A New Mycoplasma Species Isolated From Pneumonic Lungs Of Calves (Mycoplasma Dispar Sp. Nov.)

Abstract

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...

The production of mastitis in cows by the intramammary inoculation of T-mycoplasmas

“…This was similar to GS broth (Gourlay & Leach, 1970) units (c.c.u.). The titre was the highest dilution that produced a colour change after incubation at 370 C. Control inocula were either uninoculated broth or broth culture of T-mycoplasmas which had been incubated at 370 C. until viable organisms could no longer be demonstrated (dead organisms).…”

“…Four strains of T-mycoplasmas were used: the A417 strain that had been isolated from a pneumonic calf lung (Gourlay & Thomas, 1970), the D32 strain that had also been isolated from a pneumonic calf lung (calf 24, table 1, Gourlay, Mackenzie & Cooper, 1970), the REOW strain isolated from the human urogenital tract and supplied by Dr D. Taylor-Robinson, and the M 126/68 strain also isolated from the human urogenital tract and supplied by Dr B. E. Andrews. Two different substrains of the A 417 strain were used; the first was at the 10-11 dilution from the original lung tissue while the second -'cloned' -had undergone six subcultures in broth of which three had been from the terminal dilution, as a means of purification, and was at a 10-25 dilution from the original lung.…”

Serological comparison and haemagglutinating activity ofMycoplasma dispar

“…Mycoplasmas were grown in glucose calf-serum (GS) broth (Gourlay & Leach, 1970) containing ampicillin (Andrews, Leach, Gourlay & Howard, 1973) except when organisms were grown for injection into rabbits, in which case rabbit digest broth and rabbit serum, heated at 56°C. for 30 min.…”

“…M. hominis (Purcell et al 1967) M. gallisepticum (Taylor-Robinson & Berry, 1969) and M. pulmonis (Forshaw & Fallon, 1972) have shown these species to be serologically heterogeneous and to contain various subtypes. No serological comparison of a group of M. dispar strains has been made except for that of Gourlay & Leach (1970) who found that antiserum to strain 462/2 inhibited the growth on agar of 23 out of 36 strains examined. As little information is available on the serological heterogeneity or homogeneity of the species the first part of this study deals with this aspect of M. dispar.…”

“…Mycoplasma dispar was first isolated from pneumonic calf lungs (Gourlay, 1969;Gourlay & Leach, 1970) and subsequently from the respiratory tract of nonpneumonic calves (Thomas & Smith, 1972). Its role in calf pneumonia remains to be determined.…”