Changes in fibrinogen levels, platelet counts, clotting times and fibrinolytic activity in relation to thrombosis in contagious bovine pleuropneumonia

J. COUP.

PATH.

1973.

VOL.

583

85.

CHANGES IN FIBRINOGEN LEVELS, PLATELET COUNTS, CLOTTING TIMES AND FIBRINOLYTIC ACTIVITY IN RELATION TO THROMBOSIS CONTAGIOUS BOVINE PLEUROPNEUMONIA

IN

By L. C.

LLOYD,

D.

T.

M;.

PIERCY*

and J. B.

BINGLEY~

CSIRO Division of Animal Health, Animal Health Research Laboratory, Private Bag No. 1, P.O., Parkuille, Victoria, 3052. Australia INTRODUCTION

Thrombosis of blood vessels in lung lesions occurs in most, if not all, cases of contagious bovine pleuropneumonia (CBPP) (Woodhead, 1888; Turner, 1959; Bygrave, Moulton and Shifrine, 1968; Lloyd and Trethewie, 1970). Necrosis occurs when arteries are affected (Turner, 1959) and this tissue, when encapsulated, can harbour the causative organism Mycoplasma mycoides subsp. mycoides (hereafter referred to as Mycoplasma mycoides) for long periods (Turner, 1959). The stage of the disease when thrombosis occurs is not known. The histopathological appearance of early lesions is consistent with pulmonary oedema (Bygrave et al., 1968; Lloyd and Trethewie, 1970) arising from thrombosis of blood vessels and an increase in the resistance to blood flow. If it is a primary change and develops before the exudation, pneumonia and lymphatic involvement of the typical lesion then the infection with M. mycoides might be responsible for changes in blood platelets or in coagulation factors which would predispose to thrombosis. Since the thrombosis is confined to the lungs there must be local changes in that site to account for this: kidneys show similar lesions though these may be caused by emboli. An alternative explanation is that the thrombosis occurs because the vessel walls are involved in the inflammatory process; it would then be a secondary change. Piercy and Bingley (1972) showed that blood fibrinogen levels increased after killed M. mycoides organisms were injected into the joints of calves. Although a rise in fibrinogen occurs with some infections in cattle (McSherry, Horney and de Groot, 1970) there was a possibility that the increase caused by the dead organisms was a particular characteristic of M. mycoides and perhaps associated with thrombosis. An experiment was carried out to test whether fibrinogen levels, blood clotting factors, platelet numbers and the levels of other blood components were altered in animals with CBPP in such a way as to predispose to thrombosis. In part of the experiment the level of fibrin degradation products was measured to indicate when deposits of fibrin were being broken down. MATERIALS

AND

METHODS

Animals. The cattle were adult females of either Jersey or Friesian breed weighing between 306 and 404 kg. They were accommodated in pairs in isolation pens and were brought in at least 2 days before the experiment commenced. Experimental infection. Eight animals were infected by intubation using the method described by Hudson (1971) except that the inoculum was a 48 h culture of the * Present t Present

address: address:

Wellcome Veterinary C/o UNDP-FAO-INTA,

Research Laboratories, C.C. 276, Balcarce,

Berkbamstead Argentina.

Hill,

Herts,

England.

