A gel diffusion precipitin test for Contagious Bovine Pleuropneumonia

J.

COMP.

PATH.

1965.

A GEL

VOL.

223

75.

DIFFUSION

CONTAGIOUS

PRECIPITIN

BOVINE

TEST

FOR

PLEUROPNEUMONIA BY

R. M.

GRIFFIN

INTRODUCTION

The laboratory diagnosis of Contagious Bovine Pleuropneumonia (CBPP) is based upon isolation of Mycoph-ma mycoides in culture from infected tissues and the detection of complement fixing, agglutinating and precipitating antibodia in serum samples. Earlier workers gave considerable attention to the use of tube precipitin tests in the examination of sera for specific precipitim in the diagnosis of CBPP, but the results appeared unreliable and failed to correlate with post-mortem findings. The low incidence of precipitating sera in cases of natural infection of CBPP was noted

by Dafaalla (1959). The need for a suitable field diagnostic test resulted in the development of a rapid slide agglutination test in which whole blood samples were tested against a stained antigen preparation (Newing and Field, 1953; Newing, 1955). In practice, this test also produced unreliable results. The limitations have been discussed by Lindley (1958) and Provost, Villemot, Queval and Valanza (1959) who considered the test suitable as a herd screening test but insufficiently reliable for use on individual animals. One possible explanation of the unreliability of both precipitin and agglutination tests has been suggested by Turner (1962 a, b), who followed the serological response of cattle experimentally infected with CBPP and showed the agglutinin and precipitin responses of animals to be involved in an “eclipse” phenomenon or “in viva” neutralisation due to circulating antigen, which is known to occur at certain phases of infection (Ono, 1925; Nakamura, Futamura, Watanuki, 1926). The complement fixing antibodies were only rarely involved in this phenomenon. It is likely, therefore, that the complement fixation (CF) test introduced by Campbell and Turner (1936) will remain the serological test of choice for the diagnosis of infection in the individual living animal. Complement fixation, however, cannot be regarded as ideal for field application when facilities are limited and is more suitably applied in a laboratory. Since the need for suitable field tests remains, the possible use of a precipitin test to detect specific Mycoplasma antigens in tissue samplesfrom dead animals, using anti-M. mycoides sera has been reconsidered as a diagnostic aid. The examination of tissue samplesfor specific Mycoplasma antigens might be applied : (1) to provide a rapid diagnosisfrom individual samplesand (2) to screen tissuestaken from abattoirs or slau hter slabs, in order to gain information about the incidence of CBPP in a particu [iar area. Johnston and Simmonds (1963) used the tube precipitin test to detect specific M. mycoides antigens in extracts of infected lung tissuesin a survey of macroscopically abnormal lungs and obtained 25 positive reactions from samples taken from 28 casesof CBPP, confirmed by other tests. White (1958) described the application of agar gel diffusion techniques in the diagnosisof CBPP. He showedthat a reaction, comprising three lines of precipitation, occurred between rabbit anti-M. mycoides sera and samplesof fresh and preserved lung tissuesfrom CBPP infected animals, cultures and culture extracts of M. mycoides. Gel diffusion techniques have been widely applied to diagnostic problems, particularly with virus systems,in many casesproviding more detailed qualitative informa-

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tion about the system than conventional tube precipitin tests. Assessments of the reliability and sensitivity of precipitin tests in the detection of specific Mycoplasma antigens have not so far been reported. This paper describes the development of a gel diffusion test (GD diagnosis of CBPP and an assessment of it specificity in the field. MATERIALS

