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Species Identification of Deer Blood by Isoelectric Focusing
J.F.S.S. ORIGINAL PAPERS
Deer Blood Species Identification Isoelectric Focusing -
MARGARET E. LA\VTON and J. G. SUT1'ON Home O$ce Central Research Establishment, Aldermaston, lieading, Berkshire, United Kingdom KG7 4 P N
Abstract A method of identifying the blood from various spccies of deer found in the United Kingdom is described. 'I'he rncthod is based on the simultaneous electrofocusing of haemoglobin and the red cell enzyme, superoxide dismrrtase, in a pH 5 to pH 9 gradient. Journal of the Forensic Science Societ3, 1982; 22 : .?(il-.?(ili Received I September 1981
Introduction Species identification of bloodstains has been carried out by forcnsic scientists for a number of years with the application of specific antisera. 'I'he methods used are based on electrophoresis ( 1 ) or a diffusion technique (2, 3 ) . These techniques are limited by the specificity of the antisera used so that the level of identification generally achieved is limited to the generic order or family. O n many occasions this degree of differentiation will suffice but in other circumstances it would be advantageous to identify or eliminate a particular species. The forensic scientist is generally involved in the identification of blood from deer in the investigation as a result oF poaching offences. Deer keeping, and in particular farming for venison, has increased steadily in the last few years and this trend has been paralleled by an increase in the incidence of poaching. Of a total of 40 species of deer six species roam freely or are confined in deer parks or farms in Britain. Of these six species, Red and Roe are indigenous while the Fallow, Sika, Muntjac and Chinese Water Deer have been introduced into Britain in the past and are now feral (4). These species, and their general areas of distribution in Britain, are listed in Table 1. With the exception of a managed herd of Reindeer in the Cairngorms, other species of deer found in Britain are contained in zoos and are less accessible to poachers, and therefore not likely to be encountered in casework.
TABLE 1 T H E S I X SPECIES O F DEER FOUND I N BRITAIN AND THEIR GENERAL LOCATlON
Common N a m e Red
Geueric Nnrne C e ~ u u elaphus t
Roe Fallow
Cnf~reoluscnpreo1ti.t D a m n damn
Sika Muntjac
Cervus nippun Muntincu.r reeaesi
Chinese Water Deer
Hydropote.r inermi.~
Arenr of 1)istrihutiou Scotland, North-\Vest Enqland. Few feral breeds in parks scattrred throuqhout Midland\ and Southern England. Scotland, North, South and South-West England. South and South-East England. T o a lescer degree Slidlands and Wales. Scattered in small areas throughout Britain. South and South-East England particularly Bedfordshire. Redfordshirr and East Anglin.
Haemoglobin patterns in members of the Cervidae (Deer) family have previously been studied by isoelectric focusing on a pH6-pH7 gradient (5, 6). Marked heterogeneity in the haemoglobin patterns in this family was found as were variations within and between species. While providing a good basis, haemoglobin patterns alone proved insufficient for separating all the species and the observed polymorphism within some species made the results somewhat ambiguous. T o provide further discrimination we examined some red cell enzyme systems in the deer blood samples and found that superoxide dismutase (SOD*), which is also known as indophenol oxidase, showed considerable potential for species differentiation. This study is primarily concerned with identifying British deer but includes several other species found in zoos. All the species examined are listed in Table 2. The electrofocusing technique employed is relatively fast and simple and could possibly be applied to other animal families. TABLE 2 DEER SPECIES EXAMINED IN THIS STUDY Species Reindeer Moose Sika Red Swamp Indian Muntjac Reeves Muntjac Pere David Hog Axis Fallow Roe Chinese Water Deer
Genus Rangzifir Alces Ceruus Cervus Ceruus Muntiacus Muntiacus Elaphurus Axis Axis Dnma Cafireolus Hvdropetes
Number Studied 3 3 6 5 5 1 2 4 1 5 26
7 1
