(From the 'Laboratory Animal Services, Institute of Medical and Veterinary Science,. Frome Road, Adelaide, South Australia 5000. and tC.S.I.R.O., Division ...
228
TAM, FREWIN, ELLIOTT, LUKE
AND
AJEBAK 55 (Pt. 2) 229.232 (1977)
DOWNEY
increasing peripheral blood flow and sweating ( Woodbury and Fingl, 1975). In their study of the finger heat elimination response to core temperature change, Rosendofi and Cranston (1968) found that aspirin had no effect on the response in afebrile young males. However, in the present study, the increased sweating response has been demonstrated in non-pyrexial subjects. The experiments in the present study were conducted during the month of October and it seems unlikely that an acclimatization effect would have influenced the results. Whether this variance in findings from the two studies can be explained on the basis of age and/or sex differences warrants further investigation. The present results could also serve as a basis for further sweating studies on older females who have heat intolerance, menopausal flushing or are on high doses of aspirin for musculo-skeletal disorders. Acknowledgements. The authors wish to express their gratitude to the subjects who participated in this study and particularly to the Volunteer Department of the Columbia Presbyterian Medical Center for assistance with the project.
Brief Communication:
LACK OF GENETIC DIFFERENTIATION BETWEEN DOMESTIC DOGS AND DINGOES AT A FURTHER 16 LOCI by S. R. COLE', P. R. BAVERSTOCK' and B. GREEN+ (From the 'Laboratory Animal Services, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000. and tC.S.I.R.O., Division Wildlife Research, P.O. Box 84, Lyneham, A.C.T. 2601.)
REFERENCES. AMEn1cAs COLLEGEOF SURGEONS, SUBCOMMITTEE ON B U ~ N(1967): S Am. Coll. Stug. Bull., 52, 196.
PALMES,E. D., and PARK,C. R. (1947): Fort Knor, Ky: U.S. Army Med. Dept, Field Research Lab. Rept 2-17-1.
BANERJEE, M. R., ELIzONDO, R., and BuLLARD,R. W. (1969): J. Appl. Phusiol., 26. 787.
ROSENDORFF, C., and CRANSTON,W. I. (1968): Clin. Sci., 35, 81.
BULLARD, R. W. (1962): J. Appl. Pht~siol., 17, 735.
NADEL,E. R. SToLwlJK, 1. A. (1973): Int. Fed. Auto Control, 221.
1. A. and DA~LING, R. C. (1962): DOWNEY, J. Appl. Physiol., 17, 323.
\VACNE~, J. A., ROBINSON, S., and M ~ n m o , R. P. (1974): J. Appl. Physiol., 37, 562.
HUCKABA, C. E., FREWIS,D. B., DOWNEY, J. A., TAM, H. S., DARLING, R. C., and CHEH, H. Y. (1976): Arch. Phys. Med. Rehab., 57, 268. NADEL,E. R., BULLARD, R. W., and STOLWIJK,J. A. J. (1971): J. Appl. Physiol., 31, 80.
J.2
WOODBURY, D. M. and FINGL,E. (1975): 'Analgesic-antipyretics, anti-inflammatory agents, and drugs employed in the therapy of gout.' In: "The Pharmacological &is o f ~ h e r a ~ e u t i c s ,5th " Ed., L. S. Goodman and A. Gilman, Ch. 17, Macmillan.
(Accepted for publication Februay 24, 1977.)
Summary. Clarke, Ryan and Czuppon (1975) failed to detect electrophoretic differences between domestic dogs and dingoes at 14 loci. We have found that domestic dogs and dingoes are monomorphic for the same electrophoretic alleles at a further 15 loci, and polymorphic for the same alleles at a 30th locus.
INTRODUCTION. As part of a study of the biology of the dingo (Canis familiaris dingo) the C.S.I.R.O. Division of Wildlife Research have attempted to assess the extent of hybridization between dingoes and domestic dogs (Cf. familia*). This task would be appreciably simplified if an easily scorable genetic difference could be found between the two taxa. Clarke et al. (1975) failed to find a constant electrophoretic difference between the taxa at 14 loci. Shaughnessy, Newsome and Corbett (1975) also failed to differentiate the taxa at 5 of the loci studied by Clarke et al. (1975). We have extended the study to a further 16 loci.
The animals used in the study were members of a colony which has been maintained in Canberra for about 6 years. Four types of animals were studied, a Central Australian type, an Eastern Highlands type, dingo/domestic hybrid type and domestic type. There were 4 animals of each type. About 2 m1 of blood were taken from the brachial vein of each animal, using a heparinised syringe. The blood samples were transferred to heparinised plastic vials and centrifuged. The plasma was removed from each sample and stored individually at -17". The red cell fractions were added to an equal volume of chilled ethylene glycol and also stored at -17-. Electrophoresis was conducted within 10 weeks of collection of blood. A total of 20 enzyme systems were stained for an either Starch or Cellogel (Table 1 ) .
l
230
COLE, BAVERSTOCK
ELECTROPHORESIS I N DOGS AND DINGOES
GREEN
AND
RESULTS AND DISCUSSION. The 20 enzyme systems stained for yielded excellent resolution for a minimum of 16 loci (Table 2 ) but only one system revealed variability, uiz. peptidase using phe-pro as substrate. However, the variability failed to correlate with the taxa (Table 2 ) and it appears o is polymorphic for the same alleles in both that the locus encoding the ~ h e - ~ rpeptidase domestic dogs and dingoes.
The failure to discover an enzyme system that differentiates between domestic dogs and dingoes means tbat it may be extremely di5cult to determine the degree of hybridization that has occurred between these subspecies in the wild. It also hinders any attempt to study gene flow between packs and larger populations of wild dingoes.
TABLE 2. Blood enzymes of dingoes, dogs and hybrids.
TABLE 1. Eleetrophoretic gel media, buffer and staining systems employed in rhe study.
I
Buffer
Enzyme
Gel
6-Phosphogluconatedehydrogenase EC1.1 .l .44 (6PGD) Leucine aminopeptidase EC3.4.1.1. (LAP)
I
S
I1
Brewer (1970) Selander i t 01. (1971) ~- -,
S S
Brewer (1970) Brewer (1970)
Glyceraldehyde 3-Phosphate dehydrogenase EC1.2.1.12. (GA3PD) Suoeroxidase dismutase (SOD)
Gahne (1966) Gahne (1966)
Esterases (Est) Ng (see Birdsall, Redfield and Cameron, 1970) Adenosine deaminase EC3.5.4.4. (ADA)
Acid phosphatase EC3.1.3.2. (AcP) Adenylate kinase EC2.7.4.3. (AK) 3-Phosphoglycerate kinase EC2.7.2.3. (PGK) Peptidase (L-Valyl-L-Leucine as substrate) (Pep A) Peptidase (L-Leucylglycylglycine as substrate) (Pep B) Peptidase (L-Phenylalanyl-L-Proline as substrate) ,l,".. P, L,
,=:v
Pept~dase(L-Glycyl-L-Leucine as substrate) (Pep A) Glutamate-oxaloacetate transaminase EC2.6.1.1. (GOT) Phosphorylase a EC2.4.1.1. (Pa) Isocitrate dehydrogenase ECl.I.1. 42 (IDH) Hexokinase EC2.7.1.1. (HK) Nucleoside phosphorylase EC2.4.2.1. (NP) a-Glycerophosphate dehydrogenase EC1.1.1.8. (GPD:
S
Spencer, Hopkinson and Harris (1968) Brewer (1970) Richardson* Richardson* Richardson* Richardson*
1
I
231
-.
Stain Brewer (1970) Brewer (1970) Brewer (1970) Scored from gels stained for GA3PD Gahne (1966) Scored from gels stained for Est. Spencer et al. (1968) Brewer (1970) Richardson* Richardson* Richardson* Richardson*
Richardson* Richardson*
Richardson* Richardson*
Richardson* Richardson* Richardson* Richardson* Richardson* Richardson*
Richardson" Richardson* Richardson* Richardson* Richardson*
* Pers. comm. Dr. B. J. Richardson, Research School Biological Sciences, Australian National University. S = Starch gel. C = Cellogel (Chemetron, Milan) Shaughnessy et al. (1975) commented that it was surprising that neither they nor Clarke et al. (1975) found electrophoretic differences between domestic dogs and dingoes at 14 loci. The extension of this result to a further 16 loci is even more surprising. The obvious conclusion to be drawn from the homogeneity of dingo and dog blood enzymes is tbat they have very similar gene pools. However, Clarke et al. (1975) and Simonsen (1976) have found very little biochemical differentiation in a wide range of canid species and the similarity of dingoes and domestic dogs may merely reflect the conservative nature of biochemical evolution in the Cauidae, rather than a very close taxonomic relationship between dingoes and domestic dogs.
Enzyme system
Authors
I. Variable Pep C (using phe-pro as substrate) Designating single fast band as F, single slow baud as S and double band as FS, the following was obtained: Domestic dogs IF, IFS, 2S Central dingoes IF, ZFS, IS Eastern Highland dingoes 3F, IFS Crosses 3F, IFS
Present study
-.