AND

test) for the

METHODS

Preparation of Immune Serum Strains of M. Mycoides. Strain KHS/J : An attenuated strain at the 85th broth culture passage, obtained originally from Khartoum and used for vaccine production. Strain V/5 : a strain received from Australia, where it is use dfor vaccine production. Production of antigenic material. Strains of M. mycoides were grown in a serum broth medium based upon that of Bennett (1932) with the additional ingredients and bacteriostats described by Edward (1947). Th e medium was dispensed in 3 Iitre volumes in 4 litre flasks. Seed inocula were prepared in the same medium. Before use the cultures were checked for purity by dark-ground microscopy, Gram stained films and plate cultures on 10 per cent ox-blood agar. Flasks were inoculated with 25 ml. of a 72-hour growth of the seed culture and incubated at 37°C. with aeration (Newing, 1955) for 5 days. Samples from the flasks were checked for purity of growth. The bulk growth was centrifuged in a Sharples continuous flow centrifuge at 65 x lo3 “g” at a feed rate of 150 ml./min. The organisms were harvested in 50 ml. of 0.01 M. sterile phosphate buffered saline at pH 7.3 (PBS). The suspensions were washed once in 50 ml. of PBS, recentrifuged at 20 x lo3 “g” for 30 min. The supernatant fluids were decanted and the sediments resuspended in 10 ml. of sterile PBS. Suspensions were stored at -20°C. Zmmunisation of rabbits. New Zealand White rabbits, of about 2 kg. body-weight, were immunised in groups of four as follows : each rabbit was given a course of intravenous inoculations on alternate days of 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, and 2.0 ml. of a washed culture suspension at a density equivalent to Brown’s opacity tube No. 3 (Burroughs Wellcome). They were bled at the 5th day after the last inoculation. Pre-inoculation serum samples were examined with the 5th day post-inoculation samples for agglutinating antibody by the tube agglutination test of Edward (1950) and for complement fixing antibody bv the plate technique of Lindley (1960). Sera from individual rabbits were screened in gel diffusion systems. Sera with an agglutination titre of l/160 or above were acceptable for gel diffusion studies. The specificity of the sera was examined by testing for residual specific precipitins in GD tests, after absorption with suspensions of CBPP infected and normal bovine lung, following the technique of Darbyshire (1962). Preparation of Antigens for the GD Test Crude infected lung tissue (IL). Small portions of known CBPP infected lung were eluted in PBS in the basins of diffusion plates. Infected lung extract (ILE). Infected lung tissues were cut up with scissors and ground in 10 ml. of sterile PBS with sterile sand in a mortar. The resultant suspension was kept at 4°C. for 24 hours. After centrifugation at 1000 “g” (10 mm.), the supernatant fluid was decanted and used as antigen . Washed culture suspensions (C). Concentrated suspensions of the antigenic materials as prepared for rabbit immunisation. Filter paper strips soaked in lung exudate. Sheets of standard filter paper (Whatman No. 1, 15 cm. diameter) were soaked in a bacteriostatic solution of merthiolate (1 : 5,000) or phenol (0.5 per cent w/v) in normal saline and dried in air. Sheets were cut into arcs (approx. 22”) and booklets of 10 sample papers prepared. Samples of lung exudate were collected as blots on the filter papers from the cut-surface of freshly incised lesions in lung tissue (Fig. 1). The same process was used for sampling bronchial

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M.