Materials Collection of Samples Blood samples from thirteen species of deer representing nine genera were collected from deer parks and the Nuffield Laboratories a t Regents Park Zoo as dried stains, fresh whole blood or frozen lysates. Upon receipt the whole blood samples were washed three times with physiological saline and the red cells frozen until required. A portion of each bloodstain was examined while fresh by isoelectric focusing and the remainder stored a t room temperature (8-16°C). A bloodstain was made from at least one blood sample of each species to assess the stability of the systems with age. Some of the frozen lysates obtained from Regents Park Zoo were approaching two-years old. I n addition a number of goat, sheep and human lysates were made and compared with the deer samples. The goat and sheep samples were included as they are closely related genetically to the deer family with whom they can cross-react immunologically. Methods Isoelectric Focusing Reagents and Apparatus All electrofocusing work was conducted on 1mm gels (7), pH5-9, using 2117 Multiphor apparatus (LKB Instruments, LKB House, Croydon, Surrey). Gels were made from 36ml stock acrylogel (66g/L), 3.6g sucrose, 0.7ml each of pH5-pH7 and pH7-pH9 ampholine, and 0.2ml riboflavin 1OmgX. The electrode solutions (1M phosphoric acid, anode, and 1N sodium hydroxide, cathode) were applied using Whatman 17 Chroma grade 1cm wide strips. 362
Application of the Red Cell Lysates and Stains Lysates were absorbed onto filter paper strips (5mm x 3mm) while stains (5mm x 5mm) were initially moistened with water and applied directly to the gel. Lysates and stains were applied in a line along the gel lcm from the anode. Running Conditions Electrofocusing was conducted for 3 hours at maximum settings of 1100 volts and 7 watts; these settings produced an initial current of 5mA. After one hour's electrofocusing the filter paper inserts and stains were removed. Visualisation Haemoglobin The haemoglobin focused sharply after 3 hours when the results could be recorded. SODA A solution containing 2mg of M T T tetrazolium and lmg of phenazine methosulphate was dissolved in 20ml of 0.3M Tris buffer (pH8.0). Th'1s was added to 20ml of 2% agar solution which had been cooled to 56OC. The mixture was poured evenly over the surface of the gel and allowed to set. The plate was exposed briefly to light to initiate the background reduction of M T T to formazan and then incubated in a 37°C oven for 1-2 hours. The resulting enzyme activity appeared as pale yellow bands on a dark blue background.
Results and Discussion Haemoglobin The haemoglobin patterns obtained from the lysates of the thirteen species of deer studied are shown in Figure 1 and compared with lysates from human, goat and sheep blood. No difference in the degree of partial haemoglobin oxidation was noticed between fresh lysates and two-year-old lysates of any one species. The brown oxidised methaemoglobin bands focused nearer the cathodic end of the gel than did the non-oxidised bands. Bloodstains up to two-weeks old had identical haemoglobin patterns to the lysates. After two weeks storage and subsequent analysis, the anodic haemoglobin bands appeared to be less intense until after two months only the methaemoglobin bands were visible. No extra bands appeared as the stains deteriorated. a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
Figure I. Isoelectric focusing patterns of haemoglobin, pH gradient 5-9. (a) Human, (b) Sheep, (c) Goat, (d) Reindeer, (e) Moose, ( f ) Sika, (g) Red, (h) Swamp, (i) Indian Muntjac, (j) Reeves Muntjac, (k) Pere David, (1) Hog, (m) Axis, (n) Fallow, (0) Roe, (p) Chinese Water Deer.
Using lysates it was possible to differentiate between the nine genera by their haemoglobin patterns although in some cases the similarity was extremely close, for example Reindeer (Rangifer) and Moose (Alces). In addition it was possible to identify all the species within their genera except Axis and Hog (Axis) and Swamp, Sika and Red (Cervus). An identical polymorphism was observed between Swamp and Sika deer. I n old bloodstains the differentiation between genera and species became less distinct because of the disappearance of some of the haemoglobin bands.
