Immunoprophylaxis of experimental Mycoplasma bovis arthritis in calves. Protective efficacy of live organisms and formalinized vaccines

Veterinary Microbiology, 5 (1980) 113--122 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

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IMMUNOPROPHYLAXIS OF EXPERIMENTAL MYCOPLASMA BOVIS ARTHRITIS IN CALVES. PROTECTIVE EFFICACY OF LIVE ORGANISMS AND FORMALINIZED VACCINES

J.C. CHIMA.1 , B.N. WILKIE~ , H.L. RUHNKE s , R.B. TRUSCOTT* and R.A. CURTIS4 1Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ont. (Canada) 2 Ontario Ministry of Agriculture and Food, Veterinary Services Laboratory, Guelph, Ont. (Canada) SAnimal Pathology Division, Health of Animais, SackviUe, N.B. (Canada) 4Department of Clinical Studies, University of Guelph, Guelph, Ont. (Canada) *Present address: National Veterinary Research Institute, Vom, Plateau State (Nigeria) (Accepted 16 November 1979) ABSTRACT Chima, J.C., Wilkie, B.N., Ruhnke, H.L., Truscott, R.B. and Curtis, R.A., 1980. Immunoprophylaxis of experimental Mycoplasma boris arthritis in calves. Protective efficacy of live organisms and formalinized vaccines. Vet. Microbiol., 5: 113--122. Live Mycoplasma boris (M. boris) organisms given subcutaneously or intraperitoneally protected nine of ten calves and eight of nine calves, respectively, from clinical arthritis, while the formalinized vaccine given subcutaneously protected eight of ten calves. In contrast, clinical arthritis was induced in all non-vaccinated calves that were challenged intravenously. The arthritic lesion was more severe in non-vaccinated calves than in the few vaccinated calves that developed clinical arthritis. Unlike formalinized vaccine, live M. boris culture given subcutaneously provoked a local reaction at the site of injection in most calves in the form of oedematous plaques of about 7--8 cm in diameter. Results suggest that the formalinized vaccine may offer a practical approach to the control of Mycoplasma boris arthritis in calves.

INTRODUCTION M y c o p l a s m a agalactiae subsp, bovis (Hale et al., 1 9 6 2 ) n o w k n o w n as M y c o p l a s m a boris ( A s k a a and E r n o , 1 9 7 6 ) is responsible f o r m y c o p l a s m a arthritis in cattle in N o r t h A m e r i c a (Hjerpe a n d K n i g h t , 1 9 7 2 ) . S u b s e q u e n t t o t h e initial r e p o r t o f M. bovis arthritis in f e e d l o t cattle in C a n a d a (Singh et al., 1 9 7 1 ) , e p i z o o t i c s w i t h v e r y high m o r b i d i t y (10 t o 85%) a n d m o r t a l i t y (3 t o 50%) have also been r e p o r t e d (E.V. L a n g f o r d , p e r s o n a l c o m m u n i c a t i o n , cited by Stalheim, 1 9 7 6 ) . As a result o f v e r y p o o r r e s p o n s e t o t r e a t m e n t w i t h t h e antibiotics in c o m m o n use (Stalheim, 1 9 7 6 ) v a c c i n a t i o n m a y be a r a t i o n a l a p p r o a c h t o t h e c o n t r o l o f disease p r o d u c e d b y M. bovis. A r e c e n t s t u d y b y H o w a r d and co-

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workers (1977) appears to be the only reported a t t e m p t to vaccinate against M. bovis infection. Their work, which was to determine whether mycoplasma infections of the respiratory tract of the calf could be prevented by vaccination, showed that calves inoculated intramuscularly and then intratracheally with formalin inactivated M. boris were better protected against respiratory infection with M. boris than calves inoculated twice intramuscularly. In three separate experiments, we have attempted to evaluate the potential of live organisms or formalinized vaccine for the prevention of M. bovis arthritis in calves. MATERIAL AND METHODS

Mycoplasma Mycoplasma boris, strain 427 was used throughout. This strain was originally isolated from the joint of a calf involved in a field outbreak of pneumonia and arthritis. Calves A total of 42 conventionally reared male calves, ranging in age from 1 to 41/~ months, obtained from local farms, were used in the experiments. They were randomly divided into groups and kept in pairs in isolation pens with concrete floor and sawdust bedding. Each pair of calves in a pen received the same type of vaccine and were challenged by the same route. Clinical observations were made on all calves beginning 1 week prior to vaccinations. Rectal temperatures were recorded twice daffy throughout the experiments. All of the calves were screened for antibodies against M. bovis using the passive haemagglutination test (Cho et al., 1975). They "allhad a pre-inoculation titre of less than 1 : 20 except one calf with a titre of 1 : 640. The latter was considered to have been exposed to a natural M. bovis infection. Details of the immune response of the animals following vaccinations and challenge is the subject of another paper. However, generally, there was no correlation between protection and immune response. Media The liquid medium for preparing the vaccine and challenge inoculum was essentially as described by Davies (1967). It was based on Difco PPLO broth 1 to which was added 15% unheated horse serum 2 , 1% yeastolate 1 , 0.1% glucose, 0.002% phenol red and 1000 units/ml penicillin. The medium was adjusted to pH 7.6. 1 Difco Laboratories, Detroit, MI, U.S.A. g Grand Island Biological Company, Grand Island, NY, U.S.A.

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The solid medium used for viable counts and mycoplasma isolations was prepared by substituting PPLO agar I for PPLO broth. Also the agar base contained 0.02% thallium acetate but no phenol red and the pH was n o t adjusted.

Live cultures and challenge inoculum The same procedure was followed in preparing live cultures and challenge inoculum. The seed culture stored in liquid nitrogen and containing 3.0 × l 0 s colony forming units (c.f.u.) was inoculated into 10 ml of mycoplasma broth and incubated aerobically at 37°C for 48. Then 1.5 ml of the 48-h culture was transferred to 25 ml of broth and incubated aerobically at 37°C for 24 h. Finally, each litre of broth in a 2-1itre Erlenmeyer screw-cap flask was inoculated with 50 ml of the 24-h culture and incubated aerobically ~at 37°C for 48 h with occasional shaking. A b o u t 20 ml o f the culture was centrifuged and the sediment Gram stained and examined microscopically for possible bacterial contamination. The challenge inoculum was prepared by concentrating the culture by centrifugation at 16319 × g for 40 rain at 4°C in a Sorval RC2-B refrigerated centrifuge 3 . The sediment was reconstituted with the supernatant broth to the desired concentration.

Formalinized vaccine The initial steps were as outlined above for the live culture and challenge inoculum. Finally, 3 1 of the 48-h culture were concentrated by centrifugation at 16319 × g for 40 min at 4°C. The sediment was resuspended in 60 ml of sterile 0.15 M phosphate buffered saline (PBS) pH 7.2. After determining the purity as described above and the viable c o u n t by the Miles et al. (1938) method, 1% formalin was added to the suspension to give a final concentration of 0.025% and the mixture was incubated aerobically at 37°C for 18 h. A loopful of the inactivated suspension was plated on mycoplasma agar and incubated for 7 days at 37°C in a humidified incubator 4 automatically supplied with 10% CO2. This was to make sure t h a t no viable mycoplasma remained after the inactivation. The vaccine was then stored a t - - 7 0 ° C pending the result of the viable c o u n t initiated prior to formalinization. From this count, the dose of the vaccine was calculated and dispensed into small vials. The volume of each dose was made up to 5 ml with PBS pH 7.2 and stored at --70°C until required.