584

L. c. LLOYDet al.

Gladysdale strain of M. mycoides in BVFOS broth (Turner, Campbell and Dick, 1935). One animal developed an unrelated enteric disease during the experiment and died; the data from this animal were not used. Four sham-operated control animals received sterile BVFOS broth only, by the same procedure. The temperatures of the animals were taken and recorded daily for the period from 2 days prior to, until 28 days after, intubation. Sampling procedure. Blood samples were collected daily commencing 2 days prior to intubation and then, on the day the cattle were intubated (day 0), on day 1 to day 3, on day 5 to day 10 and then on days 12, 14, 16, 19, 21, 23, 26 and 28 or until the animal died. Samples were taken at the same time each day. Nine samples were taken in the same order from each animal at each sampling. These included 2 titrated samples, taken by syringe for the thrombin clotting time (TCT) and the prothrombin time (PT) tests and a sample for fibrin degradation products estimation, also taken by syringe, into a tube containing epsilon amino caproic acid (EACA) (Ekaprol, Difrex (Aust.) Lab. Sydney, Australia) to inhibit further fibrinolysis; the latter from 2 control and 2 infected animals only. Other samples, collected from a freely flowing stream of blood, were (a) into a tube containing EDTA for the kaolin partial thromboplastin time (KPTT) test and platelet count, (b) 2 into Wintrobe tubes for fibrinogen, haemoglobin, plasma protein estimations and packed cell volume, (c) one in BVFOS broth to culture for M. mycoides (2 infected animals only), (d) one in a tube containing oxalate for the slide agglutination blood test and (e) a clotted sample for the complement fixation test. Analytical procedures. The following blood components were measured by published procedures, or as recommended by the reagent manufacturers; packed cell volume (microhaematocrit method, Schalm, 1965) ; haemoglobin (cyanhaemoglobin method at 1:251 dilution, Stigbrand, 1967); fibrinogen (Piercy and Bingley, 1972); total protein (Lowry, Rosebrough, Farr and Randall, 1951); platelet counts (Dacie and Lewis, 1968) ; prothrombin time (Owren and Aas, 195 1, with modifications suggested by Dade Division, American Hospital Supply Corp., Miami, Florida); thrombin clotting time and kaolin partial thromboplastin time (manufacturer, Behringwerke AG, Marburg Lahn, W. Germany). Fibrin degradation products were estimated by the tanned red-cell haemagglutination inhibition immunoassay (TRCHII) described for man by Merskey, Lalezari and Johnson (1969) and modified by us for cattle. Bovine red cells were collected into an equal volume of modified Alsever’s solution (Bukantz, Rein and Kent, 1946), dispensed into 100 ml. amounts and stored at 4 “C. for up to 1 month. Normal bovine serum was obtained by collecting 10 ml. of blood in a tube containing 0.3 ml. of EACA, adding thrombin at the rate of 1 unit per ml., taking the serum off the clot and storing it at 4 “C. Normal bovine plasma was obtained by collecting blood into sodium citrate and EDTA, centrifuging, taking off the plasma and storing it at 4 “C. Lyophilized aged bovine serum was prepared by adding sodium azide to normal bovine serum and then “ageing” it at room temperature (approximately 22 “C.) for 24 h. It was then lyophilized and stored in an airtight container. Antifibrinogen antiserum was prepared by the method of Merskey, Kleiner and Johnson (1966) except that bovine fibrinogen (Commonwealth Serum Laboratories, Melbourne) was used. The methods of Merskey et al. (1969), with minor modifications, were used to determine the concentration of the antiserum, for sensitizing the red blood cells and for titrating the fibrin degradation products. In the test the antiserum was used at a concentration of 1 in 2000 and it was carried out in Cooke roundbottomed plastic microtiter plates with microdiluter loops. The last well to show inhibition of agglutination with normal bovine plasma was that containing a dilution of 1 in 3200. The fibrinogen level of the normal bovine plasma was 429 mg./ml. so that dilution of plasma contained l-3 l.tg./ml. of fibrinogen. Thus, if a test sample diluted 1 in 32 caused a similar amount of inhibition then the sample would contain 32 ?< 1.3 or 41.6 pg./ml. and so on through the series. Blood samples were tested for antibody to M. mycoides by the slide agglutination