Of the six common deer species found in Britain only Red and Sika had indistinguishable haemoglobin patterns as seen in the patterns obtained from red cell lysates (Figure 2). I t should be pointed out that human, sheep and goat haemoglobins focused in a slightly more cathodic position to those of any of the deer species examined.
3
b
c
d
r
f
Figure 2. Isoelectric focusing patterns of haemoglobin from species of British deer on a pH 5-9 gradient. (a) Fallow, (b) Red, (c) Sika, (d) Reeves Muntjac, (e) Roe. (f \ Chinese Water Deer
SODA The subsequent development of SODAenzymes (Figure 3) provided increased discrimination as the bands developed over a wide p H range. Each species showed two or sometimes three SODAbands except human blood which had a single band that focused at about pH5 and was not always evident on the plate. Generally the SODApatterns consisted of one strong band, with a weaker band situated anodally to the strong band. I t was observed that, despite using the same running conditions each time, the separation and relative positions of the SODA and haemoglobin bands varied slightly with each batch of ampholines.
Figure 3. Diagram showing the isoelectric focusing patterns of SODAonly on pH 5-9 gradient. (a) Human, (b) Sheep, ( c ) Goat, (d) Reindeer, (e) Moose, ( f ) Sika, (g) Red, (h) Swamp, (i) Indian Muntjac, (j) Reeves Muntjac, (k) Pere David, (1) Hog, (m) Axis, (n) Fallow, (0) Roe, (p) Chinese Water Deer. 364
No deterioration was observed in the haemoglobin and SODA patterns of the lysates despite thawing and refreezing of the samples on several occasions throughout this study. The strong SODAbands were still visible in two-monthold bloodstains while the weaker bands were often absent after a month. I t must be pointed out that in some instances only a few species of deer were examined so it is not known whether different polymorphic forms exist. In the event that this system is used in case work it would be advisable to use a number of different control bloods from the same species of deer within the crime area. The development of SODA distinguished between those species whose haemoglobin patterns were almost identical; for example, Sika and Red, Axis and Hog, Reindeer and Moose could now be easily distinguished from each other as shown in Figure 4. It can also be seen that some species such as Roe and Chinese Water Deer which are in different genera have virtually identical SODA. Only deer of the Red and Swamp species showed identical haemoglobin and SODA patterns and could not be distinguished. None of the six British species showed the same combination of SODa and haemoglobin patterns, (Figure 2 and Figure 4). (Blood samples of these six species will be available from HOCRE in due course).
Figure 4. Diagram showing the isoelectric focusing patterns of SODA from the species of British deer on a pH5-9 gradient. (a) Fallow, (b) Red, (c) Sika, (d) Reeves Muntjac, (e) Roe, (f ) Chinese Water Deer.
Conclusion Using the isoelectric focusing patterns of haemoglobin and SODA combined, a high degree of differentiation can be obtained between species of the Cervidae family. In particular the six species of British deer can be identified from their bloodstains.
Acknowledgements We would like to thank Dr. Rachel Fischer of the Nuffield Laboratories, Mr. Street and the staff at the Bedford Estate, Mr. Pepper of the Forestry Research Station, Farnham and Dr. McDiarmid of the British Deer Society for their valuable discussions and assistance in obtaining blood samples from the various species of deer.
References (1) Culliford BJ. Nature 1964; 201 : 1092. (2) Ouchterlony 0. Acta Pathologica et Microbiologica Scandinavica 1949; 26: 507. (3) Hewitt R and Fish JL. Journal of the Forensic Science Society 1973; 13 : 97. (4) Corbett GB and Southern HN. The Handbook of British Mammals. London : Blackwell Scientific Publications, 1977. (5) Butcher PD and Hawkey CM. Comparative Biochemistry and Physiology 1977; 56B: 335. (6) Meadows RW. Proceedings of Forensic Science Symposium Calgary Alberta, 1977 : 39-46. (7) Sutton JG and Burgess R. Vox Sanguinis 1978; 34: 97.
