Progressive serum protein changes in experimental infections of calves with Trypanosoma vivax

J.

COMP.

PATH.

1975.

VOL.

8.5.

397

PROGRESSIVE EXPERIMENTAL

SERUM PROTEIN INFECTIONS OF TR YPANOSO

MA

CHANGES CALVES

IN WITH

VIVA-7

BY M. Department

of Veterinary

J.

CLARKSON

Parasitology, Chiversi[v

Liverpool School of Liverpool

of Tropical

Medicine.

and

W. J. PENHALE* Department

of Veterinary

and R. B. MCKENNA

Pathology, Royal (Dick) School of Veterinary lJniversi& of Edinbuqh

Studies,

INTRODUCTION

A great increase in serum IgM concentration is characteristic of infection of man and experimental animals with pathogenic trypanosomes (Mattern, 1964; Seed, Cornille, Risby and Gam, 1969; Takayanagi and Enriquez, 1973). Luckins (1972) demonstrated increases in IgM concentrations of 2 to 9 times preinfection levels after Zebu cattle had been introduced into an area of Africa in which trypanosomiasis was enzootic. He subsequently published a brief account of immunoglobulin changes in experimental infections of Zebu cattle with T?;ypanosomacongolenseand T. vivax (Luckins, 1974). In 9 animals IgM concentration increased shortly after infection and IgG, and IgG, showed little change. These animals were treated with diminazene aceturate (“Berenil”, Hoechst) 50 days after infection and the IgM concentration fell. The IgM level also decreased in one animal which died 47 days after infection. The present study was undertaken to follow serum protein changes, including immunoglobulin concentrations, in susceptible British calves, infected with strains of T. vivax of differing virulence, under conditions in which no other tropical infections were present. Frequent serum samples were taken in an attempt to relate the serum protein changes to the course of infection. A brief report has already been presented (Clarkson and Penhale, 1973). MATERIALS

AND

METHODS

Calves.These were purchased from British farms within a few days of birth after receiving colostrum and were of various breeds. They were kept in a fly-proof room in a controlled environment and fed a diet of Bibbys’ “Superstart” calf meal for 4 weeks and progressively weaned to a diet of crushed oats, flaked maize and B.O.C.M. “Sheepmix” crumbs. They were given hay and water ad lib. Infection procedures.Ten calves (Nos. 1 to 10) were infected at 1 to 4 months old with strain 64123 of T. vivax originally isolated in Nigeria and subsequently maintained in Liverpool by passage through Glossinamorsitans.Infections were either by * Present

address:

Immunology

Laboratories,

2 Forrest

Road,

Edinburgh.

398

M.

J.

CLARKSON

et

d.