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blood test (SABT) (T urner and Etheridge, 1963). Complement fixing (CF) antibody was determined by the method of Campbell and Turner (1953) modified by Etheridge and Lloyd ( 1968). Mycoplasmaemia was detected by inoculating 10 ml. of blood into 90 ml. of BVFOS broth containing penicillin and thallium acetate (BVF-OS-PTA) (Turner and Trethewie, 1961). These cultures were then incubated at 37 “C. and examined under dark-ground illumination each day for 7 days or for a shorter time if characteristic filaments appeared. .Necropsy procedure. The experiment was terminated 26 to 28 days after intubation when the cattle remaining in the group that had been infected with M. mycoides were killed. Necropsieswere carried out on these and also on those that died prior to 28 days. The lungs, in particular, were examined and all lesions of CBPP were measured, samplestaken for culture for M. mycoides into BVFOS-PTA broth (Turner and Trethewie, 1961) and sown onto blood plates to determine whether other organisms were present. Specimens for histological examination were fixed in 10 per cent. buffered formalin and in Zenker acetic. Sections cut after paraffin embedding were stained with haematoxylin and eosin (HE) and phosphotungstic acid haematoxylin (PTAH). Thrombi were classified according to whether they were in arteries or veins by the location of elastic laminae in sections stained with Verhoeff’s strain. The severity of the diseasein each animal was scored according to the system described by Hudson and Turner (1963). Control cattle were killed during the 3 months period after the infected cattle were killed and necropsieswere carried out. They were blood tested regularly between the termination of the experiment and necropsy. RESULTS

The reaction of the cattle to the infective inoculum was typical. All showed serological reactions to both the SABT and the CF test by the 8th day (Table 1). Six of the 7 had febrile reactions and these were first seen on the 3rd day ( 1 animal), 8th day (3 animals) and 9th day (2 animals). Organisms were recovered from the blood on day 2 from one and day 3 from the 2nd of the 2 TABLE EFFECT

K44 K45 K47 K48 K49 K50 K59 * KPTT

OF EXPERIMENTAL

6

6 6

5

7 10

: 7 8 7

: 6 kaolin

9

!i

:

17

C.B.P.P. IN CATTLE-CLINICAL, HAEMATOLOGICAL ASPECTS

19 26 28 28

x11:; ill 6 partial

18

1

22

thromboplastin

Necrosis ::3,

Hyperacute Acute >,

::

Hypeyacute Acute

,,

Hyperacute

time.

PATHOLOGICAL

1025112 1415/14 1520/16 1455/12 1455112 775114 1680/12

AND

1*7/14 I.912 2.413 2.713 2.3116 3.112 3.913

SELECTED

5.212 5.9121 12.4126 6.5123 6.8/12 1 8.512 9.3/19

6019 54112 56/10 51/10 69/10 5412 5919

L. c. LLOYD et al.

586

Fig.

1 . Thrombus in a vein HE. x 110.

in a focus

adjacent

to this focus.

that were sampled for this purpose. Two animals died, one was killed and another was severely affected but survived to the end of the affected. At necropsy the the other 3 animals were moderately forrr led to the description given by Hudson (1971). In 3 animals

in extl -emis experin lent; lesions con(K44, K49

Fig. 2 . Thrombus infiltrated

fibrocyte!

in an artery the thrombus.

of early

organization

where the lumen HE. x 450.

is almost

and in an artery

completely

obliterated;

s have

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BOVINE

587

PLEUROPNEUMONIA-THROMBOSIS

and K59) the disease was hyperacute; varying degrees.

the others were acutely affected, but to

Lesions were confirmed by histopathological examination and thrombosis was observed in lung sections from all affected animals (Figs 1, 2 and 3). There was necrosis in some parts of the lung lesion in each animal indicating that the blood supply to that area had been interrupted and this was compatible with thrombosis. Of 59 thrombi in HE stained sections of lung from 3 cattle, 30 were in arteries (Figs 1 and 2) and 18 in veins (Fig. I), as determined by the

Fig. 3. Artery with two elastic laminae, left, and vein with one, lower occupies much of the field. VerhoeX’s stain. x 260.

right;

a degenerating

bronchiole

position of elastic laminae in vessels in Verhoeff stained slides (Fig. 3) ; there were less in the Verhoeff stained slides because thrombi were sometimes not visible in adjacent sections. The controls showed no evidence of CBPP during the experiment, on repeated testing after the experiment or at necropsy. Packed Cell Volume and Haemoglobin Estimation Analyses of variance of PCV values and haemoglobin 9, 11 and 12 showed there was no significant differences and control groups for either parameter.

levels on days 0, 3, 6, between the infected

Fibrinogen The mean level of fibrinogen in the 4 controls over the 18-day period before deaths began to occur in the infected cattle was 612.4 mg./lOO ml. From the 7th day after inoculation, a significant difference (P < O-05) existed between the fibrinogen levels for the control and infected animals after log,, transformation of the results. The variation due to time and time/treatment interaction was very highly significant (P < 0.001). The mean values for control and infected animals are given in Fig. 4. Some infected animals showed a

588

L. C. LLOYD

et Ul.

marked rise in fibrinogen levels and in one case this reached a value of 1680 mg./lOO ml.; in 3 the levels rose to two and a half times the levels at the beginning of the experiment. 1600 -T E

. :. : 1 I : .:’ . . . . _,.. . . . ... II i -1 / ER ---&-J-&) M1 1.1:

1400

8 T E c

I200

. .. .

....

3 .c L e

1000

j

800

E E %

600

400 0

L

,

I

I

I

I

I

I

I

I

I

-2

0

2

4

6

8

IO

12

14

16

Days

Fig. 4. Changes uninfected Control.

0

in fibrinogen levels in 7 cattle controls. Mean and standard

from

inoculation

developing artificially induced deviation are given. (0 . . . 0)

C.B.P.P. C.B.P.P.;

and in 4 (O-O)

Total Serum Protein

There was little change in the serum protein levels except that in the hyperacute cases that died early (K44 and K49) the level began to fall about 6 days before death. Platelet Counts

Platelet counts showed considerable variation. The mean for 3 of the 4 controls for all samples taken over a 37-day period, 8 days prior to and 28 days during the experiment, was 4.23 xlOs with a standard deviation of 3.27 x IO5 per cmm. The count in the 4th animal (K60) fell to O-8 ~10~ per cmm. before the experiment began and rose to 7.3 x lo6 per cmm. during the experiment. Least significant difference analysis indicated a time effect in that there were significant differences (P < 0.05) between the mean platelet counts of the infected animals at day 0 and days 2, 3 and 5 and between the means at days 1 and 3. The difference between the means of the counts of the 4 control and 7 infected animals at any time from day 0 to day 16 was not significant by the usual criteria (P < 0.1) ; animals died after this period and there were insufficient data to continue the analysis. The means of counts of the control and infected animals are given in Fig. 5. There was a notable rise in the platelet count of 3 infected cattle after the period covered by the analysis. This commenced at about 8 days and exceeded levels of 8.5 x lo5 cmm. on days 16, 19 and 21.

CONTAGIOUS

a: -2

BOVINE

0

2

4

6 Days

Fig.

8

from

IO

Thromboplastin

I2

14

I6

inoculation

5. Changes in platelet counts in 7 cattle developing artificially infected controls. Mean andstandard deviation are given. (0.

Kaolin Partial

589

PLEUROPNEUMONIA-THROMBOSIS

induced C.B.P.P. and . -0) C.B.P.P.; (0-O)

in 4 unControl.

Time

The mean value for the KPTT in the 4 controls over a period of 19 days, which included 17 days of the experiment, was 45.3 with a standard deviation of 2.6 s. The mean values for the 7 infected animals were significantly higher

1

0’

1 -2

t 0

I 2

I 4 Days

Fig. 6. Changes C.B.P.P. C.B.P.P.;

in the kaolin partial and in 4 uninfected (e-0) Control.

I 6

1 8

from

inoculation

, IO

I 12

I 14

thromboplastin time in 7 cattle developing controls. Mean and standard deviation

I 16

artificially are given.

I

induced (0.. .a)

L. c. LLOYD et al.

590

(P < 0.05) than those of the 4 controls from day 8 on. The rise reached a peak at day 10 then tapered off until day 16 when the period under review ceased. The mean values for the infected and control groups are given in Fig. 6. Thrombin Clotting Time The mean value for the controls over a period of 31 days was 12.15 s. with a range from 10.0 to 14.5 s. There was no significant difference between the control- and infected cattle nor between severely and mildly affected infected cattle. Prothrombin

Time

The mean value for the controls over a period of 31 days was 11.75 s. with a range from 11.0 to 12.5 s. There was no significant difference between these and the infected experimental cattle nor between the severely and mildly affected animals. Fibrin .Degradation Products The mean value for the amount of fibrin degradation products in 2 controls over a 22-day period, which included 19 days during the time of the experiment, was 5.1 with a standard deviation of 2.1 pg./ml. From day 9 there was a I 7 2 2 i In 5 z h 6 .z i & $ .s b.-

I

I

I

I

I

I

I

I

I

I

I

70 . ,. 60-

.

. .

: ,:.

50-

:

.’

:

:. ).

40-

.

. ..’

30i ZO-

Days

from

inoculation

Fig. 7. Changes in fibrin degradation products in 2 cattle developing artificially in 2 uninfected controls. ( l . . .@) C.B.P.P.; (0-O) Control.

induced

C.B.P.P.

and

significant difference (P < 0.05) between the mean levels of fibrin degradation products in the infected and control groups. The mean values for the groups are given in Fig. 7. DISCUSSION

The method used to induce pleuropneumonia in this experiment produces lesions which are similar to those in the natural disease (Campbell, 1938). There are major differences in the time of incubation; in the intubated animals

CONTAGIOUS

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PLEUROPNEUMONIA-THROMBOSIS

591

this is about 7 to 8 days whereas in the natural disease cases can occur between 29 and 64 days after exposure and sometimes longer (Turner, 1959). Lloyd and Trethewie (1970) reported that lesions were present when the CF test was positive. In the present experiment 2 of the 7 gave a positive CF reaction on the 6th day, 4 on the 7th and one on the 8th so it can be assumed that 6 of the 7 had lesions on the 7th day. The subsequent post-mortem examinations did not contradict this view although these were carried out up to 3 weeks later. There were no significant changes in the packed cell volume or in haemoglobin levels in the diseased animals. Total serum protein levels were similar except in the severely affected cattle where a fall preceded death. The levels of circulating fibrinogen in the experimental cattle though high, were not unusual for this species (McSherry et al., 1970; Ek, 1972). That there were reduced levels of circulating platelets in the infected animals between day 2 and day 5, indicating that platelets were being consumed, is confirmed by the subsequent large increases in the counts of those severely affected cattle that survived. This rise, in terms of the explanation offered by Ebbe (1971), was apparently due to megakaryocytopoesis and increased production of platelets which was stimulated by a low level of circulating platelets, thrombocytopenia, and superimposed on it was a non-specific thrombocytosis occurring in response to the tissue damage. There were no corresponding rises in the controls. Because of variability the counts of the infected animals at the time of the fall were not significantly different from those of the controls; the fall was confirmed by the compensatory rise. The mean value for the KPTT test in the controls was comparable to that reported by Osbaldiston, Stowe and Griffith (1970). The rise in the values of the infected animals suggested that any of factors, I, II, V, VIII, IX, X, XI and XII (Denson, 1966) could have been either depleted, perhaps when thrombosis took place, or inhibited when fibrin degradation products increased; the second aspect is discussed later. The mean prothrombin time of 11.75 s. for the controls was less than the 17.8 s. reported by Didisheim, Hattori and Lewis (1959) and the 14.3 s. obtained by Osbaldiston et al. (1970). The similar values of the control and infected cattle indicated the extrinsic clotting system factors, II, V, VII and X (Macfarlane, 1968) were normal. The mean thrombin clotting time was 12.15 s. for the controls which was not significantly different from that of the infected animals. This normal value is not compatible with extensive intravascular coagulation since in that condition the level of fibrin degradation products is high and some of these are antithrombins. The increase in the KPTT times suggests that factors in that system, in particular those outside the scope of the PT test which was normal here, were depleted. Thus factors VIII, IX, XI and XII could have been involved, but not factor I, fibrinogen, of which there were high levels. There was a significant increase in the quantity of fibrin degradation products in the infected cattle from day 9. There is strong evidence that the fibrinolytic process in cattle may differ from that in other species. Hawkey (1970) reported that plasmin was absent from cattle plasma and fibrinolytic activity could not be detected in lung tissue (Astrup, 1956) nor in blood vessel walls (Astrup and