Deer Blood Species Identification Isoelectric Focusing -
MARGARET E. LA\VTON and J. G. SUT1'ON Home O$ce Central Research Establishment, Aldermaston, lieading, Berkshire, United Kingdom KG7 4 P N
Abstract A method of identifying the blood from various spccies of deer found in the United Kingdom is described. 'I'he rncthod is based on the simultaneous electrofocusing of haemoglobin and the red cell enzyme, superoxide dismrrtase, in a pH 5 to pH 9 gradient. Journal of the Forensic Science Societ3, 1982; 22 : .?(il-.?(ili Received I September 1981
Introduction Species identification of bloodstains has been carried out by forcnsic scientists for a number of years with the application of specific antisera. 'I'he methods used are based on electrophoresis ( 1 ) or a diffusion technique (2, 3 ) . These techniques are limited by the specificity of the antisera used so that the level of identification generally achieved is limited to the generic order or family. O n many occasions this degree of differentiation will suffice but in other circumstances it would be advantageous to identify or eliminate a particular species. The forensic scientist is generally involved in the identification of blood from deer in the investigation as a result oF poaching offences. Deer keeping, and in particular farming for venison, has increased steadily in the last few years and this trend has been paralleled by an increase in the incidence of poaching. Of a total of 40 species of deer six species roam freely or are confined in deer parks or farms in Britain. Of these six species, Red and Roe are indigenous while the Fallow, Sika, Muntjac and Chinese Water Deer have been introduced into Britain in the past and are now feral (4). These species, and their general areas of distribution in Britain, are listed in Table 1. With the exception of a managed herd of Reindeer in the Cairngorms, other species of deer found in Britain are contained in zoos and are less accessible to poachers, and therefore not likely to be encountered in casework.
TABLE 1 T H E S I X SPECIES O F DEER FOUND I N BRITAIN AND THEIR GENERAL LOCATlON
Common N a m e Red
Geueric Nnrne C e ~ u u elaphus t
Roe Fallow
Cnf~reoluscnpreo1ti.t D a m n damn
Sika Muntjac
Cervus nippun Muntincu.r reeaesi
Chinese Water Deer
Hydropote.r inermi.~
Arenr of 1)istrihutiou Scotland, North-\Vest Enqland. Few feral breeds in parks scattrred throuqhout Midland\ and Southern England. Scotland, North, South and South-West England. South and South-East England. T o a lescer degree Slidlands and Wales. Scattered in small areas throughout Britain. South and South-East England particularly Bedfordshire. Redfordshirr and East Anglin.
Haemoglobin patterns in members of the Cervidae (Deer) family have previously been studied by isoelectric focusing on a pH6-pH7 gradient (5, 6). Marked heterogeneity in the haemoglobin patterns in this family was found as were variations within and between species. While providing a good basis, haemoglobin patterns alone proved insufficient for separating all the species and the observed polymorphism within some species made the results somewhat ambiguous. T o provide further discrimination we examined some red cell enzyme systems in the deer blood samples and found that superoxide dismutase (SOD*), which is also known as indophenol oxidase, showed considerable potential for species differentiation. This study is primarily concerned with identifying British deer but includes several other species found in zoos. All the species examined are listed in Table 2. The electrofocusing technique employed is relatively fast and simple and could possibly be applied to other animal families. TABLE 2 DEER SPECIES EXAMINED IN THIS STUDY Species Reindeer Moose Sika Red Swamp Indian Muntjac Reeves Muntjac Pere David Hog Axis Fallow Roe Chinese Water Deer
Genus Rangzifir Alces Ceruus Cervus Ceruus Muntiacus Muntiacus Elaphurus Axis Axis Dnma Cafireolus Hvdropetes
Number Studied 3 3 6 5 5 1 2 4 1 5 26
7 1
