ENHANCEMENT OF PROPAGATION OF MYCOPLASMA HYOPNEUMONIAE BY CULTURE IN A BIPHASIC MEDIUM

Fischer, 0.: Enhancement of Propagation of Mycoplasma hyopneumoniae by Culture in a Biphasic Medium. Acta vet. Bmo, 1995,64:243-247. Mycoplasma hyopneumoniae (strain J) was propagated in a liquid and a biphasic media. The solid phase of the biphasic medium was obtained by the addition of 1.9% or 0.19% of agar to the liquid medium. Compared with the liquid medium, a more rapid propagation of mycoplasmas was observed in the biphasic medium after two weeks of incubation. After three weeks of incubation in the liquid and the biphasic medium containing 0.19% of agar in the solid phase, the numbers of mycoplasmas rose 2.7fold and 4.7fold, respectively. The biphasic medium, simulating partly the environment of the porcine respiratory tract, supported better the propagation of M. hyopneumoniae than the liquid medium. The biphasic medium containing 0.19% of agar in the solid phase should be preferred when a mass of Mycoplasma cells is to be obtained. Porcine enzootic pneumonia, culture media, biomass The causal agent of porcine enzootic pneumonia Mycoplasma hyopneumoniae ranks with Mycoplasma species which are difficult to propagate (Friis 1975, 1979). On solid media, this species does not form the "fried eggs" colonies typical of a majority of mycoplasmas, but rather small, dispersed bodies resembling poppy-seeds. The propagation in liquid media is very slow, taking several weeks. Larger amounts of Mycoplasma cells can be obtained only by gradually increasing the volume of the culture medium (Kuksa et al. 1987). M. hyopneumoniae lives on the surface of porcine respiratory mucosae (MeyIing 1971; Kobisch et al. 1993). The mucus of the respiratory tract protects M. hyopneumoniae cells from drying in the environment thus supporting the spread of porcine enzootic pneumonia by droplet infection (Goodwin 1972; Friis 1973). As a typical mucosal pathogenic agent, M. hyopneumoniae is adapted to an environment which is more viscous than are the conventional liquid media used for its culture in vitro. A biphasic culture medium, simulating partly the conditions prevailing in the porcine respiratory tract, was used to provide more favourable conditions for the culture of M. hyopneumoniae in vitro. Materials and Methods The strain] of Mycoplasma hyopneumoniae (type strain NCI'C 10110, ATCC 25934) was obtained from the Collection of Animal Pathogenic Microorganisms, Bmo (CAPM M-38).The biphasic culture medium consisted of solid and liquid phases. Liquid phase The medium FF (Friis, 1971) was modified as follows: Saline A: 160 g NaCI, 8 g KCI, 2 g MgS04.7H20, 2 g MgC~.6H20, and 2.8 g anhydrous CaC~ were dissolvt;ld in 1 L bidistilled water. Saline B: 3 g N~HP04.12~0 and 1.2 g ~P04 were dissolved in 1 L bidistilled water.

The causal agent of porcine enzootic pneumonia Mycoplasma hyopneumoniae ranks with Mycoplasma species which are difficult to propagate (Friis 1975(Friis , 1979)).On solid media, this species does not form the "fried eggs" colonies typical of a majority of mycoplasmas, but rather small, dispersed bodies resembling poppy-seeds.The propagation in liquid media is very slow, taking several weeks.
Larger amounts of Mycoplasma cells can be obtained only by gradually increasing the volume of the culture medium (Kuksa et al. 1987).
M. hyopneumoniae lives on the surface of porcine respiratory mucosae (MeyIing 1971; Kobisch et al. 1993).The mucus of the respiratory tract protects M. hyopneumoniae cells from drying in the environment thus supporting the spread of porcine enzootic pneumonia by droplet infection (Goodwin 1972;Friis 1973).As a typical mucosal pathogenic agent, M. hyopneumoniae is adapted to an environment which is more viscous than are the conventional liquid media used for its culture in vitro.
A biphasic culture medium, simulating partly the conditions prevailing in the porcine respiratory tract, was used to provide more favourable conditions for the culture of M.