GRIFFIN

225

and mediastinal lymph nodes, pleural effusions and adhesions. The sample papers were then dried in air. In field experiments, sample papers were attached to record sheets and dispatched to the laboratory by post. Storage of samples in a refrigerator before dispatch was advised, but was not found to be essential. Sample papers were examined for Mycoplasma antigens by allowing small squares (c. 25 sq. mm.) to elute in PBS in the basins of diffusion plates. Technique of the G.D. Test The double diffusion plate technique of Ouchterlony (1948) was used. A 1.0 per cent agar gel (Oxoid Ionagar No. 2) prepared in veronal-NaCl buffer at pH 7.4 (Mayer, Osler, Bier, Heidelberger, 1946) was found to give optimal results. Merthiolate (1 : 10,008) was added as a bacteriostat. The pattern of basins most frequently used was a circle of cups of 7-O mm. external diameter, cut at 11.0 mm. centre to centre distance. Basins were sealed with a drop of melted agar medium after removal of the agar plugs. For routine purposes plates were incubated in a moist atmosphere at room temperature (c. 22°C.) in an adapted desiccator jar. In some experiments they were incubated in a refrigerator at 4.0%. Plates were examined after 6 hours incubation and again at 24 hours. Thereafter they were examined at 24-hour intervals up to 7 days. Photographic records of positive reactions were made following the technique of Hunter (1959). Materials used in Evaluating the Technique Samplesof lung tissue,sera and filter paper samplesof lung exudate together with reports of clinical and macroscopic findings, were obtained from Zebu cattle of various breeds from selected abattoirs and slaughter slabs. Animals were grouped and the samplesexamined by routine bacteriological and histopathological methods. Methods used to Diagnose CBPP The following tests were used to diagnoseCBPP in samplesfrom animals included in Group I of the trial. Since sampleswere not always received in a sufficiently fresh state for cultural examination, a final diagnosisof each casewas based on the results obtained in at least two of the tests. Isolation of M. mycoides. Tissueswere ground with sterile sand in a sterile mortar with 5-Oml. serum-broth. After light centrifugation the supernatant fluid was seeded to 10 per cent ox-blood agar plates and to tubes of serum-broth media using a lo-fold dilution technique. Plates were examined after 5 days incubation in a moist atmosphere at 37°C. Tubes were incubated at 37X., examined daily and not discarded before the 10th day. Dark-ground microscopy. Preparations from lesion and pleural fluids, ground tissue supernatant fluids and broth cultures were examined by dark-ground microscopy. Preparations from broth cultures were examined daily after 24 hours incubation. Serological tests. Complement fixation tests (CF) were carried out by the plate method of Lindley (1960) for the screening of bovine sera. Tube agglutination tests were carried out by the technique of Edward (1950) using a standard antigen prepared from Strain KHS/J. Rapid slide agglutination tests (RSAT) employed the technique of Newing and Field (1953). Gel diffusion testswere performed on bovine seraboth for precipitating antibodies and for the precipitating antigens, demonstrable at certain phasesof infection. RESULTS

Typical Precipitin Reactions The precipitin pattern observed with rabbit immune sera varied with the antigen used. The usual pattern, seen best with the ILE antigen, consisted of a

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maximum of 3 specific lines of precipitation, usually made up of 2 strong lines and a third weaker line (Figs. 2 & 3). The same pattern was observed with crude lung antigen but occasionally only 2 lines of precipitation were seen. Culture suspensions used as antigens generally produced weaker reactions and many preparations gave only 2 lines of precipitation. Specificity

of

the Precipitin

System

Ideally, cultures grown in rabbit serum enriched medium should have been used when preparing rabbit immune serum for use in this GD test. Since this was not practicable and as horse serum was used as routine, an assessment of the specificity of the system was carried out. No precipitation reactions were observed between the horse sera used in the preparation of media and either normal or CBPP infected lung tissues. No reactions were observed between the rabbit immune set-a and normal bovine lung tissues. Rabbit immune sera were absorbed with suspensions of CBPP infected and normal lung tissue and then tested for residual precipitins against crude tissue antigens prepared from normal and CBPP infected lungs in GD tests. Absorption of rabbit immune sera with CBPP infected lung tissues removed all precipitins, whereas absorption with normal lung tissue did not affect the precipitin reaction. Precipitin

Reactions

z&h Filter Paper Samples

of

Lung Exudates

The reactions between rabbit immune serum and filter paper samples of lung exudates from CBPP infected and normal animals were investigated. Typically, 2 strong lines of precipitation developed between the sera and samples from infected lungs, but some gave 3 lines of precipitation and occasionally there was only a single strong line (Figs. 4, 5 & 6). Samples from normal animals gave no reaction. In diagnostic tests, reactions were normally visible after 2 to 6 hours and never required more than 48 hours incubation to develop. The effect of environmental temperature and storage on the antigenic potency of filter paper samples of lung exudates was studied. Samples retained their antigenie potency for at least 8 weeks during storage at room temperature. Samples stored in the refrigerator and at room temperature for up to 12 months lost 50 per cent and 75 per cent of their original strength, respectively. Samples stored in the refrigerator invariably gave stronger reactions than those stored at room temperature, but in all cases detectable reactions were obtained. Field Use of the G.D. Test