592

L. C. LLOYD et d.

Buluk, 1963) of cattle. Further, Gallimore and Shaw (1969) have shown that increased levels of fibrinogen slowed the lysis time of clots in vitro and Hickman, Gordon-Smith, Whitfield and Godfrey (1973) have suggested that this mechanism might operate in vivo and inhibit fibrinolysis. Thus the advent of fibrin degradation products need not indicate the time of thrombus formation and therefore the rise at 9 days does not contradict our view that deposition of both platelets and fibrin first occurred between 2 and 5 days after inoculation. Disseminated intravascular coagulation is seen in association with the Schwartzman reaction, injection of endotoxin, injection of thrombin, shock caused by reduction in blood volume, some generalized viral and bacterial infections and a variety of other clinical conditions (McKay, 1965). In extensive intravascular coagulation there is consumption of coagulation factors shown by increased prothrombin and partial thromboplastin times (Quick, 1966) and a fall in fibrinogen level (McKay and Shapiro, 1958; Rapaport, 1959). In this study only the KPTT was altered which suggeststhat there was only moderate or localized consumption of factors and as mentioned above, the normal TCT could not have occurred with extensive vascular involvement. It can be concluded that the lung thrombosis was not part of a disseminated intravascular coagulation. The questions posed, whether thrombosis preceded the other histopathological changes such as exudation, pneumonia and lymphatic involvement, or was caused by them and whether the blood was in a hypercoagulable state at this time of thrombosis, were only answered in part. There was evidence that thrombosis occurred before the other histological changes. Hypercoagulability could not be demonstrated; the consumption of factors from the intrinsic clotting system before the 8th day and the fall in platelet counts between the 2nd and 5th day support the histological evidence for intravascular clotting but, on the results presented, thrombosis could not be attributed to increased blood coagulability. SUMMARY

Changes in the packed cell volume, haemoglobin, fibrinogen and total protein levels, in the prothrombin, thrombin clotting and kaolin partial thromboplastin times, platelet numbers and fibrin degradation products were investigated in 7 cattle in which contagious bovine pleuropneumonia (CBPP) was induced by inoculating Mycoplasma mycoidesby the endobronchial intubation method. Thrombosis of lung vessels was confirmed in all animals at necropsy. The results of the complement fixation and slide agglutination blood tests indicated that typical lesions of CBPP were present at 7 days. There was a rise in fibrinogen levels soon after inoculation and the difference between the means of the levels of the infected and control animals was significant from the 7th day. Platelet counts in the infected animals were reduced on the 2nd to the 5th day and, although a significant difference between these and the controls could not be demonstrated, this was confirmed by a compensatory rise in the severely affected animals that survived. The kaolin partial thromboplastin time test

CONTAGIOUS

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593

showed a significant rise in the infected animals from the 8th day, but there was no increase in the prothrombin time nor the thrombin clotting time indicating that depletion of factors in the intrinsic clotting system had commenced by the 8th day and that factors, VIII, IX, XI and XII could have been involved. Fibrin degradation products increased significantly in the infected animals from the 9th day. We conclude that thrombosis is an early event in the pathogenesis of CBPP, it contributes to the subsequent pathology of the lung lesions and platelets are involved to a substantial degree in the formation of the thrombi. ACKNOWLEDGMENTS

The experiment was planned with the help of Professor D. G. Penington, Department of Medicine, University of Melbourne at St Vincent’s Hospital. The blood clotting tests and determination of fibrin degradation products were carried out by MS M. Choda, the chemical analyses by MS K. Burman, the serology and microbiology by Mr J. R. Etheridge and Mr B. Heggie and the histological preparations by Mr A. Rowlatt. The statistical analyses was done by Mr P. J. Pahl, MS V. Lord and Dr A. Trajstman. We thank these people for their skilled assistance and generous contribution to the project. REFERENCES

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Gallimore, M. J., and Shaw, J. T. B. ( 1969). Th e influence of various plasma components on the lysis of dilute human blood clots. Thrombosis et diathesis haemorrhagica,