Materials Collection of Samples Blood samples from thirteen species of deer representing nine genera were collected from deer parks and the Nuffield Laboratories a t Regents Park Zoo as dried stains, fresh whole blood or frozen lysates. Upon receipt the whole blood samples were washed three times with physiological saline and the red cells frozen until required. A portion of each bloodstain was examined while fresh by isoelectric focusing and the remainder stored a t room temperature (8-16°C). A bloodstain was made from at least one blood sample of each species to assess the stability of the systems with age. Some of the frozen lysates obtained from Regents Park Zoo were approaching two-years old. I n addition a number of goat, sheep and human lysates were made and compared with the deer samples. The goat and sheep samples were included as they are closely related genetically to the deer family with whom they can cross-react immunologically. Methods Isoelectric Focusing Reagents and Apparatus All electrofocusing work was conducted on 1mm gels (7), pH5-9, using 2117 Multiphor apparatus (LKB Instruments, LKB House, Croydon, Surrey). Gels were made from 36ml stock acrylogel (66g/L), 3.6g sucrose, 0.7ml each of pH5-pH7 and pH7-pH9 ampholine, and 0.2ml riboflavin 1OmgX. The electrode solutions (1M phosphoric acid, anode, and 1N sodium hydroxide, cathode) were applied using Whatman 17 Chroma grade 1cm wide strips. 362
Application of the Red Cell Lysates and Stains Lysates were absorbed onto filter paper strips (5mm x 3mm) while stains (5mm x 5mm) were initially moistened with water and applied directly to the gel. Lysates and stains were applied in a line along the gel lcm from the anode. Running Conditions Electrofocusing was conducted for 3 hours at maximum settings of 1100 volts and 7 watts; these settings produced an initial current of 5mA. After one hour's electrofocusing the filter paper inserts and stains were removed. Visualisation Haemoglobin The haemoglobin focused sharply after 3 hours when the results could be recorded. SODA A solution containing 2mg of M T T tetrazolium and lmg of phenazine methosulphate was dissolved in 20ml of 0.3M Tris buffer (pH8.0). Th'1s was added to 20ml of 2% agar solution which had been cooled to 56OC. The mixture was poured evenly over the surface of the gel and allowed to set. The plate was exposed briefly to light to initiate the background reduction of M T T to formazan and then incubated in a 37°C oven for 1-2 hours. The resulting enzyme activity appeared as pale yellow bands on a dark blue background.
Results and Discussion Haemoglobin The haemoglobin patterns obtained from the lysates of the thirteen species of deer studied are shown in Figure 1 and compared with lysates from human, goat and sheep blood. No difference in the degree of partial haemoglobin oxidation was noticed between fresh lysates and two-year-old lysates of any one species. The brown oxidised methaemoglobin bands focused nearer the cathodic end of the gel than did the non-oxidised bands. Bloodstains up to two-weeks old had identical haemoglobin patterns to the lysates. After two weeks storage and subsequent analysis, the anodic haemoglobin bands appeared to be less intense until after two months only the methaemoglobin bands were visible. No extra bands appeared as the stains deteriorated. a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
Figure I. Isoelectric focusing patterns of haemoglobin, pH gradient 5-9. (a) Human, (b) Sheep, (c) Goat, (d) Reindeer, (e) Moose, ( f ) Sika, (g) Red, (h) Swamp, (i) Indian Muntjac, (j) Reeves Muntjac, (k) Pere David, (1) Hog, (m) Axis, (n) Fallow, (0) Roe, (p) Chinese Water Deer.
Using lysates it was possible to differentiate between the nine genera by their haemoglobin patterns although in some cases the similarity was extremely close, for example Reindeer (Rangifer) and Moose (Alces). In addition it was possible to identify all the species within their genera except Axis and Hog (Axis) and Swamp, Sika and Red (Cervus). An identical polymorphism was observed between Swamp and Sika deer. I n old bloodstains the differentiation between genera and species became less distinct because of the disappearance of some of the haemoglobin bands.