for Diagnosis

The utility of the GD test for the field diagnosis of CBPP was assessed on the results obtained in the examination of materials and filter paper samples of lung exudates from animals classified in three groups. Group I: animals with macroscopically abnormal lungs. Materials from 52 animals with macroscopically abnormal lungs and no history of CBPP vaccination were examined for CBPP infection by routine diagnostic methods and by the GD test (Table I). Twenty-four cases of CBPP were diagnosed in these animaJs. In the GD test, filter paper samples from all these cases gave positive reactions. Twelve strains of M. mycoides were isolated from 17 of the cases in which cultural

R.

M.

GRIFFIN

TABLE DIAGNOSTIC

TESTS

FOR

CBPP

227

1

ON

CONFIRMED

CASES

IN

GROUP

Serological CD test onjilter

PW sws lung cdte

of

Cultural isolaiiow M. mycoiah from tissue samples

of D.G. minoscojy tissuc&& and broth cultures

I

t&s

on RSAT

CF test

GD test for precipitating Antigen Antibody

24*

12

15

18

20

23

0

ii

17

17

21

21

23

23

* The numerator samples examined

indicates the number of positive by the particular test.

sample-a and the denominator

the total

number

of

isolations were attempted. Six strains were subsequently inoculated subcutaneously into susceptible cattle and shown to be virulent. All routine tests for CBPP and the GD test carried out on materials from the remaining 28 cases were negative. In 13 of the negative cases, bacteria to which the lung lesions could be attributed were isolated and identified as follows : Pasteurella multocida, 3 cases; Corynebacterium pyogenes, 4 cases; Escherichia co& 3 cases; Bacillus anthracis, 1 case; Pseudomonas pyocyanea, 2 caseg. Routine

bacteriological cultures from the remaining 15 negative cases produced no significant isolates, but histopathological examination showed lesions of lung abscesses in 2 cases; fibrinous pneumonia in 1 case; purulent broncho-pneumonia in 3 cases; puhnonary congestion and alveolar oedema in 5 cases; aspiration pneumonia in 2 cases; chronic interstitial fibrosis in 2 cases. Group ZZ: vaccinated animals. Filter paper samples of lung exudate, taken at slaughter from macroscopically normal lungs from 61 animals vaccinated against CBPP in Nigeria, were examined by the GD test. Forty of these animals from the laboratory herd in Vom, slaughtered from 1 to 6 months post-vaccination, were sampled from incised bronchial and mediastinal lymph nodes in addition to lung exudate. Samples from the remaining 21 animals were obtained from the field. The GD test gave negative reactions in every case. Group ZZZ: survey of animals with macroscopically normal lungs. 1,127 filter paper samples of lung exudate collected from Zebu trade cattle of varying breeds, slaughtered for the butcher or meat processing trade at 6 major abattoirs and slaughter slabs in those provinces where CBPP had previously occurred, were examined by the GD test. AR animals had macroseopically normal lungs. Positive reactions were detected in 3 cases whereas the other 1 ,124 samples were negative. It was not possible to investigate further those animals from which positive reactions were obtained. DISCUSSION

The field control of CBPP in Nigeria, based upon extensive vaccination of susceptible animals in selected areas, rapid detection and elimination by slaughter of infected animals and strict control of cattle movements, has produced encouraging results. Further progress towards eradication, using the same general methods, will only be practicable if applied interterritorially. The rapid diagnosis of CBPP under field conditions is based on cultural or

