Temperature Sensitivity Studies on Selected Strains of Mycoplasma gallisepticum R. B. Truscott and 1. R. Patel*
ABSTRACT
Mycoplasma gallisepticum (MG324), a tylosin resistant strain of low virulence, was com. pared with four other strains with respect to their survival at temperatures from 46.1 to 48.9°C. MG324 was found to be more resistant than the other strains tested.
RESUME Cette experience visait a comparer entre elles la souche MB 324 de Mycoplasma gallisepticum, resistante a la tylosine et peu virulente, et quatre autres souches de ce mycoplasme, relativement 'a leur survivance 'a des temperatures allant de 46.1 a 48.9°C. Ia souche MG 324 s'ave'ra plus resistante que les autres.
normal incubator temperature and incubated. Field reports have suggested that this procedure is quite effective. In 1970 we began an investigation of suspicious serum plate reactions in a flock that had previously been tested and found negative. This flock had originated from an MG-free flock and had been considered to be free of this infection. On retest of the flock positive serum plate reactions were present as well as some low hemagglutination-inhibition (HI) reactions. Following culture of several seropositive birds an MG was recovered from the tracheas of some and the air sacs of others. When compared to MGS6 these isolates were shown to be tylosin resistant (4). The following study was carried out to compare the effect of elevated temperatures on a representative isolate, designated MG324, with other strains of M. gallisepticum.
MATERIALS AND METHODS INTRODUCTION In general Mycopla.sma gallisepticum (MG) has been eliminated from infected chicken flocks in one of two ways. In one, an antibiotic such as tylosin is used either for treatment of the laying flock and/or for the treatment of eggs either by dipping (2) or injection (5) while the other is the heating procedure developed by Yoder (6). In this latter procedure fertile eggs are heated from room temperature to 45.8°C over a twelve hour period, cooled to *Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario, Canada. Submitted July 4, 1975.
Volume 40
-
January, 1976
The experimental protocol is shown in Fig. 1. CULTURES The S6 and Schg7 were obtained from L. Ruhnkel who had received the strains from Dr. Bigland and Dr. Adler respectively, while strains 5969 and 801 were obtained from Dr. Yoder'. MEDIA The mycoplasma broth and agar used throughout were formulated as described by Davies (1). 1V.terinary Services
Laboratory, Ontario Ministry of Agriculture and Food, Guelph, Ontario. 'Southeast Poultry Research Laboratories, 934 College Station Road, Athens- Georgia.
89
TABLE I. Survival of Two Strains of M. galliepticum at 46.1°C Dilution Min 0 20 40 60 *No. positive of 9
10-5
S6
324 10-6
10-7
9a
9 9 6 9 3 3 0 1 (triplicate tubes x 3 tests) 9 3 3
Total
l0-
10-
10-7
Total
27 24 9 4
9 7 4 2
9 3 1 0
0 3 0 0
27 13 5 2
TABLE II. Survival of Two Strains of M. gallisepticum at 46.7 - 48.9°C 324 Time - Min 5 20 Total CFU/ML& 15 10 5 Temp °C 14 2.9 52 10 12 15 15b 46.7 8 1.2 22 1 3 11 7 47.2 12 2.7 11 0 1 1 9 47.8 2 2.7 11 0 2 2 7 48.3 2 2.9 3 0 0 0 3 38.9 38 99 11 18 25 45 Total Colony forming units, count obtained from primary broth x 108 bNo. positive of 15 (triplicate tubes x 5 dilutions, 105 - 109)
20 Hr Broth Culture
Dilutions Made (10
to 10
)
4 ml (x3) to Heated Bath Time
Exposure (5 - 60 Min )
Cooled Incubated Positive Reactions (Colour Change)
Questionable Colour Change Plated or at 3 Wks
if Negative Plated
Negative for Colour Change at 4 Wks
Discarded
Fig. 1. Protocol for temperature sensitivity studies.
90
S6 Time - Min 20 15 10 9 9 14 3 3 2 3 3 4 0 1 1 0 1 1
22
17
15
Total CFU/ML
46 16 22 4 4 92
4.7 1.3 5.8 3.5 2.2
DILUTIONS Dilutions in broth (Fig. 1) of 20 hour broth cultures were dispensed in triplicate four ml amounts in screw capped tubes. Whenever plate counts were made, 0.02 ml of the appropriate dilution was transferred with an Eppendorf pipette to agar plates which-were incubated in 10% CO2 at 370C. TEMPERATURE STUDIES The tubes containing the various dilutions were placed in a Labline3 shaking water bath after the water obtained a temperature of 0.60 C above the test temperature (46.1, 46.7, 47.2, 47.8, 48.3 or 48.9°C) and were shaken at the rate of 120 oscillations per minute. The increase of 0.6° was found to reduce the equilibration time to about one minute. Temperature was monitored using a thermoelectric potentiometer4 with thermocouple leads in two control tubes and the bath. The time period commenced when the control tubes reached the desired temperature and the temperature was maintained within + 0.30 by manual operation of the heater switch. Following heating the tubes were cooled to 370C in a water bath and then incubated aerobically at this temperature. Survival was in3Canadian ario.
Laboratory
Supplies Limited, Toronto, Ont-
4Thermo Electric, Brampton, Ontario.
Can. J. comp. Med.
TABLEIIII. Estimated Numbers of Organisms in Broth Cultures Used in Heating Trials Temp. (0C) 47.2 47.8 48.3 48.9 a x 108
324
6.5a
3.8 4.2 2.9
S6 9.7 11.0 7.3 4.2
Strain Schg7 5.7 7.6 5.2 5.5
5969 801 11.0 1.2 9.2 0.9
10.0 8.0 8.7 3.6
dicated by a definite colour change in the medium. Questionable growth was checked by plating on agar. All tubes negative at three weeks were plated before being discarded at four weeks. STATISTICAL ANALYSIS The results were analyzed using Duncan's analysis of variance as described by Malik and Mullen (3).
RESULTS In the first series of experiments 324 and S6 were compared at 46.1°C for 20, 40 and 60 min. A statistically significant difference (p < .05) in the survival of the two strains was apparent at 20 min (Table I) and a linear relationship was shown between time and survival. The questions that arose concerned the number of organisms present in the higher dilutions and the effect of this on the results. An attempt was made to evaluate this in the second series of experiments where the same strains were compared at 46.7°-48.9°C for five, ten, 15 and 20 min. The results presented in Table II were analyzed with respect to differences between temperatures, time and dilutions and indicated highly significant differences (p
ABSTRACT
Mycoplasma gallisepticum (MG324), a tylosin resistant strain of low virulence, was com. pared with four other strains with respect to their survival at temperatures from 46.1 to 48.9°C. MG324 was found to be more resistant than the other strains tested.
RESUME Cette experience visait a comparer entre elles la souche MB 324 de Mycoplasma gallisepticum, resistante a la tylosine et peu virulente, et quatre autres souches de ce mycoplasme, relativement 'a leur survivance 'a des temperatures allant de 46.1 a 48.9°C. Ia souche MG 324 s'ave'ra plus resistante que les autres.
normal incubator temperature and incubated. Field reports have suggested that this procedure is quite effective. In 1970 we began an investigation of suspicious serum plate reactions in a flock that had previously been tested and found negative. This flock had originated from an MG-free flock and had been considered to be free of this infection. On retest of the flock positive serum plate reactions were present as well as some low hemagglutination-inhibition (HI) reactions. Following culture of several seropositive birds an MG was recovered from the tracheas of some and the air sacs of others. When compared to MGS6 these isolates were shown to be tylosin resistant (4). The following study was carried out to compare the effect of elevated temperatures on a representative isolate, designated MG324, with other strains of M. gallisepticum.
MATERIALS AND METHODS INTRODUCTION In general Mycopla.sma gallisepticum (MG) has been eliminated from infected chicken flocks in one of two ways. In one, an antibiotic such as tylosin is used either for treatment of the laying flock and/or for the treatment of eggs either by dipping (2) or injection (5) while the other is the heating procedure developed by Yoder (6). In this latter procedure fertile eggs are heated from room temperature to 45.8°C over a twelve hour period, cooled to *Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario, Canada. Submitted July 4, 1975.
Volume 40
-
January, 1976
The experimental protocol is shown in Fig. 1. CULTURES The S6 and Schg7 were obtained from L. Ruhnkel who had received the strains from Dr. Bigland and Dr. Adler respectively, while strains 5969 and 801 were obtained from Dr. Yoder'. MEDIA The mycoplasma broth and agar used throughout were formulated as described by Davies (1). 1V.terinary Services
Laboratory, Ontario Ministry of Agriculture and Food, Guelph, Ontario. 'Southeast Poultry Research Laboratories, 934 College Station Road, Athens- Georgia.