2. Invariable AK Pep A (identical mobility was obtained using as substrate val-leu or gly-leu) Pep B (using leu-gly-gly as substrate) 6PGD GOTl Pa IDH, IDHz HK NP SOD GA3PD ADA GPD Ng
Present study
I
1
I!
I
LDH, LDH. MDH G6PD GP1 PGM, PGM, Alb Trf Hp (2 loci) ~b (2 loci)
Clarke er al. (1975)
Clarke et a? (1975) and Shaughnessy et al. (1975)
I
3. Loci stainedfor but not scorable PGK AcP LAP Plasma Est GOT,
Present study
I
29 loci invariant 1 locus variable but polymorphic 5 loci not scorable 35 loci total
230
COLE, BAVERSTOCK
ELECTROPHORESIS I N DOGS AND DINGOES
GREEN
AND
RESULTS AND DISCUSSION. The 20 enzyme systems stained for yielded excellent resolution for a minimum of 16 loci (Table 2 ) but only one system revealed variability, uiz. peptidase using phe-pro as substrate. However, the variability failed to correlate with the taxa (Table 2 ) and it appears o is polymorphic for the same alleles in both that the locus encoding the ~ h e - ~ rpeptidase domestic dogs and dingoes.
The failure to discover an enzyme system that differentiates between domestic dogs and dingoes means tbat it may be extremely di5cult to determine the degree of hybridization that has occurred between these subspecies in the wild. It also hinders any attempt to study gene flow between packs and larger populations of wild dingoes.
TABLE 2. Blood enzymes of dingoes, dogs and hybrids.
TABLE 1. Eleetrophoretic gel media, buffer and staining systems employed in rhe study.
I
Buffer
Enzyme
Gel
6-Phosphogluconatedehydrogenase EC1.1 .l .44 (6PGD) Leucine aminopeptidase EC3.4.1.1. (LAP)
I
S
I1
Brewer (1970) Selander i t 01. (1971) ~- -,
S S
Brewer (1970) Brewer (1970)
Glyceraldehyde 3-Phosphate dehydrogenase EC1.2.1.12. (GA3PD) Suoeroxidase dismutase (SOD)
Gahne (1966) Gahne (1966)
Esterases (Est) Ng (see Birdsall, Redfield and Cameron, 1970) Adenosine deaminase EC3.5.4.4. (ADA)
Acid phosphatase EC3.1.3.2. (AcP) Adenylate kinase EC2.7.4.3. (AK) 3-Phosphoglycerate kinase EC2.7.2.3. (PGK) Peptidase (L-Valyl-L-Leucine as substrate) (Pep A) Peptidase (L-Leucylglycylglycine as substrate) (Pep B) Peptidase (L-Phenylalanyl-L-Proline as substrate) ,l,".. P, L,
,=:v
Pept~dase(L-Glycyl-L-Leucine as substrate) (Pep A) Glutamate-oxaloacetate transaminase EC2.6.1.1. (GOT) Phosphorylase a EC2.4.1.1. (Pa) Isocitrate dehydrogenase ECl.I.1. 42 (IDH) Hexokinase EC2.7.1.1. (HK) Nucleoside phosphorylase EC2.4.2.1. (NP) a-Glycerophosphate dehydrogenase EC1.1.1.8. (GPD:
S
Spencer, Hopkinson and Harris (1968) Brewer (1970) Richardson* Richardson* Richardson* Richardson*
1
I
231
-.
Stain Brewer (1970) Brewer (1970) Brewer (1970) Scored from gels stained for GA3PD Gahne (1966) Scored from gels stained for Est. Spencer et al. (1968) Brewer (1970) Richardson* Richardson* Richardson* Richardson*
Richardson* Richardson*
Richardson* Richardson*
Richardson* Richardson* Richardson* Richardson* Richardson* Richardson*
Richardson" Richardson* Richardson* Richardson* Richardson*
* Pers. comm. Dr. B. J. Richardson, Research School Biological Sciences, Australian National University. S = Starch gel. C = Cellogel (Chemetron, Milan) Shaughnessy et al. (1975) commented that it was surprising that neither they nor Clarke et al. (1975) found electrophoretic differences between domestic dogs and dingoes at 14 loci. The extension of this result to a further 16 loci is even more surprising. The obvious conclusion to be drawn from the homogeneity of dingo and dog blood enzymes is tbat they have very similar gene pools. However, Clarke et al. (1975) and Simonsen (1976) have found very little biochemical differentiation in a wide range of canid species and the similarity of dingoes and domestic dogs may merely reflect the conservative nature of biochemical evolution in the Cauidae, rather than a very close taxonomic relationship between dingoes and domestic dogs.