Materials and Methods
The strain] of Mycoplasma hyopneumoniae (type strain NCI'C 10110, ATCC 25934) was obtained from the Collection of Animal Pathogenic Microorganisms, Bmo (CAPM M-38).The biphasic culture medium consisted of solid and liquid phases.
The stock salines A and B were diluted by adding to 7.5 ml125 ml and 17.5 ml bidistilled water, respectively.Then the salines were mixed and 2.6 g Mycoplasma Broth Base (Oxoid) and 2.5 g Brain Heart Infusion (Oxoid) were added.The mixture was completed with 225 ml bidistilled water and autoclaved at 121°C and 110 kPa for 30 min.After cooling, 78 ml horse blood serum, 18 ml yeast extract, 5 mllO% (w/v) glucose and I ml5% (w/v) thalium acetate solutions and 200 mg ampicillin were added.pH was adjusted to 7.6.

Solid phase
The liquid medium FF was completed with 4.5 or 0.45 g Noble agar (Difco) before autoclaving, supplemented with the same components as the liquid medium FF after autoclaving and cooling to 50°C and poured into culture vessels.The agar concentrations in the solid media were 1.9% and 0.19%, respectively.
The solid medium for counting M. hyopneumoniae bodies was prepared by dissolving 4 g PPLO agar (Difco) in 105 ml bidistilled water.The medium was completed with 30 ml horse blood serum, 15 ml yeast extract, 100 mg ampicillin and 0.75 ml5% thalium acetate after autoclaving and cooling to 50°C.The hot medium was poured into plastic 60mm Petri dishes to form a 2-mm layer.
Comparison of M. hyopneumoniae propagation in liquid and biphasic media Experiment 1 Forty ml of the liquid medium or forty ml of the hot solid phase containing 1.9% agar were poured into lOO-ml glass bottles.The agar was left to solidify in slant position (Fig. 1).After warming to 37°C, the media were inoculated with 5 ml of aM.hyopneumoniae suspension containing 2.9 x lOS bodies per I ml and the cultures were incubated at 37°C.Forty ml offresh liquid medium were added after 3 days incubation and 0.2 ml of the suspension were collected and inoculated onto the solid medium for the counting of M. hyopneumoniae bodies after another 14 days.

Experiment 2
Forty ml of the liquid medium or forty ml of the hot solid phase containing 0.19% agar were poured into lOO-ml glass bottles.The agar was left to solidify in slant position (Fig. 1).After warming to 37°C, the media were inoculated with 5 ml of a M. hyopneumoniae suspension containing 1.2 x lOS bodies per 1 ml and the cultures were incubated at 37°C.Forty-five ml of fresh liquid medium were added after 3 days of incubation and 0.2 ml to 0.4 ml of the suspension were collected and inoculated onto the solid medium for the counting of M. hyopneumoniae bodies at weekly intervals during the three-week incubation period.The experiment was repeated twice.

Counting of M. hyopneumoniae bodies
A known volume (0.2 ml to 0.4 ml) of a well shaken Mycoplasma suspension was applied onto the solid medium in a 60-mm Petri dish to cover the whole surface of the medium.The dishes were left to stand for 30 min and then incubated at 37°C for 3 days.
M. hyopneumoniae bodies were counted at 10 randomly selected sites at the magnification 630x using an eyepiece screen.

Sterility checks
A sample of the suspension was collected at each opening of a culture vessel, inoculated onto blood agar and incubated at 37°C.Attention was paid to Mycoplasma colonies suggestive of contamination at the counting of the M. hyopneumoniae bodies.

Results
Mycoplasma hyopneumoniae propagated in both the liquid and the biphasic media and M. hyopneumoniae bodies resembling dispersed poppy-seeds were observed with a light microscope after reinoculation onto the solid medium.