89
TABLE I. Survival of Two Strains of M. galliepticum at 46.1°C Dilution Min 0 20 40 60 *No. positive of 9
10-5
S6
324 10-6
10-7
9a
9 9 6 9 3 3 0 1 (triplicate tubes x 3 tests) 9 3 3
Total
l0-
10-
10-7
Total
27 24 9 4
9 7 4 2
9 3 1 0
0 3 0 0
27 13 5 2
TABLE II. Survival of Two Strains of M. gallisepticum at 46.7 - 48.9°C 324 Time - Min 5 20 Total CFU/ML& 15 10 5 Temp °C 14 2.9 52 10 12 15 15b 46.7 8 1.2 22 1 3 11 7 47.2 12 2.7 11 0 1 1 9 47.8 2 2.7 11 0 2 2 7 48.3 2 2.9 3 0 0 0 3 38.9 38 99 11 18 25 45 Total Colony forming units, count obtained from primary broth x 108 bNo. positive of 15 (triplicate tubes x 5 dilutions, 105 - 109)
20 Hr Broth Culture
Dilutions Made (10
to 10
)
4 ml (x3) to Heated Bath Time
Exposure (5 - 60 Min )
Cooled Incubated Positive Reactions (Colour Change)
Questionable Colour Change Plated or at 3 Wks
if Negative Plated
Negative for Colour Change at 4 Wks
Discarded
Fig. 1. Protocol for temperature sensitivity studies.
90
S6 Time - Min 20 15 10 9 9 14 3 3 2 3 3 4 0 1 1 0 1 1
22
17
15
Total CFU/ML
46 16 22 4 4 92
4.7 1.3 5.8 3.5 2.2
DILUTIONS Dilutions in broth (Fig. 1) of 20 hour broth cultures were dispensed in triplicate four ml amounts in screw capped tubes. Whenever plate counts were made, 0.02 ml of the appropriate dilution was transferred with an Eppendorf pipette to agar plates which-were incubated in 10% CO2 at 370C. TEMPERATURE STUDIES The tubes containing the various dilutions were placed in a Labline3 shaking water bath after the water obtained a temperature of 0.60 C above the test temperature (46.1, 46.7, 47.2, 47.8, 48.3 or 48.9°C) and were shaken at the rate of 120 oscillations per minute. The increase of 0.6° was found to reduce the equilibration time to about one minute. Temperature was monitored using a thermoelectric potentiometer4 with thermocouple leads in two control tubes and the bath. The time period commenced when the control tubes reached the desired temperature and the temperature was maintained within + 0.30 by manual operation of the heater switch. Following heating the tubes were cooled to 370C in a water bath and then incubated aerobically at this temperature. Survival was in3Canadian ario.
Laboratory
Supplies Limited, Toronto, Ont-
4Thermo Electric, Brampton, Ontario.
Can. J. comp. Med.
TABLEIIII. Estimated Numbers of Organisms in Broth Cultures Used in Heating Trials Temp. (0C) 47.2 47.8 48.3 48.9 a x 108
324
6.5a
3.8 4.2 2.9
S6 9.7 11.0 7.3 4.2
Strain Schg7 5.7 7.6 5.2 5.5
5969 801 11.0 1.2 9.2 0.9
10.0 8.0 8.7 3.6
dicated by a definite colour change in the medium. Questionable growth was checked by plating on agar. All tubes negative at three weeks were plated before being discarded at four weeks. STATISTICAL ANALYSIS The results were analyzed using Duncan's analysis of variance as described by Malik and Mullen (3).
RESULTS In the first series of experiments 324 and S6 were compared at 46.1°C for 20, 40 and 60 min. A statistically significant difference (p < .05) in the survival of the two strains was apparent at 20 min (Table I) and a linear relationship was shown between time and survival. The questions that arose concerned the number of organisms present in the higher dilutions and the effect of this on the results. An attempt was made to evaluate this in the second series of experiments where the same strains were compared at 46.7°-48.9°C for five, ten, 15 and 20 min. The results presented in Table II were analyzed with respect to differences between temperatures, time and dilutions and indicated highly significant differences (p
