SHORT COMMUNICATION
Br. vet.}. (197 7), 133, 3 18
RECOVERY OF TRICHINELLA SPIRALIS LARVAE By G.jACKSON*
Veterinary Investigation Centre, Moulton, Northampton
SUMMARY
It is suggested that muscle samples submitted for the demonstration of Trichinella spira/is by the peptic digestion method should not be frozen .
INTRODUCTION
Veterinary Investigation Centres of the Ministry of Agriculture, Fisheries and Food (MAFF) have been engaged in a survey of pig diaphragmatic muscle for evidence of T. spira/is . The receipt of muscle which had been frozen at minus 8°C for three to six days prompted enquiry into the effects of such procedures on the demonstration of T. spira/is using a peptic digestion method, since it is known that freezing (20 days at -15°C, 10 days at-23·3°C, and six days at-37 ·2°C ) kills encysted T. spira/is (Ransom, 1916). . .
MATERIALS AND METHODS
T. spira/is isolated at Penrith in 1939, and maintained at the London School of Hygiene and Tropical Medicine, was used to infect a pig at the Central Veterinary Laboratory, Weybridge. The pig received an oral dose of 700 larvae per kg bodyweight. Seven weeks later the pig was slaughtered. A portion of hind leg was received two days later. Skeletal muscle was cut into pieces (approximately 25 mm cubes) and these were well mixed and divided into four samples of approximately 150 g each and then weighed. The samples were stored in plastic bags in four separate sites ; namely in a deep freeze (- 18°C), in a refrigerator (-8°C and +4°C) and in a store room (+ 18°C). After six days both frozen samples were thawed overnight at + 4°C. The following morning the samples were processed using a digestion method (MAFF, 1971). Incubation was for 5 h 10 min. After initial examination of the sediments for presence and morphology of larvae the samples were individually fixed in buffered 10% formalin and the final volumes recorded. One millilitre samples, taken by a graduated pipette from a suspension stirred magnetically were examined in a McMaster slide and all larvae in the sample were counted. The mean of three larval counts per treatment sample was obtained and the number of larvae per gram of muscle calculated . • Present address: Veterinary Investigation Centre, Woodthorne, Wolverhampton WV6 8TQ..
3 19
TRI CHI NE LLA S PIR A LIS
RES ULTS
Larvae obtained from frozen muscle were usually uncoiled and frequently transpa rent. A few larvae were of typical morphology but invariably these had reta ined a cover of muscle fibres . Larvae recovered from unfrozen muscle were typical of co iled T. spira/is larvae . TA BLE ! LARVA LCO UNTSFO R EAC H TREATMENTSA MPL E
Storage temperature °C
La rvae per gram o f muscle
- 18
-8
+4
+ 18
8· 9
8 ·2
29·2
3 1·9
DI SCUSSION
Seventy eight per cent of previously frozen larvae were lost compared to previously unfrozen larvae. The frozen samples not only yielded far fewer larvae, but those remaining, which were not ensheathed in muscle fibres, appeared to be disintegrating. It is concluded that storage of encysted larvae for six days at -Soc and -I SOC reduces the chances of their being demonstrated by peptic diges tion m ethods. Therefore such storage is not to be recommended when the obj ective is to demo nstrate the presence of T. spiralis larvae in muscle b y peptic digestion . ACKNOWLEDGEMENTS
Gratitude is expressed to Mrs G. Blake, Mr C. L. Davidson, Mrs V. Griffiths, Mrs J. Mori ng and Mr D. A. Wassal for technical assistance. REFERENCES MAFF, (197 j) . Manual of Veterinary Parasitological Laboratory Techniques, Technical Bulletin N o. 18, p .54. Lo nd o n : H .M .S.O . RANS OM, B. H . (1916).]. agric. Res. 5,819 .
(Acceptedfor publication 18 November 1976)
Br. vet.}. (197 7), 133, 3 18
RECOVERY OF TRICHINELLA SPIRALIS LARVAE By G.jACKSON*
Veterinary Investigation Centre, Moulton, Northampton
SUMMARY
It is suggested that muscle samples submitted for the demonstration of Trichinella spira/is by the peptic digestion method should not be frozen .
INTRODUCTION
Veterinary Investigation Centres of the Ministry of Agriculture, Fisheries and Food (MAFF) have been engaged in a survey of pig diaphragmatic muscle for evidence of T. spira/is . The receipt of muscle which had been frozen at minus 8°C for three to six days prompted enquiry into the effects of such procedures on the demonstration of T. spira/is using a peptic digestion method, since it is known that freezing (20 days at -15°C, 10 days at-23·3°C, and six days at-37 ·2°C ) kills encysted T. spira/is (Ransom, 1916). . .
MATERIALS AND METHODS
T. spira/is isolated at Penrith in 1939, and maintained at the London School of Hygiene and Tropical Medicine, was used to infect a pig at the Central Veterinary Laboratory, Weybridge. The pig received an oral dose of 700 larvae per kg bodyweight. Seven weeks later the pig was slaughtered. A portion of hind leg was received two days later. Skeletal muscle was cut into pieces (approximately 25 mm cubes) and these were well mixed and divided into four samples of approximately 150 g each and then weighed. The samples were stored in plastic bags in four separate sites ; namely in a deep freeze (- 18°C), in a refrigerator (-8°C and +4°C) and in a store room (+ 18°C). After six days both frozen samples were thawed overnight at + 4°C. The following morning the samples were processed using a digestion method (MAFF, 1971). Incubation was for 5 h 10 min. After initial examination of the sediments for presence and morphology of larvae the samples were individually fixed in buffered 10% formalin and the final volumes recorded. One millilitre samples, taken by a graduated pipette from a suspension stirred magnetically were examined in a McMaster slide and all larvae in the sample were counted. The mean of three larval counts per treatment sample was obtained and the number of larvae per gram of muscle calculated . • Present address: Veterinary Investigation Centre, Woodthorne, Wolverhampton WV6 8TQ..
3 19
TRI CHI NE LLA S PIR A LIS
RES ULTS
Larvae obtained from frozen muscle were usually uncoiled and frequently transpa rent. A few larvae were of typical morphology but invariably these had reta ined a cover of muscle fibres . Larvae recovered from unfrozen muscle were typical of co iled T. spira/is larvae . TA BLE ! LARVA LCO UNTSFO R EAC H TREATMENTSA MPL E
Storage temperature °C
La rvae per gram o f muscle
- 18
-8
+4
+ 18
8· 9
8 ·2
29·2
3 1·9
DI SCUSSION
Seventy eight per cent of previously frozen larvae were lost compared to previously unfrozen larvae. The frozen samples not only yielded far fewer larvae, but those remaining, which were not ensheathed in muscle fibres, appeared to be disintegrating. It is concluded that storage of encysted larvae for six days at -Soc and -I SOC reduces the chances of their being demonstrated by peptic diges tion m ethods. Therefore such storage is not to be recommended when the obj ective is to demo nstrate the presence of T. spiralis larvae in muscle b y peptic digestion . ACKNOWLEDGEMENTS
Gratitude is expressed to Mrs G. Blake, Mr C. L. Davidson, Mrs V. Griffiths, Mrs J. Mori ng and Mr D. A. Wassal for technical assistance. REFERENCES MAFF, (197 j) . Manual of Veterinary Parasitological Laboratory Techniques, Technical Bulletin N o. 18, p .54. Lo nd o n : H .M .S.O . RANS OM, B. H . (1916).]. agric. Res. 5,819 .
(Acceptedfor publication 18 November 1976)