Experiment 1
The number of M. hyopneumoniae bodies increased 1.5fold (from 2.9 x 10 5 bodies per 1 ml of inoculum to 4.5 x 10 5 bodies per 1 ml, P < 0.01) and 4.8fold (to 1.4 x 10 6 bodies per 1 ml, P < 0.01) during the 14-day incubation in the liquid and the biphasic media, respectively.The medium containing 1.9% agar disintegrated when the culture vessels were shaken, but large fragments of solid agar remained in the liquid phase.During the second week, the numbers of mycoplasmas rose twofold (from 1.2 x lOS to 2.5 X 10 5 bodies per 1 mI, P < 0.01) and threefold (to 3.9 x 10 5 bodies per 1 mI, P < 0.01) in the liquid and the biphasic media, respectively.Further 2.7fold (3.3 x 10 5 bodies per 1 mI, P < 0.01) and 4.7fold (5.7 x 10 5 bodies per 1 mI, P < 0.01) increases were recorded after three weeks of incubation in the liquid and the biphasic media, respectively.The 0.19% agar dissolved almost completely in the liquid medium.No contamination by bacteria or colony-forming Mycoplasma species was demonstrable.Bruggmann et al. (1977) demonstrated M. hyopneumoniae in frozen lung sections collected from swine suffering from enzootic porcine pneumonia by the immunoperoxidase reaction.M. hyopneumoniae cells were visible as reddish brown pleiomorphic spots on the mucosa of the respiratory tract and on the surface of scraped-off epithelial cells.Zielinski et al. (1990) observed adhesion of M. hyopneumoniae to porcine and human cell cultures.The adhesion to the porcine cells was more intensive.De Bey and R 0 s s (1994) described M. hyopneumoniae-induced ciliostasis and loss of ciliae in cells of organ cultures prepared from porcine tracheal rings.
The culture in a biphasic medium combines the advantages of the liquid and the solid culture media and partly simulates the conditions prevailing in the porcine respiratory tract.While mycoplasmas grown in a liquid medium can adhere only to the glass surface of culture vessels, in a biphasic medium they can fIrst propagate in the nutrient-rich agar and the continuing growth is supported by the addition of fresh liquid medium providing further nutrients and diluting metabolic products of mycoplasmas.The semisolid agar swells in the liquid medium and eventually dissolves almost completely.Mycoplasma bodies are thus released into the liquid phase of the medium and can be harvested by washing and the centrifugation.The remains of the undissolved agar area very suitable substrate for the propagation of mycoplasmas.
M. hyopneumoniae propagates slowly in liquid media, fermenting glucose and trehalose, but not saccharose, lactose and mannitol (Bannerman and Nicolet 1971), and can be distinguished from the non-pathogenic, slowly growing and glucose fermenting Comparative morphologic studies of M. hyopneumoniae and M. flocculare were made by Hovind-Hougen and Friis (1991).Both the species carried on their surfaces fIne, 30 to 100 nm long hairs, were surrounded by a simple membrane consisting of three layers and propagated by binary fIssion.The size of the spherical or oval bodies of M. hyopneumoniae varied between 0.8 and 2.5 /lm, being within the considerably broader range 0.4 to 4.0 /lffi of M. flocculare.Unlike M. hyopneumoniae, M. flocculare formed fIlamentous cords between cells measuring 140 x 30 to 90 nm.However, special microscopic techniques are necessary for the recognition of this difference.Tajima and Yagihashi (1982), Blanchard et al. (1992), Jacques et al. (1992) in their electron microscopic studies of mycoplasmas found M. hyopneumoniae apparently lying free in the bronchiolar lumen of pigs, but in close contact with tips of several microvilli.Very fIne fIbrils, measuring 0.005 /lffi in diameter and 0.25 /lm in length, linked mycoplasmas with microvilli or cilia of host cells and interconnected also individual Mycoplasma bodies.Mycoplasmas propagated in a liquid medium lacked the fIbrils.The size of the bodies grown in the liquid medium and found in the porcine respiratory tract ranged from 0.4 to 1.2 /lffi and from 0.5 to 1.0 /lffi, respectively.While spherical and oval forms prevailed among mycoplasmas propagated in vitro, those observed in vivo among host cell cilia were elongated.
All the media were warmed to 37°C prior to inoculation to avoid cold shock.The modifIed medium FF has proven effective for the propagation of M. hyopneumoniae.Although the counts decreased in the initial phase of incubation due to dilution and death of a part of the inoculum, a marked increase could be recorded after the adaptation of mycoplasmas to the culture conditions in the 2nd and 3rd weeks of culture.The experiments were discontinued after two or three weeks of incubation, because the quality of the media decreased owing to the accumulation of toxic metabolic products of mycoplasmas and dead, disintegrating cells after prolonged incubation.
Although a part of the mycoplasma culture adhered to undissolved remains of agar gel upon the harvest, the yield from the propagation in the biphasic medium was higher than that from cultures in liquid media.