Proceedings for Pain Management and Humane Endpoints
Endpoints in Infectious Disease Animal Models
Ernest D. Olfert1, DVM, MSc, Dale Godson2, DVM, PhD, and Monique Habermehl2, DVM
1Animal Resources Centre, and 2Veterinary Infectious Diseases Organization, University of Saskatchewan
The CCAC guidelines on: choosing an appropriate endpoint in experiments using animals for research, teaching, and testing (CCAC, 1998) were developed to assist with the process of selecting an appropriate (more humane) endpoint. The endpoint guideline relating to infectious diseases in the CCAC document is stated as follows: "For all infectious disease research, including virulence tests in animal models, endpoints should be established that minimize the potential for pain and/or distress in the animals."
With the move to earlier endpoints, there is a valid scientific concern that significant differences in experimental treatments might be masked; earlier endpoints should not alter the outcome of the experiment. Endpoints should be scientifically valid as well as meet the obligation to minimize distress and pain in the animals.
Endpoints for some experimental animal use (including infectious disease models) are currently set at a point when the animals may already have undergone significant pain and/or distress as part of the disease process (Morton and Griffiths, 1985; Morton, 1990; Morton and Townsend, 1995; Workman, et al, 1998). Measuring some of the biological changes that occur early in the immune response can serve as objective indicators of the presence of disease as well as early predictors of the severity of infectious disease, and outcome. This approach has been used, for other reasons, in human medicine and veterinary medicine, and it promises to have application in selecting earlier, more humane endpoints in infectious disease animal models.
Activation of the Immune System by Infection, and the Acute Phase Response
Research during the past decade has revealed a great deal about how the body responds to infection. Infectious organisms invading the body stimulate the immune system into rapid action, initiating (up-regulating) a complex array or cascade of activities that include biochemical, endocrinological, physiological, behavioral and pathological changes. This activation of the body's immune system-mediated defense mechanisms is termed the acute phase response (APR).
The initial response to the foreign agents (e.g., bacteria, fungi, parasites, viruses) is usually local, at the site of infection, and involves the neutrophils, the macrophages and other immune cells, and includes the release of small proteins known as the cytokines from these cells. The cytokines act as regulatory proteins that orchestrate the development and regulation of the immune response, both locally and systemically (van Deuren, et at, 1992; Godson, et al, 1995; Godson, et al, 1997; Schijns and Horzinek, 1997; Gregory, 1998). Interleukin-1 (IL-1), Interleukin-6 (IL-6), and Tumor Necrosis Factor (TNF) are the cytokines whose effects have been most studied.
The local effects occur rapidly and include neutrophilia, increased capillary permeability, increased leucocyte adhesiveness (e.g., to endothelium), increased production and release of other cytokines, up-regulating production of antibodies, up-regulation of the MH-II complexes on monocytes, and so on (van Deuren, et al, 1992; Schijns and Horzinek, 1997; Gregory, 1998). These actions all tend to be positive with respect to helping the body deal with the invading organisms.
If the infection becomes general or prolonged the cytokines reach the circulation and have effects in other organs, turning on other biological functions and processes. In the central nervous system the effects include producing fever, lethargy/sleep and inappetance (van Deuren, et al, 1992; Schijns and Horzinek, 1997; Gregory, 1998), hyperalgesia (increased sensitivity to pain) (Dray, 1995), and increase in some hormone levels (van Deuren, et al, 1992). In liver cells the cytokines stimulate up-regulation and release of a group of proteins collectively known as the acute phase proteins (APP).
Sickness Behaviour Associated with the Acute Phase Response
The behavioural effects produced by the cytokines acting in the brain (lethargy, sleep, inappetance/anorexia) have been termed "sickness behaviour" (Gregory, 1998). It is the resulting deviations from normal behaviour and physiology that are currently measured with the checklists used to set endpoints.
In laboratory animal models of infection, primary indicators of severity of disease or progression to moribundity are weight loss and hypothermia. Secondary signs include those listed in the Rodent Protection Test Guidelines (Acred, et al, 1994) and other publications dealing with signs of pain and distress in laboratory animals.
Temperature Change as an Endpoint in Infectious Disease Animal Models
The initial body temperature response to infection is hyperthermia, or fever. The fever stage may be transient, however, particularly in small animal (rodent) infectious disease models. Fever is not included as a cardinal sign of infection in the Rodent Protection Tests, for example, (Acred, et al, 1994). Lowered body temperature, or hypothermia, however, can be an important indicator of a deteriorating condition in the animal, in specific disease or toxic states. Animals in a septic state lose the ability to maintain body temperature. Decrease in body temperature beyond a certain point (e.g., > 4-6°C) has been correlated with death as an outcome in several infectious disease models (Soothill, et al, 1992; Siems and Allen, 1989; Wong, et al, 1997; Kort, et al, 1998).
Measuring body temperature should therefore be part of the monitoring of any infectious disease animal model. Frequency of monitoring will depend on the progression of the infection, and anticipated time of severe disfunction. The drop in body temperature that can be used as the endpoint depends on the specific infectious organism / animal model under study. Hypothermia by itself is not necessarily predictive of mortality, since sedation and anesthesia can result in reduction of body temperature not associated with mortality. Monitoring body temperature in the small laboratory animals without undue disturbance can readily be accomplished with the use of infrared temperature scanners or implanted thermistors or microchips (Kort, et al, 1998).
Weight Loss as an Endpoint in Infectious Disease Animal Models
As noted above, one effect of the cytokines is to produce inappetance or anorexia in the animal. Thus, weight loss is a cardinal indicator of the severity of infectious disease in animal models. A number of scoring systems in use include monitoring body weight, and recommend using a pre-determined amount of weight loss (e.g., 10-20%, 20%) as an endpoint (Morton and Townsend, 1995; Workman, et al, 1998). Prolonged inappetance can lead to cachexia. The total amount of the weight lost, as well as the duration and consistency of the weight loss should be used to determine the endpoint for infectious disease animal models.
Cytokine Levels as Indicators of Disease, Severity of Disease, and Outcome
Increased cytokine levels can allow early detection of the disease process prior to manifestation of severe clinical illness, can be related to the severity of infection and can thus serve as a prognostic indicator, and can be used to assess the response to therapy (Espersen, et al, 1991). Assays for human cytokines are being used not only to detect disease, but to determine disease severity and predict outcome in a number of disease syndromes (Hack, et al, 1989; Waage, et al, 1989; Cannon, et al, 1990; Kwiatkowski, et al, 1990; Sawada, et al, 1991; Braegger, et al, 1992; Suputtamongkol, et al, 1992; Damas, et al, 1992; Sullivan, et al, 1992; Girardin, et al, 1992; Suter, et al, 1992; Casey, et al, 1993; Sehgal, 1996). In veterinary medicine, the relationship between levels of cytokine and disease has also been studied (Bielefeldt Ohmann and Babiuk, 1985; Bielefeldt Ohmann, et al, 1989; Morris and Moore, 1991; Morris, et al, 1991; Sordillo and Peel, 1992; Horadagoda, et al, 1994; Nakajima, et al, 1997).
The predictive ability of cytokine monitoring, however, has not been consistent. Time of sampling may be a critical feature due to the relatively short half-life of the cytokine in circulation, and the production of cytokine binding factors which is initiated fairly shortly after the production of the cytokine.
The Acute Phase Proteins (APP) and the Acute Phase Response
The production and release of the acute phase proteins (APP) into the blood is an important part of the acute phase response. In normal healthy animals, acute phase proteins are either not detectable, or occur at very low levels in the plasma. Plasma levels rise rapidly (within hours) following cytokine up-regulation of the hepatocytes in response to an infection or inflammatory process. The main interest in the APP in human and veterinary medicine has been their diagnostic value as indicators of the presence of infection and inflammation (Eckersall and Connor, 1988; Connor, et al, 1989; Eckersall, 1992; Eckersall, 1995, Godson, et al, 1996). High levels of the major APP correlate well with presence and severity of infectious disease. They can thus be used for diagnosis (the presence and extent of inflammatory lesions), for prognosis in infectious disease cases, for measuring the response to treatment, and in experimental situations can be used as predictors of outcome (Godson, et al, 1996).
Using the Acute Phase Protein - Haptoglobin - for Diagnosis, Prognosis, Treatment Evaluation, And Endpoint Determination in Cattle Infectious Diseases The study of haptoglobin levels in cattle serves as an excellent example of how an acute phase protein can be useful for diagnostic, prognostic, and treatment evaluation purposes, (Spooner and Miller, 1971; Makimura and Suzuki, 1982; Conner, et al, 1986; Conner, et al, 1988; Conner, et al, 1989; Skinner, et al, 1991; Kent, 1992; Faulkner, et al, 1992; Morimatsu, et al, 1992; Uchida, et al, 1993; Yoshino, et al, 1992; Murata and Miyamoto, 1993; Hofner, et al, 1994; Horadogoda, et al, 1994; Stokka, et al, 1994; Eckersall, 1995; McNair, et al, 1995; Godson, et al, 1996; Hirvonen, et al, 1996; Salonen, et al, 1996; Fisher, et al, 1997; Young, et al, 1996; Wittum, et al, 1996), and can serve as an early indicator suitable to determine the endpoint in experimental infectious disease models.
Haptoglobin has very low values in normal healthy cattle. The increase in haptoglobin following infection can be in the order of a 1000-fold, and is associated with the severity of the insult. In a controlled experimental situation, haptoglobin levels are an excellent monitor of the inflammatory response (Godson, et al, 1996) and can be used in research trials as an objective measure of disease severity. Studies on a bovine respiratory disease model in dairy calves indicated that elevations in haptoglobin levels correlated with the subjective clinical sickness score, elevated body temperature, weight change, and could be used to discriminate between clinical outcomes. Haptoglobin levels began increasing 24-48 hours after the calves were exposed to the bacterial agent Pasteurella haemolyticus and were significantly higher by 72 hours, in calves that were euthanized (or died) (Godson, et al, 1996).
Because the high haptoglobin levels occur early in the development of clinical signs of disease, and correlate with the severity of the lesions, they can serve as early and objective indicators that a severe infection is developing in an experimental animal, on which a decision to terminate the animal can objectively be based.
Summary and Conclusions
Clinical signs of sickness behaviour associated with infectious processes are produced by the systemic effects of cytokines. The cardinal clinical signs in infectious disease animal models are changes in body temperature, weight loss (due to inappetance or cachexia), and lethargy. An understanding of the acute phase response is essential to supporting the rationale for selection of pertinent clinical signs in infectious disease animal models, and for the correlation of sickness behavior with biochemical and physiological changes occurring in the body.
Increases in blood levels of cytokines, and major acute phase proteins (APP) precede severe clinical signs. Cytokine levels are associated with infectious disease and its severity, however the transient nature of higher cytokine levels, and the inconsistency of predictive ability of cytokine monitoring limit the value of using elevated cytokine levels to determine earlier endpoints in infectious disease animal models.
Elevated acute phase protein (APP) levels occur early in a variety of infectious diseases, remain high throughout the infection, and decrease in response to treatment. Measuring the species-typical major acute phase proteins has good potential for use as a diagnostic tool, for prognosis, and for evaluating treatment. Such measurements can also be valuable in selecting scientifically objective, earlier endpoints in infectious disease research animal models.
- Acred, P., Hennessey, T. D., MacArthur-Clark, J. A., et al. (1994). Guidelines for the welfare of animals in rodent protection tests - A report of the Rodent Protection Test Working Party. Lab. Anim, 28: 13-18.
- Bielefeldt Ohmann, H. and Babiuk, L. A. (1985). Viral bacterial pneumonia in calves: Effect of bovine herpesvirus-1 on immunological functions. J. Inf. Dis. 151: 937-947.
- Bielefeldt Ohmann, H., Campos, M., Griebel, P. J., et al. (1989). 2',5' oligoadenylate synthetase activity in bovine peripheral blood leukocytes and alveolar macrophages exposed to recombinant interferons and tumor necrosis factor-alpha. Can. J. Vet. Res. 53: 161-166.
- Bielefeldt Ohmann, H., Campos, M., Harland, R., et al. (1989). 2',5' oligoadenylate synthetase activity in bovine peripheral blood mononuclear cell following bovine herpes type-1 induced respiratory disease: A prognostic indicator? J. Interferon Res. 9: 159-166.
- Braegger, C. P., Nicholls, S., Murch, S. H., et al. (1992). Tumour necrosis factor alpha in stool as a marker or intestinal inflammation. Lancet 339: 89-91.
- Brailly, H., Montero Julian, F. A., Zuber, C. E., et al. (1994). Total interleukin-6 in plasma measured by immunoassay. Clin. Chem. 40(1): 116-23.
- Campos, M., Godson, D. L., Hughes, H. P. A., et al. (1994). Cytokine applications in infectious disease. In Cell-Mediated Immunity in Ruminants. (Eds. Goddeeris, B. M. and Morrison, W. I.). CRC Press, Inc., Boca Raton.
- Cannon, J. G., Gelfand, J. A., Stanford, G. J., et al. (1990). Circulating interleukin-1 and tumor necrosis factor in septic shock and experimental endotoxin fever. J. Inf. Dis. 161: 79-84.
- Casey, L. C., Balk, R. A. and R. C. Bone (1993). Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome [see comments]. Ann. