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An increase in deaths in calves from respiratory disease from an average of 9.7 per year to 36.5 per year corresponded with the isolation of Mycoplasma bovis from the lungs. It is suggested that this mycoplasma enhanced the severity of the disease which was normally present on the farm. The characteristic microscopic lesion and demonstration of M bovis by immunoperoxidase labelling could be useful aids to diagnosis.

PLATES VIII-X T Y P I c A L large-colony mycoplasmas are prevalent in the upper respiratory tract of normal cattle and in the lungs of calves with pneumonia (Davies, 1967; Gunther et al., 1967;Leach, 1967) and mycoplasma T-strains have also been isolated from the latter source (Gourlay, 1968).These organisms can be cultivated by the use of the appropriate conventional mycoplasma medium, but such media are not suitable for cultivation of all types of mycoplasma, and several mycoplasmas found in animals require special media for primary isolation. A relevant example is the mycoplasma causing enzootic pneumonia in pigs, named Mycoplasma hyopneumoniae (Mare and Switzer, 1965) or Mycoplasma suipneumoniae (Goodwin, Pomeroy and Whittlestone, 1965). This organism can be cultivated in a special acellular medium modified from tissue-culture fluids, but cannot be grown in conventional mycoplasma medium of the type described by Chanock, Hayflick and Barile (1962). This suggested that there might be unrecognised mycoplasmas in other animals, and that they might be detected by the use of similar media. With this in mind we used a modification of the medium of Goodwin et al. (1965) in attempts to isolate mycoplasmas from the lungs of calves, as part of a current microbiological and pathological investigation of calf pneumonia at Compton. Strains of an unusual type of organism were isolated in this medium. They have the general characteristics of mycoplasmas, but with some atypical features, and do not appear to have been described before. A preliminary report on the isolation of these organisms has been published (Gourlay, 1969). The pH was adjusted to 7.8 with N-NaOH. Solid medium (GS agar) was prepared by the addition of These solutions were made in glass-distilled double-deionised water. Tests were carried out in the acellular fluid or solid media for M. suipneumoniae described by Goodwin et al. (1967), which will be referred to as the Cambridge media. Cambridge broth differs from GS broth in containing pig serum and yeast extract but not foetal calf serum or calf thymus DNA, and in the absence of added glucose. The corresponding solid medium is made by the addition of Oxoid Ion Agar to Cambridge broth.Isolation procedure. Pieces of lung from a pneumonic area when this was present (about 0.5 cm3) were triturated in 5 ml phosphate-buffered saline (pH 7-4) and serial ten-fold dilutions were prepared; 0.2 ml quantities were inoculated into 1.8 ml amounts of GS broth. The inoculated broths were incubated at 37°C for at least 3 wk and were examined at intervals for evidence of growth, as indicated by an acid change in pH. A drop of culture from each of the tubes in which such a colour change occurred was plated on GS agar, which was examined microscopically for colonies during a similar period of incubation. Solid media were incubated in a mixture of 5 per cent. CO2 in nitrogen.Staining methods. Films prepared from centrifuged deposits of broth cultures (25,0009 for 30 min.) were fixed in methanol and stained for 2 hr at 56...

SUMMARYSix milking cows were inoculated with bovine and human T-mycoplasmas and control materials into the udder via the teat canal. Control materials produced only a slight transient cell response in the milk. Bovine T-mycoplasmas produced clinical mastitis in nine out of ten quarters inoculated. Seven developed clinical mastitis together with visible changes in the milk, excretion of T-mycoplasmas and greatly increased cell counts in the milk. In three of these quarters, in two different cows, milk secretion ceased completely. Two quarters in a different cow showed visible milk changes, excretion of T-mycoplasmas and increased cell counts. Two quarters were inoculated with human T-mycoplasmas and neither produced any signs of mastitis.Infection of the udder with T-mycoplasmas did not stimulate high-titre serum antibody levels as measured by the metabolic inhibition test, but whey samples gave high titres in two of the cows that were able to control and resolve the infection.

A field trial to assess the ability of two vaccines to protect calves against respiratory disease was carried out on a large beef rearing unit in southern England over the two winters of 1983 to 1984 and 1984 to 1985. A quadrivalent vaccine containing the killed antigens of respiratory syncytial virus, parainfluenza virus type 3, Mycoplasma bovis and M dispar or a vaccine containing only the respiratory syncytial virus component were inoculated into 246 and 245 calves, respectively; 245 calves remained as unvaccinated controls. The calves were reared in seven batches and outbreaks of disease occurred in five; significant protection was achieved in the four batches in which disease was associated with respiratory syncytial virus and M bovis infection, together or independently. The death rate from pneumonia was 9 per cent in the control group, 2 per cent in the calves inoculated with the quadrivalent vaccine (P less than 0.001), a protection rate of 77 per cent, and 3 per cent in the calves inoculated with the respiratory syncytial virus vaccine (P less than 0.01), a protection rate of 68 per cent. The proportion of calves receiving treatment for respiratory disease was 38 per cent in the control group, 25 per cent in the calves inoculated with the quadrivalent vaccine (P less than 0.001) and 27 per cent in the calves inoculated with the respiratory syncytial virus vaccine (P less than 0.01). The results show that protection against respiratory disease can be achieved by parenteral vaccination of calves with the appropriate inactivated microorganisms.