3 Du Pont Company, Newton, CT, U.S.A. 4 Hotpack (Canada) Limited, Waterloo, Ont., Canada.

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Vaccination and challenge procedures The live organisms were given in doses of about 5 X 109, 1 X 10 '° and 2 X 10 '° c.f.u, for the first, second and third vaccinations, respectively, at 10-day intervals. The formalinized vaccine was given in three repeated doses with an equivalent of 4.5 X 1 0 " organisms also at 10~lay intervals. In the first experiment, twelve calves were randomly divided into four groups (A, B, C and D) of three animals each. Calves in groups A and B were vaccinated intraperitoneally with live organisms, while calves in groups C and D were left unvaccinated. Then 18 days after the third vaccination, calves in groups A and C were challenged intravenously, while groups B and D were challenged intraperitoneaUy with a concentrated suspension of M. boris containing 6.5 X 10 l° c.f.u, and 6.0 X 1 0 " c.f.u., respectively. In the second experiment, 16 calves were randomly divided into four groups (E, F, G and H) of four animals each. Calves in group E were given formalinized vaccine subcutaneously; group F received live organisms subcutaneously, while group G animals were given live organisms intraperitoneally. Animals in group H were left as unvaccinated controls. Animals in all the groups were challenged intravenously at 18 days after the third vaccination with a concentrated suspension of M. bovis containing 9.5 X 10 l° c.f.u. The third experiment was carried out with 15 calves. Six of t h e m in group J received the formalinized vaccine subcutaneously, while another six animals (group K) were given live organisms subcutaneously. Two calves {group L), w i t h o u t prior contact with M. bovis, and one calf (group M) with antibodies against M. bovis were left unvaccinated. All of the animals were challenged intravenously with 5.5 X 10 '° c.f.u, of M. bovis at 60 days after the third vaccination. The vaccine and challenge doses in the trials were based on the experience of one of the authors (R.B. Truscott, personal communication, 1976), who found that 5 X 10 '° M. bovis did n o t produce any adverse reaction when given intraperitoneally, while this dose induced clinical arthritis when given intravenously.

Evaluation by scoring system In that M. bovis infection rarely leads to death within a reasonable time, the evaluation of vaccination efficacy must depend upon other criteria. The object of the vaccination, however, was to prevent arthritis and the accompanying lameness. In the scoring system adopted, considerable weight was invariably given to qualitative factors such as the severity of the lameness. This was, however, balanced with quantitative factors such as the number of joints involved and the frequency of mycoplasma isolations. The following system was employed to assign a numerical index of severity to each M. bovis-challenged calf:

117 A s s e s s m e n t factors

Score

A n t e mortem Severe lameness, killed in extremis

6

Examination Moderate lameness, obviously lame but able t o stand unassisted Mild lameness, o n l y n o t i c e a b l e o n c l o s e

4

examination 2 Post mortem Each joint grossly affected 2 1 E x a m i n a t i o n Each joint with histological lesion Each isolation of M. boris from joint and other sites 1

The scores were t h e n accumulated for each group. RESULTS

Acceptibility o f the vaccine Systemic reaction to vaccinations in t he f o r m of elevation of rectal temperatures was minimal in all the groups. T e m p e r a t u r e s of 39.4°C t o 39.8°C were r e c o r d e d on t he second or third day after the first vaccination, lasting for a b o u t 2 days before returning t o normal. Subsequent vaccinations usually did n o t elicit any appreciable rise in rectal temperatures. One calf in group G t h a t was vaccinated intraperitoneally with live organism maintained a persistently high t e m p e r a t u r e of between 40 ° C and 41.6 ° C for a b o u t 21 days following the first vaccination. This animal became emaciated and lameness was apparent 18 days after the first vaccination. T r e a t m e n t with chloramphenicol lowered the t e m p e r a t u r e but t he lameness persisted. The calf was killed bef or e challenge and necropsy revealed severe peritonitis, periorchitis and arthritis involving two joints from which M. bovis was isolated. Serum collected f r om this calf before, and 2 weeks after immunization with M. bovis showed a n t i b o d y titres t o infectious bovine rhinotracheitis (IBR) virus o f 1 : 96 and 1 : 8, respectively. A b o u t 2 days after t he first vaccination, most o f t he calves t hat received live M. bovis subcutaneously developed a local reaction at the site of injection in the form of o e d e m a t o u s plaques o f a b o u t 7 t o 8 cm in diameter. T he swelling however, disappeared spontaneously a b o u t 9 days later. Subsequent vaccinations did n o t p r o d u c e any further reaction. Reactions of this t y p e were n o t observed in calves given formalinized vaccine.