syringe passage of cattle blood containing trypanosomes or by infected G. morsitans, produced from pupae supplied by the Tsetse Research Laboratory, Langford, Bristol. Two calves (11, 12) were infected at 2 months old with the “Desowitz” strain and one (13) at 1 month old with a strain isolated from Colombia, South America. Infection of these strains was by syringe passage of cattle blood containing trypanosomes because neither strain is infective to tsetse flies. Three infected calves (6, 7, 8) and 1 control (14) were treated with diminazene aceturate and 2 other infected calves (9, 10) with a nitroimidazole drug (“2190”) which was on test for Eli Lilly (Ross, Jamieson and McCowen, 1973). Two calves (14, 15) were used as uninfected controls, the first blood sample being taken at 2 months old. Sampling techniques. Blood samples were taken into a sterile syringe from the jugular vein before and at frequent intervals after infection. The serum was separated by centrifugation after allowing the clotted blood to stand at 4 “C. for 2 h., and stored at -20 “C. A daily estimate of the parasitaemia was made by examination of blood taken from an ear vein into a heparinized capillary tube. A drop was placed on a slide, a cover slip applied and the number of parasites in 30 fields ( x 10 eyepiece, x40 objective) counted. The results were expressed as log equivalent values (LEV) (Walker, 1968). Estimation of serum protein levels. Antisera specific to bovine p and y heavy chains were raised as described by Penhale and Christie (1969). Antiserum to bovine cl-chain was raised as described by Logan and Penhale ( 1972). Antiserum to a serum protein, provisionally designated 7Sy,, was prepared as described by Spooner, Penhale, Burridge and Brown (1973). This protein has been described as the X-component by Sullivan, Prendergast, Antunes, Silverstein and Tomasi (1969). IgM and IgG concentrations were determined on all samples by the single radial diffusion method (Fahey and McElvey, 1965) as described by Penhale and Christie (1969). IgA concentration was determined on a small number of samples and the concentration of the 7Sy, protein was assayed on many samples by a similar method. It was not possible to express the concentration of this protein in absolute values because it has not been purified and characterized. All values are given as a percentage of a standard serum which was obtained from a single animal, not involved in the present experiments, which had a high serum concentration of 7Sy, protein. Immuno-electrophoresis was performed on microscope slides using 1 per cent. ionagar (Oxoid) and barbiturate buffer (pH 8.6, 0.01 ionic concentration). Titration of natural antibodies. Natural agglutinating antibody levels to washed red blood cells of man, sheep, rabbit and fowl were measured by mixing 0.025 ml. of a 1 per cent. suspension of cells in phosphate buffered saline (pH 7.2) with 0.025 ml. of serum dilutions, in disposable perspex plates. Fractionation of sera. One ml. serum samples were dialyzed overnight against trisHCl buffer (pH 8.0) containing 1 M NaCl and subsequently passed through a column (30 cm. x 4.4 cm.2) containing Sephadex G200 (Pharmacia, Sweden) at a flow rate of approximately 5 ml./h. Effluent samples were collected in 1 ml. volumes and pooled into a series of fractions according to the protein separation profile at 280 nm. The fractions were concentrated to 1 ml. by ultrafiltration.

RESULTS

An outline of the experiment given in Table 1.

involving

13 infected

and

2 control

calves

Course of Infection Strain 64123 produced a high parasitaemia with frequent relapses. produced a rapidly developing anaemia with the haematocrit falling from

is

It the

T.

k&Y

INFECTION

IN CALVES:

SERUM

PROTEIN

399

CHANGES

preinfection value of about 38 per cent. to 15 per cent. by day 28 after infection. Loss in condition was severe and death occurred in 4 of 6 untreated animals from 24 to 122 days after infection. In animals treated with either drug the parasites were destroyed rapidly, but relapses occurred in 3 of 4 calves which were kept for over 10 days after treatment. The Desowitz strain was not as TABLE OUTLINE

Calf No. Breed and sex

1 2 3 4 5 6

Jersey Friesian Jersey Ayrshire Jersey Hereford

M. F. M. M. M. M.

7 Hereford

Age at infection (months)

OF INFECTIONS

Strain of T. vivax

: 4 3

64123 64123 64123 64!23 64123 64j23

M.

2

64123

8 Jersey

M.

2

64123

9

Hereford

M.

2

64123

10

Hereford

M.

2

64123

11 Jersey 12 Jersey 13 Ayrshire 14 Friesian

M. M. M. M.

15

M.

2 2 1 Uninfected old at start Uninfected old at start

Hereford

T = Tsetse,

:

S = Syringe,

Desowitz Desowitz Colombian control. 2 months of experiment control. 2 months of experiment

D = Died,

1 AND

THEIR

Method of infection

-

OUTCOME

Drug

treatment

None None None None None Diminazene aceturate day 20 Diminazene aceturate davs 34 and 47 Diminkene aceturate day 18 Lilly 2 190 davs 7. 21. 43 iill; 2 190 days 7, 22, 43, 44, 45, 46 None None None Diminazene aceturate days 40 and 54 None

outcome of infection (d4 D. D. D. D. K. K.

24 26 53 122 84 30

K. 100 K. 90 K. 88 K. 88 D. 33 K. 90 K. 154 K. 130

K = Killed.

pathogenic and produced a relapsing infection with distinct peaks of parasitaemia. The haematocrit did not fall below 25 per cent. and there was only slight loss in condition. Although one of the 2 calves died this was surprising as death did not occur in 6 other calves which had been infected for another purpose. The Colombian strain produced a chronic relapsing infection in which distinct peaks of parasitaemia occurred with often several days between these peaks when no parasites could be seen in the blood. The haematocrit fell to 23 per cent., loss in condition was severe, but death did not occur. Changes in Serum Proteins

The preinfection IgM concentrations in all the calves were between I and 2 mg./ml. In calf 15, in which IgM was measured 7 times over 40 days, the maximum value was 2-2 mg./ml. In control 14, which was examined 15 times over 130 days, all values were between 1-O and 2-l mg./ml. The preinfection IgG concentrations varied considerably with a mean of 13.3 mg./ml. (standard error (s.E.) l-27). Th e mean value of 24 samples from 14 and 15 over the

400

M.

J. CLARKSON

et d.

periods mentioned above was 13-9 mg./ml. (s.E. O-92). The mean value of the 7Sy, protein of the preinfection samples was 51.3 per cent. (s.E. 4.13) and of 18 determinations in the 2 controls was 66.2 (s.E. 4.08). The fluctuations in the 3 proteins in control 14 over 130 days are shown in Fig. 1. The changes in these serum proteins during trypanosomiasis depended on the course of infection and examples have been selected as illustrations.

0

I IO

I 20

I 30

I 40

I 50

I 60

I 70

I 80

I 90

I 100

I 110

I 120

I 130

0

-0

Time (days)

Fig.

1. Serum

protein

concentrations

in uninfected

control

calf 14. (0-O)

IgM;

(0-O)

IgG;(

C;- -2)

Calf 1 illustrates an acute infection, in which death occurred 24 days after infection with strain 64/23 (Fig. 2). P arasitaemia was high from day 6 to death. IgM concentration increased between days 7 and 13 and was 2 to 3 times the I 8

I

I

I

(a)

- PO

15 $ -10

1 ” P

-5 0

0

‘I:

0

5

IO

15

Days after

Fig. 2. Parasitaemia strain 64/23 Parasitaemia.

and serum protein of I: viuax i.v. (a)

changes (0-O)

20

infection

in calf 1, which died 24 days after infection with IgM; (0-O) IgG; (b) (O-O) 7Syl; (0-O)

7Sy,

T.

ViVaX

INFECTION

IN

CALVES:

SERUM

PROTEIN

401

CHANGES

normal value at death. No significant alterations occurred in IgG levels, but the 7Sy, concentration fell markedly at 7 days and was only 3.7 per cent. on day 19.

0

1 (b)

Days

Fig. 3. Parasitaemia strain 64/23

after

mfectmn

and serum protein changes in calf of T. viva i.v. Symbols as Fig. 2.

3, which

died

53 days

a ter infection

with

Figure 3 shows the changes which occurred in calf 3, an example of sub-acute infection, which died 53 days after infection with strain 64123. The parasitaemia was sustained at a high level, but at least 9 antigenically distinct populations appeared during the infection (Jones, personal communication). IgM rose to a maximum of 16 mg./ml. 25 to 30 days after infection and then fell until at death the concentration was under 4 mg./ml. IgG concentration rose slightly, but the levels were not significantly above normal. 7Sy, concentration was low before infection, but fell to less than 10 per cent. at the time that parasites appeared in the blood in large numbers. The fall was progressive except for occasional samples which showed a slight rise, possibly associated with falls in the parasitaemia which were noted concurrently. Calf 4, an example of chronic trypanosomiasis, died 122 days after infection with strain 64/23 (Fig. 4). After a high parasitaemia between days 10 and 30, infection was characterized by a relapsing parasitaemia. The IgM concentration rose to a maximum of almost 20 times the normal 45 days after infection and then fell up to day 80 rising again to a peak at 110 days. IgG concentration was not significantly raised until 65 days after infection after which its concentration rose steadily to a maximum of over 40 mg./ml. on day 110. 7Sy, concentration showed a fall, related to the appearance of parasites in the blood, on day 12 and remained low throughout infection.