Enzyme system
Authors
I. Variable Pep C (using phe-pro as substrate) Designating single fast band as F, single slow baud as S and double band as FS, the following was obtained: Domestic dogs IF, IFS, 2S Central dingoes IF, ZFS, IS Eastern Highland dingoes 3F, IFS Crosses 3F, IFS
Present study
-.
2. Invariable AK Pep A (identical mobility was obtained using as substrate val-leu or gly-leu) Pep B (using leu-gly-gly as substrate) 6PGD GOTl Pa IDH, IDHz HK NP SOD GA3PD ADA GPD Ng
Present study
I
1
I!
I
LDH, LDH. MDH G6PD GP1 PGM, PGM, Alb Trf Hp (2 loci) ~b (2 loci)
Clarke er al. (1975)
Clarke et a? (1975) and Shaughnessy et al. (1975)
I
3. Loci stainedfor but not scorable PGK AcP LAP Plasma Est GOT,
Present study
I
29 loci invariant 1 locus variable but polymorphic 5 loci not scorable 35 loci total
It
232
COLE, BAVERSTOCK
AND
AJEBAK 55 (Pt. 3) 233-244 (1977)
GREEN
Acknowledgement. We thank Dr. B. J. Richardson for his advice on electrophoretic techniques.
REFERENCES. BIRDSALL,D. A., REDFIELD, J. A., and CAMERON, D. G. (1970): Biochem. Genet., 4, 655.
SELANDER,R. K., SMITH, M. H., YANG, S. Y., JOHNSON,W. E., and GENTRY,J. B. (1971): Stud. Genet. VI, 49. (Univ. Texas Publ. 7103).
B~EWB, G . 1; (1970): 'Introduction to Isozyme Techniques,' Academic Press, New York.
SHAUGHNESSY, P. D., NEWSOME,A. E., and CORBETT,L. K. (1975): Awt. Mammal. 1, 355.
C
~ E P,,, RYAN, G. E., and CZUPPON, A. B. (1975): Aust. J. Zool., 23, 411.
GAHNE,B. (1966): Genetics, 53, 681.
SIMONSEN,V. (1976): Hefeditas, 82, 7. SPENCER, N., HOPKINSON, D. A., and H A ~ R I SH. , (1968): Ann. Hum. Genet. Lond., 32, 9.
BRAIN DAMAGE BY EXTRACTS OF PARASITISED ANNUAL
'
RYEGRASS (LOLIUM RIGIDUM) I N NURSLING RATS
by J. E. PETERSON
AND
MARJORIE V. JAG0
(From the C.S.I.R.O. Division of Animal Health, Parkville, Victoria.)
(Accepted for publication March 4, 1977.)
Summary. Extracts of annual ryegrass (Lolium rigidum) infected with Anguina sp. and Coynebacterium sp., and associated with an outbreak of annual ryegrass toxicity in sheep, were administered to 2-week-old rats by a single intraperitonea1 injection. Rats that received a lethal dose of toxin developed neurological signs including incoordination and convulsions from the second day and most died between 2 and 7 days after injection. Histologically, the brains showed widespread lesions of focal necrosis consistent with anoxia. Peripheral circulation rate was greatly reduced after 3 days and gangrene of the tail and hind legs developed in some rats. Evidence of restricted blood flow was also seen in kidneys, lungs and brain. It was concluded that the toxin contains a long-acting vasoconstrictor.
INTRODUCTION. A neuropathy of sheep and cattle grazing annual ryegrass (Lolium ~igidum) has been associated with the ingestion of seed heads infected by a nematode, Anguina sp, and a bacterium, Corynebacterium sp. (McIntosh, Rac and Thomas, 1967; Gwynn and Hadlow, 1971; Berry and Wise, 1975). Lanigan, Payne and Frahn (1976) identified the Corynebacterium galls as the source of the toxicity. Clinically, the disease is characterised by incoordination, convulsions and other neurological signs. Morbidity and mortality rates of up to 100% and 7058, respectively, have been recorded (Berry and Wise, 1975). While investigating assay systems suitable for monitoring the extraction and purification of toxins from infected grass, nursling rats were found to display prominent neurological signs and a relatively greater sensitivity than other laboratory animals. The effects produced in the 2-week-old rats by a single intraperitoneal injection of toxic extract are described here.
,'
