomes present in the original host. The practicality of the infectivity titration m e t h o d was tested in each of four fowls that had been infected with T. brucei brucei at different times, i.e., 35, 8 3 , 2 2 0 and 247 days previously. The number of infective trypanosomes in all the birds was found to be extremely low (~ 100 m1-1 of blood).
Germinal centre development during the course o f an infection Trypanosoma brucei brucei infection, monitored by blood inoculation into mice, was found to persist in the birds t h r o u g h o u t the duration of the investigation. A progressive increase in the number of germinal centres was noticeable in the spleen sections as the infection persisted (Table I). Germinal centres increased as much as five fold by Day 84 post infection and this level was sustained thereafter w i t h o u t apparent increase in n u m b e r of trypanosomes. Gross normal appearance o f spleen during the course o f infection One of the c o m m o n e s t post mortem findings was the inability to distinguish grossly between the spleens from infected and control chickens. Despite the striking increase in the number of germinal centres in the spleens
105 TABLE I Mean
number of germinal centres per meridional section of spleen from groups of 2--3
birds Days post Germinal centres infection in spleen sections (Arithmetic mean) 7 14 21 62 84 180 225 290 310 392
37 64 72 126 202 171 177 162 233 178
o f infected birds, this organ did n o t reveal any abnormal increase in size over those o f c o n t r o l birds. The ratio o f the spleen to the live body weight of each bird was calculated. This is referred to as splenic index (S.I.) S.I. = Weight o f spleen X 1 0 0 / T o t a l live b o d y weight The splenic index of infected birds (mean = 0.145) showed no apparent increase as c o m p a r e d with the ratio of this organ in the control (mean = 0.150). DISCUSSION These experiments have shown t hat a stock of T. brucei brucei is able t o p r o d u c e a persistent infection lasting for one year in domestic chickens. It is obvious also t h a t splenic germinal centres respond to stimulation by t r y p a n o s o m e antigen during the course of an active infection. A striking feature of T. brucei brucei infected chickens, is the great increase in the n u m b e r of splenic germinal centres. Quantitatively, the n u m b e r of germinal centres present in the spleens of t r y p a n o s o m e - i n f e c t e d birds was 15--20 times greater than the n u m b e r found in the spleens of u n i n f e c t e d birds. R ecen t studies (Wallace, 1976; Herbert et al., 1981) have shown that the d e v e l o p m e n t o f large numbers of splenic germinal centres reflects the birds' i m m u n e response to the elaboration of a succession of variable antigen types. The n u m b e r o f germinal centres increased progressively in the present investigation during the first 84 days. Different variable antigen types were isolated f r o m the infected chickens within this period. This suggests t h a t germinal centres are f o r m e d in response to emerging t r y p a n o s o m e antigens.
106
There are two types of trypanosome antigens, i.e., the c o m m o n and the variable. If germinal centres are produced to the c o m m o n antigen, increase at a persistent rate would have been observed in the spleen sections during the course of the infection. It seems unlikely that the germinal centres are formed in response to the c o m m o n antigens since an exponential increase in number was not found in infections over one year. It could therefore be said that the periodic emergence of new surface coat (variable) antigen stimulates new germinal centres and that resolution later occurs in older ones. Despite the increased number of germinal centres seen, the gross architecture of the spleens of infected fowls remained undisrupted. The other lymphoid organs (thymus, bursa, bone marrow and caecaltonsils) were grossly and histologically indistinguishable from those of uninfected birds. It could be argued that the compact structure of the chicken spleen did not readily lead to splenomegaly, but this is not so for enlargement of the avian spleen is c o m m o n l y found in diseases such as visceral leucosis, erysipelas (Erysipelothrix insidiosa) and ornithosis. In the latter, spleen increases of up to 40 times the normal size have been recorded in pet birds (Blackmore, 1968). Also domestic chickens have no organized lymph nodes. The spleen would therefore be expected to suffer the whole disruptive effect of a trypanosome infection and be even more markedly affected than that of mammals where an increase of over ten times has been recorded (Assoku, 1975). This assumption was not borne out in this investigation. Hanna (1965) and White et al. (1970) showed that germinal centres appear in the white pulp of the spleen following antigenic stimulation. They are formed by the progressive agglutinative growth of antigen-bearing dendritic cells and B-cells (bursa derived lymphocytes). The increased number of germinal centres in spleen of birds infected with T. brucei brucei therefore represents a massive segregation of B-lymphocytes to the germinal centres of the spleen during the chronic trypanosome infection. A probable cause of the increased germinal centre development in the spleen of infected chickens is the persistence of internal non-variant trypanosome antigens in the bird during the chronic infection. In addition, new antigens emerging on the coat of the trypanosome could cause repeated burst of antigenic stimulation. Lack of a persisting infection is analogous to the absence of antigenic stimulus in a situation such as is found in germ-free animals (Silverstein and Lukes, 1962). The gradual return to normal level of germinal centres following the cure of the infections confirms that the numerous germinal centres are due to a persisting trypanosome infection. The normal ability of domestic chickens to control T. brucei brucei infection may, therefore, be due to their ability to elaborate numerous germinal centres. If the immune apparatus of mammals could be made to react as that
107
of birds, it might be possible for trypanosomiasis to be self-limiting in mammals. CONCLUSION
The immune response of chickens is intimately involved in the control of parasitaemia. The astonishing increase in splenic germinal centre numbers caused by T. brucei brucei infections in chickens is probably immunologically induced. These organelles probably arise in response to the sequential presentation to them of numerous trypanosome variant antigens. ACKNOWLEDGEMENT
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