228

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serological examination of tissues taken post-mortem, or on serological tests on other animals from the suspect herd. The need for reliable, simply applied serological tests for use on the individual animal, both living and dead, by field staff has yet to be met. Two important epidemiological features which raise particular diagnostic problems in the living animal and are also related to the insidious spread of infection are firstly, the occurrence of sub-clinical infection, recognised only by transient serological responses and secondly, the occurrence of clinically healthy carrier animals with chronic infections. The extent to which these types of infection might be expected to occur in natural outbreaks of disease was shown by Campbell and Turner (1936), who investigated over a period of 18 months the types of infection developing in 64 animals, closely confined in a quarantine area with a number of animals with acute CBPP infection taken from a natural outbreak of infection. Hyperacute infections developed in 8 per cent, acute infection in 14 per cent, very mild infection in 30 per cent, sub clinical infection, recognised by regular CF tests 23 per cent and the remaining 25 per cent resisted infection. Of the animals that survived infection, 10 per cent developed into clinically healthy chronic cases, as shown by post-mortem examinations up to 10 months after infection, when sequestrated lesions from which viable organisms were recovered were found. The accuracy of serological tests on living animals with these two types of infection varies markedly. CF tests based on the techniques of Campbell and Turner (1936, 1953) h ave been used extensively and have been shown to give accurate results with all types of infection. The modified technique of Huddart (1963) using plasma samples has been widely applied in East Africa with specialised mobile testing units and has given equally good results, but complement fixation tests are in general by no means ideal for use by field staff. Agglutination and precipitation tests for Mycoplarma antibodies are not only subject to the “eclipse” phenomenon demonstrated by Turner (1962 a, b), but also have failed to give positive results with sub-clinical and chronic infections and a proportion of false positive reactions have been found with the agglutination tests. Villemot and Provost (1959 a, b) reported that common agglutinating antigens between M. mycoides and other Mycoplasma spp. occur in cattle, to which these false positive reactions might be attributed. Their findings await confirmation. Allergic reactions, based on the intradermal inoculation of extracts of M. mycoides, have received attention and have been re-investigated recently and shown to give promising results (Gourlay, 1962, 1964). If shown reliable on further evaluation, a test of this type would be ideal for use by field staff. The only serological test which can be used on post-mortem samples is the precipitin reaction, in which tissues or tissue extracts are examined for Mycoplasma antigens, using known anti-M. mycoides immune sera. This test has not been widely applied, but does provide a rapid diagnosis. In the evaluation of any new diagnostic test for an infectious disease, at least three factors must be considered. Firstly, the test must be simple to perform and give reliable results, secondly, the antigenic heterogeneity of strains of the infective agent and antigenic relationships with other organisms should be minimal and thirdly, vaccination procedures in current use should not interfere with the results of the test.