402

et al.

M. J. CLARKSON 5c

I I I / I, (a)

, ( / , , , , , , / , ( ,

I , / I , r1

4c f E ‘, E

3c

cl 9

20

r s? IO

0 4 z -’ .o E = i! 2

(b)

3

2

I

0

Days after

infection

Fig. 4. Parasitaemia and serum protein changes in calf 4, which died strain 64123 of T. vivax through G. monitans. Symbols as Fig. 2.

122 days after

infection

with

”

0 4

0 (bl

60

IO

20

30

40

50

60

70

Days

Fig.

5. Parasitaemia and serum with a Colombian strain

80

after

90

100

110

I20

130

140

150

infection

protein changes in calf of T. z&ax i.v. Symbols

13, which was killed as Fig. 2.

154 days after

infection

;r,

ViVUX INFECTION

IN CALVES

: SERUM

PROTEIN

403

CHANGES

Calf 13, infected with the Colombian strain, also illustrates the changes associated with chronic trypanosomiasis, but the calf was recovering and improving in condition when it was killed on day 154 (Fig. 5). This recovery can be seen from the peaks of parasitaemia which became lower and less Treatment

-5

0

IO

20

30

40

60

50

70

Bo

90

lot

Days after infection Fig.

6. Parasitaemia and serum protein changes in calf 7, which was treated with diminazene 34 and 47 days after infection with strain 64123 of 1. vivax through G. morsitanr. Symbols

aceturate as Fig. 2.

frequent from day 75. IgM concentration rose to a maximum of only 9 mg./ml. and fluctuated, even falling to normal 30 days after infection. IgG levels were normal until day 76, after which they increased progressively to almost 40 mg./ml. 7Sy, concentration fell as soon as parasites appeared in the blood and remained low, with occasional peaks perhaps related to a fall in parasite numbers, until day 55 after which a slow steady rise occurred. 2

TABLE EFFECT

Maximum cont. (mdml.)

NO.

2 24.1 15.2 14.8

ii

1:

4.0 6.7

=

not done.

ON

IgM,

IgG

AND

W

kM

Calf

N.D.

OF TRYPANOSOMIASIS

7sY,

PROTEIN

Minimum cow as percent of standard

7SY,

Day

Maximum cont. (mdml.)

Day

21

19.0

25

23: ?i

20.1 14.0 29.0

30 50 i:

N.D. 18.0

19.1 8.0

13 35 50

12.0 20.4

887

N.D. N.D.

-


Day 15, 21, 25

404

M. J. CLARKSON

et

al.

Calf 7, infected with strain 64/23, illustrates the changes seen after unsuccessful treatment, which resulted in relapse (Fig. 6). The raised IgM level fell after treatment, but remained above normal. IgG concentration remained unchanged and the low 7Sy, level increased following treatment. Calf 8 was treated successfully on day 18 as daily blood examination showed no parasites until the calf was killed on day 90. IgM concentration was 8 mg./ml. on day 18, commenced to fall on day 24 until it was 0.9 mg./ml. on day 48 and then remained at normal levels. IgG concentration did not alter significantly. 7Sy, concentration was not measured. A summary of the changes in serum protein concentrations in the remaining calves is given in Table 2. The concentration of IgA was O-15 mg./ml. in the preinfection serum sample of calf 4, and ranged from O-1 1 to O-36 mg./ml. in 14 samples taken throughout infection. No other calf was examined.

Fig.

7. Representative immunoelectrophoresis of serial samples from calf 3, infected with strain 64/23 of T. vivux i.v. (a) Upper well, preinfection serum; middle well, 19 day serum; bottom well, 25 day serum. All 3 troughs contain rabbit anti-bovme whole serum. Anode at left. 1. IgM line, barely visible in preinfection sample (arrows). 2. 7Sy, line. (b) From left. Wells 1 to 6; preinfection, 19 day, 25 day, preinfection, 19 day, 25 day sera respectively. Troughs 1 to 3; rabbit anti-bovine 7Sy,. Troughs 4 to 6; rabbit anti-bovine IgM. Anode at top. Note progressive shortening and weakening of 7Sy, line‘and strengthening of IgM line.

7.

vivax INFECTION

IN CALVES:

SERUM

PROTEIN

CHANGES

405

Immunoelectrophoresis The increase in IgM and the fall in 7Sys protein is illustrated in Fig. 7, which shows the immunoelectrophoresis pattern of sera from calf 3 against rabbit anti-whole bovine serum and against antisera specific to individual serum components. The use of specific anti-IgM and anti-7Syl sera clearly demonstrates the striking changes occurring in the concentrations of these components as the infection progresses.

.Natural Antibodies Antibody to red blood cells from sheep were usually absent from preinfection sera and rose after infection to a maximum titre of 8. There were higher titres of antibody to the red cells from the other species both before and after infection with the highest titres occurring to chicken cells. Low titres to chicken cells, usually 2 to 8, occurred in preinfection sera and these rose after infection to maximum titres of 16 to 128 in different calves. These maximum titres occurred 30 to 60 days after infection and then usually remained at the same titre throughout infection. Titres to rabbit and human cells were about the same, intermediate between those to chicken and to sheep cells. The control calves showed titres of between 0 and 16 in the 22 samples tested.

(b)

Fig.

8. Sephadex G200 fractionation of serum from calf 4 which was infected with strain 64/23 of ‘I. uivux through G. morsituns. (a) and (c) are protein elution profiles (O.D. 280 nm) of preinfection and 41 day sera respectively, whilst (b) and (d) show the IgM content of the eluted fractions.

Fractionation of Serum from an Infected Calf on Sephadex GZOO Fractionation was carried out on the preinfection serum from calf 4 and on the sample taken 41 days after infection when the IgM concentration was 30 mg./ml. (Fig. 8). Th ere was a noticeable difference in the protein elution profile (280 nm) in that the first elution peak containing the larger molecular material ( 19s) was much greater in height in the 41 day sample. When the E

406

M.J.

CLARKSON

et

al.

individual fractions were tested for IgM content it was found that peak concentrations of IgM eluted at the same volume in both samples, indicating that no change in molecular size had occurred. DISCUSSION

The most pronounced changes which occurred in the serum proteins of calves infected with I: vivax was an immediate rise in IgM concentration and a fall in the 7Sy, concentration. There was also evidence of increased IgG concentration late in infection. The extent of the increase in IgM varied considerably between animals, but the rise normally commenced about 10 days after infection and increased progressively until days 30 to 40. IgM levels then usually fell, but remained high for many months in untreated infections. Occasionally there was a fall to normal levels (13, Fig. 5) followed by a further increase. Luckins (1972) f ound an increase in IgM in Zebu cattle kept under conditions of field exposure to trypanosomiasis and in experimental infections of Zebu cattle (Luckins, 1974). These Zebu cattle had probably been exposed to other protozoa such as Babesia and Anaplasma, of which it is known that the latter causes increase in IgM (Klaus and Jones, 1968). Luckins ( 1974) treated all his animals 50 days after infection and all except one animal produced similar changes. The present studies extend the observations over a longer period and indicate the considerable variation which can occur, at least in young animals. Increased IgM concentration has been used as a screening test in surveys for trypanosomiasis in man (Binz, Timperman and Hutchinson, 1968) and these studies indicate that trypanosomiasis in cattle is likely to be associated with increased IgM concentration. However, since other infections may cause increased IgM levels, and there are occasions when an infected animal may show a normal or only slightly raised level, it appears that IgM levels cannot be used as a certain specific diagnostic method for bovine trypanosomiasis. IgM concentration fell after treatment and when this was successful normal levels were attained after about 4 weeks. On the other hand, Luckins (1974) found that abnormal levels were still present 50 days after treatment. The high IgM concentration may be of significance in the pathogenesis of trypanosomiasis and could be the cause of the increased viscosity of blood which occurs and may lead to circulatory disturbancies (Boreham and Facer, 1975). Immune complexes may be produced and could be responsible for the haemolytic anaemia which occurs in bovine trypanosomiasis (Holmes and Mamo, 1975) or give rise to kinin production (Boreham, 1968). It also seems highly likely that the increased IgM is associated with the immunodepression which occurs in trypanosomiasis (Goodwin, Green, Guy and Voller, 1972). Only 2 calves (4 and 13) showed a significant rise in IgG concentration, from 65 and 90 days after infection respectively. This indicates that the normal sudden change from IgM to IgG production did not occur in the calves until late in infection. Treatment prevented this late rise in IgG and Luckins (1974) also found no increase in IgG in his animals after treatment.

7: vivax INFECTION IN CALVES: SERUM PROTEIN CHANGES

407

The present studies give no information about the functional significance of IgM and IgG in cattle trypanosomiasis. Seed et al. (1969), working with T. gambiense in rabbits, showed that agglutinating antibody first appeared in the IgM fraction and later in both IgM and IgG. Takayanagi and Enriquez (1973) showed that IgM was more effective than IgG in producing a variant population and in the passive transfer of immunity. Studies on the possible separation of functions by IgM and IgG are needed in cattle. These studies shed only a little light on the mechanisms behind the increased IgM concentration. The rise may be caused by the repeated stimulus of antigenic variants which appear every 2 to 5 days (Jones and Clarkson, 1974), 9 of which were isolated from calf 3 (Jones, personal communication). Seed et al. (1969) suggested that the raised IgM level in rabbits infected with r. gambiense results, at least in part, from the continuous synthesis of new specific antibody to each antigenic relapse. Antibodies to antigens other than those present in the organism such as heterophile and auto-antigens have been found in man and animals infected with trypanosomes (Houba and Allison, 1966; MacKenzie, Boreham and Facer, 1973; MacKenzie and Boreham, 1974) and it has been suggested that trypanosomes may contain a non-specific B cell mitogen (Greenwood, 1974) which may initiate immunoglobulin synthesis in immunocompetent B cells of various specificities, Natural agglutinins to red blood cells were detected in low titres in the infected calves and it is probable that the synthesis of other nontrypanosome antibodies is induced as MacKenzie et al. (1973) found anti-liver, anti-Wasserman and anti-fibrinogen antibodies in cattle infected with 7: congolense and T. brucei. In this study it was not possible to carry out absorption studies with all the antigenic variants of T. vivax which occurred in the calves to determine what proportion of the IgM was specific antibody. The increased IgM concentration was not associated with an immediate rise in IgG; this finding supports the suggestion of Terry, Freeman, Hudson and Longstaffe (1973) that trypanosomes effect T-B cell interaction, an important factor in the sudden change from IgM to IgG production. On the other hand, it is possible that the major antigens of these organisms are of the thymus independent type which do not stimulate the production of antibody within the IgG class. There was no indication of production of low molecular weight IgM as described in man and rabbits infected with trypanosomes by Masseyeff, Blonde1 and Mattern (1971) and Frommel, Perey, Masseyeff and Good (1970) which would invalidate the radial diffusion assay. Macrophages are also involved in T-B cell cooperation (Feldman, 1972) and alterations in macrophage function may occur in trypanosomiasis (Longstaffe, 1974, 1975). Further studies are needed on these aspects of the problem in cattle. The fall which occurred in the 7Sy, protein was most striking and appeared to be related to the presence of parasites in the blood. This may be of significance in the pathogenesis of trypanosomiasis but, unfortunately, the function of this protein is not yet known. Sullivan et al. (1969) described it as the “Xcomponent”, stated that it was not an immunoglobulin or transferrin, and showed that it was present in colostrum and saliva and in the serum of newborn unsuckled calves. We originally thought that it was a component of complement

408

M.

J. CLARKSON

et

al.

which was fixed in trypanosome antigen-antibody reactions (Clarkson and Penhale, 1973) and therefore removed from the circulation, but recent studies have not confirmed this assumption (Penhale, unpublished). A similar fall in this 7Sy, protein occurs in acute East Coast fever in cattle caused by Theileria parua (Spooner et al., 1973). Further studies on the characterization and function of this protein are in progress and it would be of interest to investigate the fluctuations in concentration of this component in field cases of trypanosomiasis in cattle and man. SUMMARY

Thirteen calves were each infected with one of 3 strains of TTypanosoma uivax by syringe passage of blood or by cyclical transmission with Glossina morsitans. The concentrations of IgM, IgG and a 7Sy, serum protein were measured before and at short intervals after infection. Infection was characterized by a marked rise in IgM concentration commencing about 10 days and a rise in IgG concentration commencing about 65 days after infection. IgM levels usually remained high in untreated infections, but occasionally fell to normal indicating that raised IgM was not completely reliable as a diagnostic method. Successful treatment resulted in a fall in IgM concentration to normal in about 4 weeks and IgG concentration did not rise. There was no indication of low molecular weight IgM in one calf whose IgM concentration was very high. There was a striking fall in the concentration of the 7Sy, protein which then fluctuated, perhaps related to parasitaemia, and was very low in calves which died. Natural agglutinins to foreign red cells, especially chicken cells, rose in infection. ACKNOWLEDGMENTS

We are grateful to Mr T. J. Cullingham and his staff for their care of the calves and to Mrs B. A. Cottrell and Mr G. C. Edwards for assistance with the natural agglutinin determinations. We wish to thank Miss P. J. Farrell for assistancewith the preparation of the manuscript and the Ministry of Overseas Development for financial support. REFERENCES

Binz, G., Timperman, G., and Hutchinson, M. P. (1968). Estimation of serum immunoglobulin M as a screening technique for Trypanosomiasis. World Health Organization,

38, 523-545.

Boreham, P. F. L. (1968). Immune reactions and kinin formation in chronic trypanosomiasis. British Journal of Pharmacology and Chemotherapy, 32, 493-504. Boreham, P. F. L., and Facer, C. A. (1975). Blood viscosity changes in rabbits infected with Trypanosoma brucei. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69 (in press). Clarkson, M. J., and Penhale, W. J. (1973). Serum protein changes in trypanosomiasisin cattle. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67, 273. Fahey, J. L., and McKelvey, E. M. (1965). Q uantitative determination of serum immunoglobulins in antibody-agar plates. Journal of Immunology, 94, 84-90.

T.

Z&X

INFECTION

IN

CALVES:

SERUM

PROTEIN

CHANGES

409

Feldmann, M. (1972). Cell interactions in the immune response in vitro V. Specific collaboration via complexes of antigen and thymus-derived cell immunoglobulin. Journal of Experimental Medicine, 136, 737-760. Frommel, D., Perey, D. Y. E., Masseyeff, R., and Good, R. A. ( 1970). Low molecular weight serum immunoglobulin M in experimental trypanosomiasis. Nature, 228,

1208-1210. Goodwin, L. G., Green, D. G., Guy, suppression during trypanosomiasis.