Clinical observation following challenge The vaccinated animals responded mildly to the challenge in terms o f elevation o f rectal temperatures. Generally, following the initial rise, the mean t e m p e r a t u r e o f vaccinated groups fell below 39.4 ° C 4 days after challenge. In contrast, the average t e m p e r a t u r e of t he non-vaccinated groups remained well above 39.4°C f or up t o 3 weeks after challenge.

118 The r o u te of challenge appeared t o affect t h e d e v e l o p m e n t of clinical arthritis. Thus, in the first exper i m ent , three non-vaccinated calves t h a t were challenged intravenously developed clinical arthritis, while a n o t h e r three calves t h a t received an even higher dose o f the challenge inoculum intraperitoneally did n o t (Table I). Generally, lameness was evident in non-vaccinated calves challenged intravenously between 6 and 10 days following challenge, while the onset in the few vaccinated calves t hat became lame after intravenous challenge was delayed b e y o n d 10 days, and in one case, for up t o 22 days. TABLE I Protective effect of intraperitoneal vaccination of calves with live Mycoplasma boris organisms following intravenQus and intraperitoneal challenge of vaccinated and non-vaccinated calves Group

Number of animals

Calveswith clinical arthritis

Meanarthritic score*

A

3

0

0

Vaccinated i/p

Challeng_ed i/v B Vaccinated i/p 3 0 1 Challenged i/p C Non-vaccinated 3 3 13 Challenged i/v D Non-vaccinated 3 0 2 Challenged i/p i/p ffi intraperitoneal; i/v ffi intravenous. * Increasing score is directly related to the severity of diseases.

Protective effects o f the vaccines Results of the first e x p e r i m e n t showed t h at all three calves t hat were challenged intravenously were p r o t e c t e d , while the nonvaccinated group that was similarly challenged developed clinical arthritis (Table I). On the o t h e r hand, intraperitoneal challenge o f the vaccinated and nonvaccinated calves did n o t produce a clearcut result as none of the groups developed clinical arthritis, although the nonvaccinated ones developed unilateral or bilateral periorchitis. Table II demonstrates the efficacy o f subcutaneous vaccination of calves with live or formalinized M. boris compared with intraperitoneal vaccination with live organisms. All o f the four calves t h at received live organisms subcutaneously were pr ot ect ed, while one calf from the group t h a t received formalinized vaccine and one f r o m the group vaccinated intraperitoneally with live organisms, developed clinical arthritis. In the third expe r i m e nt , one of six calves vaccinated subcutaneously with either formalinized or live organisms developed clinical arthritis following

119 TABLE

II

Protective effect of formalinized vaccine given subcutaneously or live M. boris given subcutaneously or intraperitoneally following intravenous challenge of vaccinated and nonvaccinated calves with a concentrated suspension o f M. bovis Group

No. of animals

Calves with clinical arthritis

Mean arthritic score*

E

4

1

3.25

4

0

1.5"*

3

1

4.3

4

4

17.75

F G H

Vaccinated s/c Formalinized vaccine Vaccinated s/c Live organism vaccine Vaccinated i/p Live organism vaccine Non-vaccinated

s/c = subcutaneous; i/p = intraperitoneal. *Increasing score is directly related to the severity of disease. **Histological lesion observed in some joints. TABLE III Protective effect of subcutaneous injection o f live or formalinized Mycoplasma boris vaccine into calves following intravenous challenge 60 days after the last vaccination Number