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The production of satisfactory precipitating antisera against Mycoplusma antigens was not easy since approximately only 50 per cent of rabbits produced acceptable sera. Multiple courses of injections of antigens failed to produce higher titres of complement fixing and agglutinating antibodies than single courses of injections. Weak immunising suspensions produced as good an antibody response as stronger suspensions. Other workers have used donkeys to produce immune sera with high precipitin titres against Mycoplusma spp., but very large volumes of immtmismg antigens were required (Villemot and Provost, 1959a). Some antigen preparations, particularly heavy culture suspensions, did not give the 3 lines of precipitation with rabbit immune serum regarded as normal and seen best with ILE antigens. A relative concentration of reactants giving the typical diffusion pattern could usually be obtained by serial dilution of the stronger reactant, normally the antigen. Weak reactions, obtained with some filter paper samples of lung exudate, consisting of a single weak precipitin line at the periphery of the antigen basin, were improved by retesting the samples using two or more squares from the sample paper, thus producing both a stronger precipitin reaction and displacement of the precipitin line towards the immune serum basin. The results of the field evaluation of the GD test showed that a precipitation reaction obtained with CBPP infected tissues from 24 confirmed cases did not occur with tissues from 27 cases with pulmonary lesions negative for CBPP, that could be attributed to other causes. These results indicate the specific nature of this precipitation reaction. Filter paper samples from 1,127 macroscopically normal lungs gave only 3 positive GD test reactions and it was not possible to follow up these 3. They may have resulted either from early cases of CBPP, not recognised on macroscopic examination or, possibly, from cross-reactions with common antigens of Mycoplusma or other organisms. Strains of M. mycoides are generally thought to form a single closely related antigenic group. Antigenic heterogeneity has been reported only once by Heslop (1924). My co ~2asma spp. that possess antigens in common with M. mycoides, notably M. mycoides var. Capri, M. laidlawii and M. hominis have been reported (Villemot and Provost, 1959a, b). Antigens common to M. mycoides and other organisms have been reported with vaccinia virus (Heslop, 1922; Provost, 1958), some strains of Actinobacillus Zignidresi (Turner, 1956) and Pasteurella multocida (Provost, Villemot, Queval and Valanza, 1959). These workers showed, by gel diffusion tests, a common precipitating antigen with strains of P. multocida (Carter’s Group B). The differential diagnosis of early acute CBPP infection and P. multocida infection from samples of lung tissue, where macroscopically similar lesions occur, has proved difficult in Nigeria. The 3 cases found in macroscopically abnormal lungs in animals in Group I all gave negative GD test reactions. One further cross reaction in the tube precipitin test was recorded in a survey of macroscopically abnormal lungs by Johnston and Simmons (1963), with fluids from a fertile hydatid cyst in an animal with a history of CBPP vaccination. Vaccines prepared from strain KH3/ J have been used extensively in Nigeria in the control of CBPP and the strain is considered avirulent for local Zebu cattle. No substantiated reports of undesirable reactions at the inoculation site or of the establishment of lung lesions attributable to vaccination have been recorded. The results of GD tests of filter paper samples of lung exudate and incised bronchial

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and mediastinal lymph nodes from 61 animals, vaccinated against CBPP were negative in every case. Where other CBPP vaccination procedures are used, further trials of the GD test on vaccinated animals would be necessary. Further evaluation of the GD test is needed, particularly with cases of chronic infection and also to determine the optimal site to sample with filter paper blots. In the pathogenesis of infection, rapid spread of the primary lesion to the peribronchial lymphatic vessels and drainage to bronchial and mediastinal lymphatic glands is well established. Campbell and Turner (1936) recorded cases where pulmonary foci could not be detected with certainty yet organisms were recovered from these lymphatic glands. It may well prove that lymphatic glands are the optimum site for sampling. Filter paper sampling is particularly convenient for surveying the incidence of infection in a locality. Samples may be collected from abattoirs and slaughter slabs during routine carcass inspection, lay staff requiring only simple training. The provision of suitable fresh diagnostic material from remote areas is difficult, expensive and often proves unsatisfactory. The resistance of the specific Mycoplasma antigens to high ambient temperatures is a further important advantage when the collection and transmission of large numbers of samples for a survey is considered. The gel diffusion test described is simple to perform, requiring a minimum of equipment, It is suitable for use by field staff and is considered a useful diagnostic aid in the control of CBPP. CONCLUSIONS

A technique of sampling using filter paper strips was developed to sample lung tissue exudates as blots, which were then examined for Mycoplasma antigens against rabbit anti-M. mycoides sera. Reactions were detected for up to 12 months with filter paper samples from CBPP infected animals stored at room temperature. From the results obtained in a field evaluation, the gel diffusion precipitln test used in the examination of tissues from dead animals is considered a useful addition to other serological tests for CBPP. As it requires a minimum of equipment and is technically simple to perform, the test is suitable for use by field staff. Sampling by filter paper blots at abattoirs and slaughter slabs is a convenient method of surveying the incidence of CBPP in a particular locality. ACKNOWLEDGMENTS

Thanks are due to the Chief Veterinary Officer, Northern Nigeria, for enabling the field assessment to be undertaken and to the Field Veterinary staff for their help. The author wishes to thank Mr A. L. C. Thorne, Director, Federal Department of Veterinary Research for his help in the preparation of this paper and for permission to publish. REFERENCES

Bennett, S. C. J. (1932). J. camp. Path., 45, 257. Campbell, A. D., and Turner, A. W. (1936). Bull. Count. sci. industr. No. 97, 11; (1953). Aust. vet. J., 29, 154. Dafaalla! E. N. (1959). Report XVI World Vet. Cong., Utb, 15, 539. Darbyshrre, J. H. (1962). Res. vet. Sci., 3, 125.