M. W., and Voller, A. (1972). ImmunoBritish Journal of Experimental Pathology, 53,

40-43. Greenwood, B. M. (1974). Possible role of a B-cell mitogen in hypergammaglobulinaemia in malaria and trypanosomiasis. Lance& i, 435-436. Holmes, P. H., and Mamo, E. (1975). Factors influencing the development of trypanosomal anaemia in Zebu cattle. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69 (in press). Houba, V., and Allison, C. A. (1966). M-antiglobulins (rheumatoid-factor-like globulins) and other gammaglobulins in relation to tropical parasitic infections. Lancet, i, 848-852. Jones, T. W., and Clarkson, M. J. (1974). The timing of antigenic variation in Tvpanosoma viuax. Annals of T’rofiicalMedicine and Parasitology, 68, 485-486. Klaus, G. G. B., and Jones, E. W. (1968). The immunoglobulin response in intact and splenectomized calves infected with Anaplasma marginale. Journal of Immunology, 100, 991-999. of the calf to Logan, E. F., and Penhale, W. J. (1972). St u d ies on the immunity colibacillosis. V. The experimental reproduction of enteric colibacillosis. Veterinary Record, 91, 419-423. Longstaffe, J. A. (1974). Immunodepression in trypanosomiasis: attempts to characterize the response of thymus-dependent lymphocytes in infected guinea-pigs. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 150. Longstaffe, J. A. (1975). Further studies on the effect of trypanosome infections on the macrophages of the mouse spleen. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69 (in press). Luckins, A. G. (1972). Studies on bovine trypanosomiasis. Serum immunoglobulin levels in Zebu cattle exposed to natural infection in East Africa. British Veterinary Journal, 128, 523-528. Luckins, A. G. (1974). Immunoglobulin response in Zebu cattle infected with Trypanosoma congolense and 1. vivax. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 148-149. MacKenzie, A. R., and Boreham, P. F. L. (1974). Autoimmunity in trypanosome infections I. Tissue autoantibodies in Trypanosoma (Trypanozoon) brucei infections of the rabbit. Immunology, 26, 1225-1238. MacKenzie, A. R., Boreham, P. F. L., and Facer, C. A. (1973). Autoantibodies in African trypanosomiases. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67, 268. Masseyeff, R., Blondel, J., and Mattern, P. (1971). Low molecular weight IgM in the serum and cerebrospinal fluid of patients infected with Trypanosoma gambiense. ~eitschrifi fur Immunitatsforchung, 143, 291-303. Mattern, P. (1964). Technique et interet epidemiologique du diagnostic de la trypanosomiase humaine africaine par la recherche de la P2-macroglobuline dans Ie sang et dans le L.C.R. Annates de 1’Institut Pasteur, 107, 415421, Penhale, W. J., and Christie, G. (1969). Quantitative studies on bovine immunoglobulins I. Adult plasma and colostrum levels. Research in Veterinary Science, 16, 493-501. Ross, W. J., Jamieson, W. B., and McCowen, M. C. (1973). Antiparasitic nitroimidazoles. 3. Synthesis of 2-(4-carboxystyryl)-5-nitro-I-vinylimidazole and related compounds. Journal of Medicinal Chemistry, 16, 347-352. Seed, J. R., Cornille, R. L., Risby, E. L., and Gam, A. A. (1969). The presence of

410

M.

J. CLARKSON

et

cd.

agglutinating antibody in the IgM immunoglobulin fraction of rabbit antiserum during experimental African trypanosomiasis. Parasitology, 59, 283-292. Spooner, R. L., Penhale, W. J., Burridge, M. J., and Brown, C. G. D. (1973). Some serum globulin changes in East Coast Fever. Research in Veterinary Science, 15, 368-374. Sullivan, A. L., Prendergast, R. A., Antunes, L. J., Silverstein, A. M., and Tomasi, T. B. Jr. (1969). Characterization of the serum and secretory immune systems of the cow and sheep. Journal of Immunology, 103, 334-344. Takayanagi, T., and Enriquez, G. L. (1973). Effects of the IgG and IgM immunoglobulins in Trypanosoma gambiense infections in mice. Journal of Parasitology, 59, 644-647. Terry, R. J., Freeman, J., Hudson, K. M., and Longstaffe, J. A. (1973). Immunoglobulin M production and immunosuppression in trypanosomiasis: A linking hypothesis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67,263. Walker, I’. J. (1968). Standardization of routine observation of blood for trypanosomes. East African Trypanosomiasis Research Organization Report, pp. 15-16. [Received for publication,

November 28th, 19741