Number of calves

Calves with clinical arthritis

Mean arthritic score*

J

Vaccinated s/c Formalinized vaccine Vaccinated s/c Live organism vaccine

6

1

1.16

6

1

1.0

Non-vaccinated Prior contact with M. boris

2 1

2 0

10.5 0

K

L M

s/c = subcutaneous. *Increasing score is directly related to the severity of disease. challenge (Table III), although the latter group had a lower arthritic score. The one calf (Group M) that had a preinoculation passive haemagglutinating anti-M, bovis titre of 1 : 640 was found to be solidly protected against challenge. DISCUSSION R e s u l t s o f t h e t h r e e e x p e r i m e n t s s u g g e s t t h a t a r t h r i t i s d u e t o M. b o r i s i n f e c t i o n in c a l v e s c a n b e p r e v e n t e d b y v a c c i n a t i o n . V a c c i n e s a g a i n s t a c o m -

120 parable disease caused by Mycoplasma agalactiae in sheep and goats have been used with good results in Rumania and Turkey (Popovici and De Simon, 1966; Foggie et al., 1970). The local reaction provoked b y subcutaneous inoculation of live M. boris resolved spontaneously within a few days and so presented no practical problems. Also systemic reaction in the form of elevation of rectal temperatures following vaccination was mild, thus suggesting that the vaccines were well tolerated by the calves. The reason for the development of clinical arthritis in one calf that received live organisms as a vaccine intraperitoneally was not clear as some nonvaccinated calves were given a much larger dose intraperitoneally without inducing arthritis. The efficacy of the vaccines was assessed mainly by intravenous challenge with a concentrated suspension of M. boris although it is believed that the natural route of infection is through the respiratory tract (Jasper et al., 1966). The effectiveness of the challenge was demonstrated b y induction of clinical arthritis in all nine nonvaccinated calves. It is possible that the dose may be heavier than that usually encountered by the animals in the field. Hence it seems reasonable to suggest that the few vaccinated calves that failed to withstand the intravenous challenge m a y have been protected against a more natural m e t h o d of challenge. Experiments are currently in progress to test this hypothesis. Concern over the use of inactivated mycoplasma vaccines is based not only on their frequent inability to induce protective immunity (Smith et al., 1967; Goodwin and Whittlestone, 1973), b u t also on the tendency of such vaccinated animals and man to develop a much more severe disease than the unvaccinated controls when challenged (Smith et al., 1967; R.N. Gourlay and M. Shifrine, unpublished data, 1965, cited by Gourlay, 1972). This is at variance with the result of the present study. Formalinized vaccine not only protected almost all of the calves that received it, b u t the disease induced by the challenge in two of such vaccinated calves was very mild compared with non-vaccinated controls. Both Mogabgab {1973) and Wenzel et al. (1976), in their respective field trials with inactivated Mycoplasma pneumoniae vaccines in man, did n o t observe any hypersensitivity to mycoplasma antigens in vaccinated individuals w h o developed the natural disease. It is recognized that the quality of inactivated mycoplasma vaccine may depend on the virulence of the strain used in the preparation (Fernald and Clyde, 1970), quality of the growth medium (Somerson et al., 1973), m e t h o d of inactivation (Senterfit and Jensen, 1967), and dose of the vaccine {Cole et al., 1969). The extent to which these factors may have contributed, either singly or in combination, to the apparent success of this trial is uncertain. It has been shown that live vaccines are more effective than killed vaccines in preventing some mycoplasma infections {Lloyd and Trethewie, 1970; Foggie et al., 1970) although they are not w i t h o u t some hazards. Thus, goats that were given live attenuated M. agalactiae vaccine were found to have residual infections with the vaccine strain when killed 3--4 months after vaccination (Foggie et al., 1970). Also the T1 and KH3J vaccine strains of Mycoplasma mycoides var. mycoides have been recovered from vaccinated cattle