Res. (Aust.),

R.

*

M.

GRIFFIN

4

Fig. 1. The method of taking a iiltcr paper blot sample from a frcshl>- incised lung lesion. Fig. 2. The reaction beturrn infcctetl lung extract (ILK). a cultwc suspension (Cl and a rabbit a nti~pla.mmqx0ide.r swum i1.S 1. A fycycl lung extracl (ILK), and rabbit anti-AJyco+zsma myc0ideJ seru‘ rn Fig. 3. The reaction betwec-n infictrd respectively. (IS). Sample papers I and 2 arc controls soaked In phrnol and mrrthiolate r0 face page 230

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and anti-.2&o) blasmu Fig. 4. The typical reaction between filter paper samples of lun$ ~~tlates qmides serum (IS). Samples 1, 3 and 6 show positive rt‘a cuons and samples 2, 4 and 5 are negative. Fig. 5. The reaction between infected lung tissue (II,), filter paper samples of lung exudate fimm a normal (I) and an inkcrtrtl 2 lung and rabbit anli-,2!l’c oi,lnsma~ v!r~oirle;, serum. Fig. 6. Single line precipitin reactions lxt\vecn filtrr paper samples of lung exudates and anti- .12lYCOplama mycoidesserum (IS). Sample 1 is a negative control and sample 4 gave a negative rea ction.

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GRIFFIN

Edward, D. G. ff. (1947). J. gen Microbial., 1, 238; (1950). Ibid., 4, 4. Gourlay, R. N. (1962). pet. Rec., 74, 1321; (1964); 1. camp. Path., 74, 286. Heslop, G. C. (1922). Ibid., 35,l; (1924). Proc. soy. Sot. Vict., 34 (N.S.), 83. Huddart, J. E. (1963). Anim. Hlth. Branch Monograph, 406, F.A.O. Rome. Hunter, J. R. (1959). Nature, Lond., 183, 1283. Johnston, L. A. Y., and Simmons, G. C. (1963). Aust. vet. /., 39, 290. Lindley, E. P. (1958). Bull. epit. Dis. Afr. 6, 369; (1960). Report of 1st Meeting joint FAO/OIE/CCTA Expert Panel on CBPP, Melbourne. May% If15M., Osler, A. G., Bier, 0. G., and Heidelberger, M. (1946). 1. exp. Med., Nakam&a, N., Futamura, H., and Watanuki, J. (1926). J. Jap. Sot. vet. Sci., 5, 296. Newing, C. R., and Field, A. C. (1953). Brit. vet. J., 109, 397. Newing, C. R. (1955). Ibid., 111, 378. Ono, S. (1925). J. /up. Sot. vet. Sci., 4, 245. Ouchterlony, 8. (1948). Arkiv Kemi Mineral, Geol., 26B, 1. Provost, A. (1958). Rev. Blev., 11, 5. Provos27A., Villemot, J. M., Queval, R., and Valanza, J. (1959). Bull. epic. Dis. Afr. 7, Turner,A. W. (1956). Bull. 08. Int. E&z., 46, 382; (1962a). Aust. vet. J., 38, 335; (1962b). Ibid., 401. Villemot, J. M., and Provost, A. (1959a). Rev. tilev., 12, 251; (195913).Ibid., 369. White, G. (1958). Nature, Lond., 181,278. [Received

for publication,

October

24th, 19641