Of the six common deer species found in Britain only Red and Sika had indistinguishable haemoglobin patterns as seen in the patterns obtained from red cell lysates (Figure 2). I t should be pointed out that human, sheep and goat haemoglobins focused in a slightly more cathodic position to those of any of the deer species examined.
3
b
c
d
r
f
Figure 2. Isoelectric focusing patterns of haemoglobin from species of British deer on a pH 5-9 gradient. (a) Fallow, (b) Red, (c) Sika, (d) Reeves Muntjac, (e) Roe. (f \ Chinese Water Deer
SODA The subsequent development of SODAenzymes (Figure 3) provided increased discrimination as the bands developed over a wide p H range. Each species showed two or sometimes three SODAbands except human blood which had a single band that focused at about pH5 and was not always evident on the plate. Generally the SODApatterns consisted of one strong band, with a weaker band situated anodally to the strong band. I t was observed that, despite using the same running conditions each time, the separation and relative positions of the SODA and haemoglobin bands varied slightly with each batch of ampholines.
Figure 3. Diagram showing the isoelectric focusing patterns of SODAonly on pH 5-9 gradient. (a) Human, (b) Sheep, ( c ) Goat, (d) Reindeer, (e) Moose, ( f ) Sika, (g) Red, (h) Swamp, (i) Indian Muntjac, (j) Reeves Muntjac, (k) Pere David, (1) Hog, (m) Axis, (n) Fallow, (0) Roe, (p) Chinese Water Deer. 364
No deterioration was observed in the haemoglobin and SODA patterns of the lysates despite thawing and refreezing of the samples on several occasions throughout this study. The strong SODAbands were still visible in two-monthold bloodstains while the weaker bands were often absent after a month. I t must be pointed out that in some instances only a few species of deer were examined so it is not known whether different polymorphic forms exist. In the event that this system is used in case work it would be advisable to use a number of different control bloods from the same species of deer within the crime area. The development of SODA distinguished between those species whose haemoglobin patterns were almost identical; for example, Sika and Red, Axis and Hog, Reindeer and Moose could now be easily distinguished from each other as shown in Figure 4. It can also be seen that some species such as Roe and Chinese Water Deer which are in different genera have virtually identical SODA. Only deer of the Red and Swamp species showed identical haemoglobin and SODA patterns and could not be distinguished. None of the six British species showed the same combination of SODa and haemoglobin patterns, (Figure 2 and Figure 4). (Blood samples of these six species will be available from HOCRE in due course).
Figure 4. Diagram showing the isoelectric focusing patterns of SODA from the species of British deer on a pH5-9 gradient. (a) Fallow, (b) Red, (c) Sika, (d) Reeves Muntjac, (e) Roe, (f ) Chinese Water Deer.
Conclusion Using the isoelectric focusing patterns of haemoglobin and SODA combined, a high degree of differentiation can be obtained between species of the Cervidae family. In particular the six species of British deer can be identified from their bloodstains.
Acknowledgements We would like to thank Dr. Rachel Fischer of the Nuffield Laboratories, Mr. Street and the staff at the Bedford Estate, Mr. Pepper of the Forestry Research Station, Farnham and Dr. McDiarmid of the British Deer Society for their valuable discussions and assistance in obtaining blood samples from the various species of deer.
References (1) Culliford BJ. Nature 1964; 201 : 1092. (2) Ouchterlony 0. Acta Pathologica et Microbiologica Scandinavica 1949; 26: 507. (3) Hewitt R and Fish JL. Journal of the Forensic Science Society 1973; 13 : 97. (4) Corbett GB and Southern HN. The Handbook of British Mammals. London : Blackwell Scientific Publications, 1977. (5) Butcher PD and Hawkey CM. Comparative Biochemistry and Physiology 1977; 56B: 335. (6) Meadows RW. Proceedings of Forensic Science Symposium Calgary Alberta, 1977 : 39-46. (7) Sutton JG and Burgess R. Vox Sanguinis 1978; 34: 97.