121 f o r 1 - - 3 m o n t h s a f t e r v a c c i n a t i o n ( H u d s o n , 1 9 6 5 ; Masiga a n d Mugera, 1 9 7 3 ) a n d t h e p o t e n t i a l risk o f r e v e r s i o n o f t h e s e strains t o full v i r u l e n c e h a s b e e n stressed (E.P. L i n d l e y a n d V. Pedersen, 1 9 6 8 ; cited b y D y s o n a n d S m i t h , 1 9 7 5 ) . In t h e p r e s e n t trial, in a d d i t i o n t o p r o v o k i n g a local r e a c t i o n a t t h e site o f s u b c u t a n e o u s i n o c u l a t i o n , live o r g a n i s m s given i n t r a p e r i t o n e a l l y a c t u a l l y ind u c e d clinical arthritis in o n e o f t h e calves. T h u s , a l t h o u g h live M. boris p r o t e c t e d m o r e calves t h a n did t h e f o r m a l i n i z e d vaccine, t h e l a t t e r has p o t e n t ial a d v a n t a g e s t h a t m a y r e c o m m e n d it o v e r t h e live o r g a n i s m u s e d as a vaccine. It is realised t h a t t h e r e g i m e n o f t h r e e v a c c i n a t i o n s at 10 d a y interval is n o t ideal f o r field a p p l i c a t i o n . H o w e v e r , h a v i n g e s t a b l i s h e d t h e possibility o f p r e v e n t i n g M. boris arthritis in calves b y s u b c u t a n e o u s i n o c u l a t i o n w i t h e i t h e r live o r g a n i s m s or f o r m a l i n i z e d v a c c i n e , it r e m a i n s t o be d e t e r m i n e d w h e t h e r or n o t single v a c c i n a t i o n or t w o v a c c i n a t i o n s given at l o n g e r intervals will p r o v i d e a significant a n d lasting p r o t e c t i o n . ACKNOWLEDGEMENTS T h e a u t h o r s are v e r y g r a t e f u l t o t h e O n t a r i o C a t t l e m e n ' s A s s o c i a t i o n a n d t h e O n t a r i o Ministry o f A g r i c u l t u r e a n d F o o d f o r p r o v i d i n g s u p p o r t f o r this p r o j e c t . Dr. B r u c e C h i c k k i n d l y p e r f o r m e d t h e n e c r o p s y e x a m i n a t i o n s .

REFERENCES Askaa, G. and Erno, H., 1976. Elevation ofMycoplasma agalactiae subsp, boris to species rank: Mycoplasma boris (Hale et al.). Int. J. Syst. Bacteriol., 26: 323--325. Cho, H.J., Ruhnke, H.L. and Langford, E.V., 1975. The indirect haemagglutination test for the detection of antibodies in cattle naturally infected with mycoplasma. Can. J. Comp. Med., 40: 20--29. Cole, B.C., Cahill, J.F., Wiley, B.B. and Ward, J.R., 1969. Immunological response of the rat to Mycoplasma arthritidis J. Bacteriol., 98: 930--937. Davies, G., 1967. Isolation of Mycoplasma from calf lungs. J. Comp. Pathol., 77: 353-357. Dyson, D.A. and Smith, G.R., 1975. The virulence of strains ofMycoplasma mycoides var. mycoides recovered from inoculated cattle. Res. Vet. Sci., 18: 115--116. Fernald, G.W. and Clyde, W.A., 1970. Protective effect of vaccines in experimental Mycoplasma pneumoniae disease. Infect. Immun., 1: 559--565. Foggie, A., Etheridge, J.R., Erdag, O. and Arisoy, F., 1970. Contagions agalactia in sheep and goats. Preliminary studies on vaccines. J. Comp. Pathol., 80: 345--358. Goodwin, R.R.W. and Whittlestone, P., 1973. Enzootic pneumonia in pigs. Immunization attempts inoculating Mycoplasma suipneumoniae antigen by various routes and with different adjuvants. Br. Vet. J., 129: 459--464. Gourlay, R.N., 1972. Discussion in "Pathogenic Mycoplasma". A Ciba Foundation symposium. London, 25--27 January. Elsevier Scientific Publishing Company, Amsterdam, pp. 371--372. Hale, H.H., Helmboldt, C.F., Plastridge, W.N. and Stula, E.P., 1962. Bovine Mastitis caused by Mycoplasma species. Cornell Vet., 52: 582--591. Hjerpe, C.A. and Knight, H.D., 1972. Polyarthritis and synovitis associated with Mycoplasma bovimastitidis in feedlot cattle. J. Am. Vet. Assoc., 160: 1414--1418. Howard, C.J., Gourlay, R.N. and Taylor, G., 1977. Induction of immunity in calves to Mycoplasma boris infection of the respiratory tract. Vet. Microbiol., 2: 29--37.

122 Hudson, J.R., 1965. Contagious bovine pleuropneumoniae: The immunizing value of the attenuated strain KH3J. Aust. Vet. J., 41: 43--49. Jasper, D.E., Jain, N.C. and Brazil, L.H., 1966. Clinical and laboratory observations on bovine mastitis due to mycoplasma. J. Am. Vet. Med. Assoc., 148: 1017--1029. Lloyd, L.C. and Trethewie, E.R., 1970. Contagious bovine pleuropneumonia. In: J.T. Sharp (Editor), The Role of Mycoplasrna and L-Forms of Bacteria in Disease. Thomas, Springfield, IL, pp. 172--197. Masiga, W.N. and Mugera, G.M., 1973. Fate of the T1 strain of Mycoplasma mycoides in cattle following vaccination. J. Comp. Pathol., 83: 473--479. Miles, A.A., Misra, S.S. and Irwin, J.O., 1938. Estimation of the bacterial power of blood. J. Hyg. (Camb.), 38: 732--748. Mogabgab, W.J., 1973. Efficacy of inactivated Mycoplasma pneumoniae vaccine demonstrated b y protection in large field trials. Ann. N.Y. Acad. Sci., 225: 453--461. Popovici, I. and De Simon, M., 1966. La vaccino-prophylaxie de l'agalaxie contagieuse des hrebis et des chevres en Roumanie. Arch. Vet. (Buchar.), I: 21--26. Senterfit, L.B. and Jensen, K.E., 1967. Progress in the immunoprohylaxis of Mycoplasma pneumoniae infection. Ann. N.Y. Acad. Sci., 143: 471--483. Singh, U.M., Doig, P.A. and Ruhnke, H.L., 1971. Mycoplasma arthritis in calves. Can. Vet. J., 12: 183--185. Smith, C.B., Charlock, R.M., Friedwald, W.T. and Alford, R.H., 1967. Mycoplasma pneumoniae infections in volunteers. Ann. N.Y. Acad. Sci., 143: 471--483. Somerson, N.L., Senterfit, L.B. and Hamparian, V.V., 1973. Development of Mycoplasma pneumoniae vaccine. Ann. N.Y. Acad. Sci., 225: 425--435. Stalheim, O.H.V., 1976. Failure of antibiotic therapy in calves with mycoplasma arthritis and pneumonia. J. Am. Vet. Med. Assoc. 169: 1096--1097. Wenzel, R.P., Craven, R.B., Davies, J.A., Hendley, J.C., Hamory, H.B. and Gwaltney, J. M., 1976. Field trial of inactivated Mycoplasma pneumoniae vaccine. 1. Vaccine efficacy. J. Infect. Dis., 134: 571--576.