22, 223-233. Hawkey, C. M. (1970). Fibrinolysis in animals. Symposium of the
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L. C., and Trethewie, E. W. (1970). Contagious bovine pleuropneumonia. In The Role of Mycoplasmas and L-Forms of Bacteria in Disease, J. T. Sharp, Ed., pp. 172-197. Charles C. Thomas, Springfield, Illinois. Lowry, 0. H., Rosebrough, Nira J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193,

265-275. Macfarlane, R. G. (1968). The hemostatic mechanism and its defects. International Review of Experimental Pathology, 6, 55-l 33. McKay, D. G. (1965). Disseminated Intravascular Coagulation: An Intermediary Mechanism of Disease. Hoeber Medical Division of Harper and Row Publishers Inc., New York. McKay, D. G., and Shapiro, S. S. (1958). Alterations in the blood coagulation system induced by bacterial endotoxin. I. In vivo (generalized Shwartzman reaction). Journal of Experimental Medicine, 107, 353-367. McSherry, B. J., Horney, F. D., and de Groot, J. J. (1970). Plasma fibrinogen levels in normal and sick cows. Canadian Journal of Comparative Medicine, 34, 191-197. Merskey, C., Kleiner, G. J., and Johnson, A. J. (1966). Quantitative estimation of split products of fibrinogen in human serum, relation to diagnosis and treatment. Blood, 28, 1-18. Merskey, C., Lalezari, P., and Johnson, A. J. (1969). A rapid, simple, sensitive method for measuring fibrinolytic split products in human serum. Proceedings of the Society for Experimental Biology and Medicine, 131, 871-875. Osbaldiston, G. W., Stowe, E. C., and Griffith, P. R. (1970). Blood coagulation: Comparative studies in dogs, cats, horses and cattle. British Veterinary Journal, 126, 512-521. Owren, P. A., and Aas, K. (1951). The control of dicoumarol therapy and the quantitative determination of prothrombin and proconvertin. Scandinavian Journal of Clinical and Laboratory Investigation, 3, 201-208. Piercy, D. W. T., and Bingley, J. B. (1972). F i b rinous synovitis in calves inoculated with killed Mycoplasma mycoides. Journal of Comparative Pathology, 82, 279-290. Quick, A. J. (1966). Hemorrhagic Diseases and Thrombosis, 2nd edit., pp. 43-45, 138-144 and 199. Lea and Febiger, Philadelphia. Rapaport, S. I. (1959). Possible relationships between clotting factors in vitro and intravascular clotting. Angiolopy, 10, 391-395. Schalm, 0. W. (1965). Veterinary Hematology, 2nd edit., pp. 79-81. Lea and Febiger, Philadelphia. Stigbrand, T. (1967). Molar absorbancy of cyanmethaemoglobin. Scandanavian Journal of Clinical and Laboratory Investigation, 20, 252-254. Turner, A. W. (1959). Pleuro-pneumonia group of diseases. In Infectious Diseases of Animals: Diseases Due to Bacteria, Vol. 2. A. W. Stableforth and I. A. Galloway, Eds., pp. 437-480. Butterworths, London. Turner, A. W., and Etheridge, J. R. (1963). Slide agglutination tests in the diagnosis of contagious bovine pleuropneumonia. Australian Veterinary Journal, 39,445-45 1.

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Turner, A. W., and Trethewie, E. R. (1961). Preventative tail-tip inoculation of calves against bovine contagious pleuropneumonia. I. Influence of age at inoculation upon tail reactions, serological responses and the incidence of swollen joints. Australian Veterinay Journal, 37, l-8. Turner, A. W., Campbell, A. D., and Dick, A. T. (1935). Recent work on pleuropneumonia contagiosa bovum in North Queensland. Australian Veterinary Journal, 11, 63-71. Woodhead, G. S. (1888). Some points in the morbid anatomy and histology of pleuropneumonia. Journal of Comparative Pathology, 1, 33-36, 123-l 33, 339-347. [Received for publication,

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