- Email: [email protected]
Plasma metabolites indicate energy metabolism disruption during the preclinical phase of bovine spongiform encephalopathy infection
Research in Veterinary Science 2002, 73, 191–193 doi:10.1016/S0034-5288(02)00103-0, available online at http://www.idealibrary.com on
SHORT COMMUNICATION
Plasma metabolites indicate energy metabolism disruption during the preclinical phase of bovine spongiform encephalopathy infection J. M. MOORBY ,*, P. BEGLEY , R. J. NASH , M. K. THEODOROU , A. R. AUSTINà
à
Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK, Farm Animal Research Associates, Oak Farm, Harpsden Bottom, Henley-on-Thames, RG9 4HY, UK SUMMARY
During the preclinical phase of bovine spongiform encephalopathy (BSE), significantly increased concentrations of lactic acid were measured in the blood of infected dairy cows. Other plasma metabolites, including alanine, leucine, serine, and glutamic acid, also showed significantly altered concentrations in the preclinical BSE animals compared to a control group. This appears consistent with the exaggerated stress response observed in clinical BSE, and precedes the development of clinical signs and overt behavioural changes. A number of other plasma metabolites including other amino acids and components of the plasma fatty acid content showed no association with BSE status. Ó 2002 Elsevier Science Ltd. All rights reserved.
THERE is a need for the development of diagnostic tests to detect bovine spongiform encephalopathy (BSE) in live animals prior to the development of clinical signs. One approach is to identify biochemical changes that are consistently associated with BSE infection. Previous statistical analysis of plasma samples that were opportunistically collected from a group of dairy cattle incubating BSE indicated that traditional metabolic and endocrine markers were of little value for pre-clinical diagnosis of the disease (Moorby et al 2000b). However, there were indications that the energy metabolism of cattle incubating BSE was affected, particularly during times of nutritional stress. This paper describes further analysis of blood samples with the objective of identifying abnormalities that were consistently associated with BSE status, and of assessing them for their diagnostic utility. Blood samples were collected from 47 multiparous Holstein–Freisian dairy cows maintained under welldefined conditions as detailed by Moorby et al (2000b). Briefly, six animals taking part in a nutritional experiment conducted in 1994–1995 (Dewhurst et al 2000, Moorby et al 2000a) developed clinical signs of BSE. One of the animals was confirmed as BSE positive and destroyed during the final stages of the experiment. A further four animals developed clinical signs within 100 days following the end of the experiment. The final BSE-infected cow did not develop clinical signs for a further 18 months, and was excluded from the current study. The BSE status of the animals was not known
*Corresponding author. Fax: +44-1970-823245; E-mail: [email protected] 0034-5288/02/$ - see front matter
when the experiment was designed and conducted, but the experiment provided a limited set of samples during the preclinical phase of naturally occurring BSE cases. The remaining animals from the experiment provided a matched set of control material, their control status being supported by their failure to develop the clinical signs of BSE before they were culled for other reasons, much later. Blood samples were collected from the coccygeal vessels into heparinised evacuated tubes (Vacutainers; Becton, Dickinson and Company UK, Oxford) and held on ice before centrifugation (1700g for 20 min at 4 °C) to harvest plasma, which was stored frozen until later analysed for lactic acid and a range of amino acids and fatty acids. Mean plasma lactic acid concentrations are summarised in Table 1. Visual inspection of the data led us to treat the samples taken during the dry period and during lactation as distinct populations. In comparison to the control animals, animals incubating BSE showed a statistically significant (P < 0:05) increase in plasma lactic acid throughout lactation. The plasma lactic acid concentrations for the control animals are in good agreement with a study by other workers (Ortiz et al 1988), who quote a reference interval of 0:73–2:17 mmol l 1 . Plasma lactic acid concentrations of 5–7 mmol l 1 have been recorded after transport and slaughter (Mitchell et al 1988), and up to 27 mmol l 1 after the extreme stress of a bullfight (Acena et al 1995). Lactic acid in blood can originate from a number of endogenous and exogenous sources, and plasma concentrations of lactate can be manipulated by diet (Owens et al 1998), coming from either the feed (e.g., in silage) or during its fermentation in the rumen. HowÓ 2002 Elsevier Science Ltd. All rights reserved.
192
J. M. Moorby, P. Begley, R. J. Nash, M. K. Theodorou, A. R. Austin
BSE-infected animals exhibited a significantly different mean concentration but this was comparable to the high end of the control range in other studies. For the late preclinical BSE animal, the observed plasma lactate levels exceed those which could be anticipated to result from the stress imposed by blood sampling, although it is unclear whether the measured values result purely from an exaggerated stress response at sampling time, or if basal lactate concentrations are increased in the ‘‘unstressed’’ preclinical BSE subject. Of the amino acids assayed, alanine, leucine, serine, and glutamic acid showed statistically significant differences between the BSE preclinical cases and their matched controls (Table 2). No significant correlations could be identified for any of the other amino acids assayed (glycine, valine, isoleucine, proline, methionine, threonine, phenylalanine, aspartic acid, hydroxyproline, histidine, and tryptophan). The concomitant increase in plasma alanine, decrease in plasma glutamate and the increase in plasma lactate described above may reflect a more frequent or more pronounced recourse to anaerobic respiration. Anoxia can lead to glutamate depletion and alanine production in the cytosol (Wiesner et al 1988), but this could not be confirmed from our sample set. Diabetes mellitus has been induced in hamster models of scrapie (Srinivasappa et al 1989), which, in humans at least, leads to elevated plasma amino acid concentrations (particularly of leucine and isoleucine), and elevated rates of amino acid oxidation (specifically leucine) (Nair and Shenck 1995). Our observations of significantly increased plasma leucine concentrations, accompanied by an increase in plasma isoleucine concentrations (which did not attain statistical significance), is of interest but cannot be considered conclusive without confirmation by a larger sample set. The observed increase in plasma serine is yet to be explained. No clear association could be made between BSE status and concentrations of any of the fatty acids measured in the range C14 –C18 . The work reported here is preliminary, and was limited by the material available for study. It is hypothesised that anaerobic respiration is increased as the disease progresses, and this reflects either a more exaggerated or more readily evoked ‘‘fight or flight’’
TABLE 1: Plasma lactic acid concentrations (mmol l)1) from dairy cattle incubating BSE or control animals maintained under the same management conditions
Preclinical BSE (0–50 days before restriction) Preclinical BSE (50–250 days before restriction) Lactating controls Dry preclinical BSE (120–250 days before restriction) Dry controls
No. of samples
Mean
SD
5 (1 animal)
9.75
2.817
36 (5 animals)
3.08
2.429
77 (29 animals) 9 (4 animals)
1.83 4.74
0.988 3.501
21 (17 animals)
4.09
3.632
Sample numbers were combined from different sampling times, from the number of different animals given in parenthesis.
ever, clinical signs of lactacidaemia are distinguishable from those of late-stage BSE infection. Plasma lactic acid concentrations were significantly increased in the animals developing BSE, during the 50 days or so before clinical signs were detected, and appeared sufficient, in our data set, to allow identification of individual animals as either BSE infected or otherwise normal. These increased plasma lactic acid concentrations were not caused by changes in the diet or feed intake of the animals (Moorby et al 2000b). Clearly, samples from more animals are required to establish whether the concentrations measured are typical of late preclinical BSE. Blood lactic acid not derived from the diet is generated as part of the Cori cycle, and therefore plasma lactic acid concentrations are influenced by a number of factors which lead to anaerobic respiration, including vigorous exercise and the initiation of ‘‘flight or fight’’ responses to threat stimuli. It is important to note that at the time of sampling, the BSE-infected cows appeared clinically normal and did not exhibit the obvious excitability or fear normally associated with later stages of the disease. Although the preclinical BSE cattle showed increased lactic acid concentrations throughout the lactation period, the magnitude of the lactate concentration increase was not sufficient to be diagnostically useful. Samples were taken by a single sampling team, on a group of animals that had been used to frequent handling and sampling, leading to a control mean concentration at the lower end of the range reported in other studies where samples were collected by venepuncture.
TABLE 2:
Mean (%SD) plasma concentrations of amino acids (mmol l 1 ) Control cows
Pre-clinical BSE cows
Statistical significance
Alanine
0.135 (0.0498) (98,31 animals)
0.156 (0.0562) (50,5 animals)
*
Leucine
0.104 (0.0498) (98,31 animals)
0.142 (0.1271) (50,5 animals)
*
Serine
0.083 (0.0462) (30,16 animals)
(0.062 (0.0245) (19,5 animals)
*
Glutamic Acid
0.136 (0.1122) (30,16 animals)
0.105 (0.0442) (19,5 animals)
*
Total number of specimens analysed (combined from different sampling times) is given in parenthesis, with the number of different animals sampled. P < 0:05.
Plasma metabolites in BSE infected dairy cows
stress response. Future work is underway which will test this hypothesis by more detailed study of plasma metabolites that are known to be influenced by the relevant stressors.
ACKNOWLEDGMENTS This work was funded by the Ministry of Agriculture, Fisheries and Food. We thank Mr. W.J. Fisher and his staff for care of the animals.
REFERENCES ACENA, M. C., GARCIA-BELENGUER, S., GASCON, M. & PURROY, A. (1995) Modifications hematologiques et musculaires pendant la corrida chez les taureau de combat. Revue de Medecine Veterinaire 146, 277–282 DEWHURST, R. J., MOORBY, J. M., DHANOA, M. S., EVANS, R. T. & FISHER, W. J. (2000) Effects of altering energy and protein supply to
193
dairy cows during the dry period. 1. Intake, body condition, and milk production. Journal of Dairy Science 83, 1782–1794 MITCHELL, G., HATTINGH, J. & GANHAO, M. (1988) Stress in cattle assessed after handling, after transport, and after slaughter. Veterinary Record 123, 201–205 MOORBY, J. M., DEWHURST, R. J., TWEED, J. K. S., DHANOA, M. S. & BECK, N. F. G. (2000a) Effects of altering the energy and protein supply to dairy cows during the dry period. 2. Metabolic and hormonal responses. Journal of Dairy Science 83, 1795–1805 MOORBY, J. M., DHANOA, M. S. & AUSTIN, A. R. (2000b) Aspects of the metabolism of dairy cows during the incubation of bovine spongiform encephalopathy. Veterinary Record 147, 409–412 NAIR, K. S. & SHENCK, W. F. (1995) Protein metabolism in diabetes mellitus. Diabetes Annual 9, 159–174 ORTIZ, F., LABRADA, I. & BELL, L. (1988) Niveles de acido lactico en sangre de vacas Holstein. Revista de Salud Animal 10, 138–141 OWENS, F. N., SECRIST, D. S., HILL, W. J. & GILL, D. R. (1998) Acidosis in cattle: A review. Journal of Animal Science 76, 275–286 SRINIVASAPPA, J., ASHER, D. M., POMEROY, K., MURPHY, L. J., WOOLF, A. V., YOON, J. W., GAJDUSEK, D. C. & NOTKINS, A. L. (1989) Scrapie-induced diabetes mellitus in hamsters. Microbiological Pathology 7, 189–194 WIESNER, R. J., KREUTZER, U., ROSEN, P. & GRIESHABER, M. K. (1988) Subcellular distribution of malate–aspartate cycle intermediates during normoxia and anoxia in the heart. Biochimica et Biophysica Acta 936, 114–123
SHORT COMMUNICATION
Plasma metabolites indicate energy metabolism disruption during the preclinical phase of bovine spongiform encephalopathy infection J. M. MOORBY ,*, P. BEGLEY , R. J. NASH , M. K. THEODOROU , A. R. AUSTINà
à
Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK, Farm Animal Research Associates, Oak Farm, Harpsden Bottom, Henley-on-Thames, RG9 4HY, UK SUMMARY
During the preclinical phase of bovine spongiform encephalopathy (BSE), significantly increased concentrations of lactic acid were measured in the blood of infected dairy cows. Other plasma metabolites, including alanine, leucine, serine, and glutamic acid, also showed significantly altered concentrations in the preclinical BSE animals compared to a control group. This appears consistent with the exaggerated stress response observed in clinical BSE, and precedes the development of clinical signs and overt behavioural changes. A number of other plasma metabolites including other amino acids and components of the plasma fatty acid content showed no association with BSE status. Ó 2002 Elsevier Science Ltd. All rights reserved.
THERE is a need for the development of diagnostic tests to detect bovine spongiform encephalopathy (BSE) in live animals prior to the development of clinical signs. One approach is to identify biochemical changes that are consistently associated with BSE infection. Previous statistical analysis of plasma samples that were opportunistically collected from a group of dairy cattle incubating BSE indicated that traditional metabolic and endocrine markers were of little value for pre-clinical diagnosis of the disease (Moorby et al 2000b). However, there were indications that the energy metabolism of cattle incubating BSE was affected, particularly during times of nutritional stress. This paper describes further analysis of blood samples with the objective of identifying abnormalities that were consistently associated with BSE status, and of assessing them for their diagnostic utility. Blood samples were collected from 47 multiparous Holstein–Freisian dairy cows maintained under welldefined conditions as detailed by Moorby et al (2000b). Briefly, six animals taking part in a nutritional experiment conducted in 1994–1995 (Dewhurst et al 2000, Moorby et al 2000a) developed clinical signs of BSE. One of the animals was confirmed as BSE positive and destroyed during the final stages of the experiment. A further four animals developed clinical signs within 100 days following the end of the experiment. The final BSE-infected cow did not develop clinical signs for a further 18 months, and was excluded from the current study. The BSE status of the animals was not known
*Corresponding author. Fax: +44-1970-823245; E-mail: [email protected] 0034-5288/02/$ - see front matter
when the experiment was designed and conducted, but the experiment provided a limited set of samples during the preclinical phase of naturally occurring BSE cases. The remaining animals from the experiment provided a matched set of control material, their control status being supported by their failure to develop the clinical signs of BSE before they were culled for other reasons, much later. Blood samples were collected from the coccygeal vessels into heparinised evacuated tubes (Vacutainers; Becton, Dickinson and Company UK, Oxford) and held on ice before centrifugation (1700g for 20 min at 4 °C) to harvest plasma, which was stored frozen until later analysed for lactic acid and a range of amino acids and fatty acids. Mean plasma lactic acid concentrations are summarised in Table 1. Visual inspection of the data led us to treat the samples taken during the dry period and during lactation as distinct populations. In comparison to the control animals, animals incubating BSE showed a statistically significant (P < 0:05) increase in plasma lactic acid throughout lactation. The plasma lactic acid concentrations for the control animals are in good agreement with a study by other workers (Ortiz et al 1988), who quote a reference interval of 0:73–2:17 mmol l 1 . Plasma lactic acid concentrations of 5–7 mmol l 1 have been recorded after transport and slaughter (Mitchell et al 1988), and up to 27 mmol l 1 after the extreme stress of a bullfight (Acena et al 1995). Lactic acid in blood can originate from a number of endogenous and exogenous sources, and plasma concentrations of lactate can be manipulated by diet (Owens et al 1998), coming from either the feed (e.g., in silage) or during its fermentation in the rumen. HowÓ 2002 Elsevier Science Ltd. All rights reserved.
192
J. M. Moorby, P. Begley, R. J. Nash, M. K. Theodorou, A. R. Austin
BSE-infected animals exhibited a significantly different mean concentration but this was comparable to the high end of the control range in other studies. For the late preclinical BSE animal, the observed plasma lactate levels exceed those which could be anticipated to result from the stress imposed by blood sampling, although it is unclear whether the measured values result purely from an exaggerated stress response at sampling time, or if basal lactate concentrations are increased in the ‘‘unstressed’’ preclinical BSE subject. Of the amino acids assayed, alanine, leucine, serine, and glutamic acid showed statistically significant differences between the BSE preclinical cases and their matched controls (Table 2). No significant correlations could be identified for any of the other amino acids assayed (glycine, valine, isoleucine, proline, methionine, threonine, phenylalanine, aspartic acid, hydroxyproline, histidine, and tryptophan). The concomitant increase in plasma alanine, decrease in plasma glutamate and the increase in plasma lactate described above may reflect a more frequent or more pronounced recourse to anaerobic respiration. Anoxia can lead to glutamate depletion and alanine production in the cytosol (Wiesner et al 1988), but this could not be confirmed from our sample set. Diabetes mellitus has been induced in hamster models of scrapie (Srinivasappa et al 1989), which, in humans at least, leads to elevated plasma amino acid concentrations (particularly of leucine and isoleucine), and elevated rates of amino acid oxidation (specifically leucine) (Nair and Shenck 1995). Our observations of significantly increased plasma leucine concentrations, accompanied by an increase in plasma isoleucine concentrations (which did not attain statistical significance), is of interest but cannot be considered conclusive without confirmation by a larger sample set. The observed increase in plasma serine is yet to be explained. No clear association could be made between BSE status and concentrations of any of the fatty acids measured in the range C14 –C18 . The work reported here is preliminary, and was limited by the material available for study. It is hypothesised that anaerobic respiration is increased as the disease progresses, and this reflects either a more exaggerated or more readily evoked ‘‘fight or flight’’
TABLE 1: Plasma lactic acid concentrations (mmol l)1) from dairy cattle incubating BSE or control animals maintained under the same management conditions
Preclinical BSE (0–50 days before restriction) Preclinical BSE (50–250 days before restriction) Lactating controls Dry preclinical BSE (120–250 days before restriction) Dry controls
No. of samples
Mean
SD
5 (1 animal)
9.75
2.817
36 (5 animals)
3.08
2.429
77 (29 animals) 9 (4 animals)
1.83 4.74
0.988 3.501
21 (17 animals)
4.09
3.632
Sample numbers were combined from different sampling times, from the number of different animals given in parenthesis.
ever, clinical signs of lactacidaemia are distinguishable from those of late-stage BSE infection. Plasma lactic acid concentrations were significantly increased in the animals developing BSE, during the 50 days or so before clinical signs were detected, and appeared sufficient, in our data set, to allow identification of individual animals as either BSE infected or otherwise normal. These increased plasma lactic acid concentrations were not caused by changes in the diet or feed intake of the animals (Moorby et al 2000b). Clearly, samples from more animals are required to establish whether the concentrations measured are typical of late preclinical BSE. Blood lactic acid not derived from the diet is generated as part of the Cori cycle, and therefore plasma lactic acid concentrations are influenced by a number of factors which lead to anaerobic respiration, including vigorous exercise and the initiation of ‘‘flight or fight’’ responses to threat stimuli. It is important to note that at the time of sampling, the BSE-infected cows appeared clinically normal and did not exhibit the obvious excitability or fear normally associated with later stages of the disease. Although the preclinical BSE cattle showed increased lactic acid concentrations throughout the lactation period, the magnitude of the lactate concentration increase was not sufficient to be diagnostically useful. Samples were taken by a single sampling team, on a group of animals that had been used to frequent handling and sampling, leading to a control mean concentration at the lower end of the range reported in other studies where samples were collected by venepuncture.
TABLE 2:
Mean (%SD) plasma concentrations of amino acids (mmol l 1 ) Control cows
Pre-clinical BSE cows
Statistical significance
Alanine
0.135 (0.0498) (98,31 animals)
0.156 (0.0562) (50,5 animals)
*
Leucine
0.104 (0.0498) (98,31 animals)
0.142 (0.1271) (50,5 animals)
*
Serine
0.083 (0.0462) (30,16 animals)
(0.062 (0.0245) (19,5 animals)
*
Glutamic Acid
0.136 (0.1122) (30,16 animals)
0.105 (0.0442) (19,5 animals)
*
Total number of specimens analysed (combined from different sampling times) is given in parenthesis, with the number of different animals sampled. P < 0:05.
Plasma metabolites in BSE infected dairy cows
stress response. Future work is underway which will test this hypothesis by more detailed study of plasma metabolites that are known to be influenced by the relevant stressors.
ACKNOWLEDGMENTS This work was funded by the Ministry of Agriculture, Fisheries and Food. We thank Mr. W.J. Fisher and his staff for care of the animals.
REFERENCES ACENA, M. C., GARCIA-BELENGUER, S., GASCON, M. & PURROY, A. (1995) Modifications hematologiques et musculaires pendant la corrida chez les taureau de combat. Revue de Medecine Veterinaire 146, 277–282 DEWHURST, R. J., MOORBY, J. M., DHANOA, M. S., EVANS, R. T. & FISHER, W. J. (2000) Effects of altering energy and protein supply to
193
dairy cows during the dry period. 1. Intake, body condition, and milk production. Journal of Dairy Science 83, 1782–1794 MITCHELL, G., HATTINGH, J. & GANHAO, M. (1988) Stress in cattle assessed after handling, after transport, and after slaughter. Veterinary Record 123, 201–205 MOORBY, J. M., DEWHURST, R. J., TWEED, J. K. S., DHANOA, M. S. & BECK, N. F. G. (2000a) Effects of altering the energy and protein supply to dairy cows during the dry period. 2. Metabolic and hormonal responses. Journal of Dairy Science 83, 1795–1805 MOORBY, J. M., DHANOA, M. S. & AUSTIN, A. R. (2000b) Aspects of the metabolism of dairy cows during the incubation of bovine spongiform encephalopathy. Veterinary Record 147, 409–412 NAIR, K. S. & SHENCK, W. F. (1995) Protein metabolism in diabetes mellitus. Diabetes Annual 9, 159–174 ORTIZ, F., LABRADA, I. & BELL, L. (1988) Niveles de acido lactico en sangre de vacas Holstein. Revista de Salud Animal 10, 138–141 OWENS, F. N., SECRIST, D. S., HILL, W. J. & GILL, D. R. (1998) Acidosis in cattle: A review. Journal of Animal Science 76, 275–286 SRINIVASAPPA, J., ASHER, D. M., POMEROY, K., MURPHY, L. J., WOOLF, A. V., YOON, J. W., GAJDUSEK, D. C. & NOTKINS, A. L. (1989) Scrapie-induced diabetes mellitus in hamsters. Microbiological Pathology 7, 189–194 WIESNER, R. J., KREUTZER, U., ROSEN, P. & GRIESHABER, M. K. (1988) Subcellular distribution of malate–aspartate cycle intermediates during normoxia and anoxia in the heart. Biochimica et Biophysica Acta 936, 114–123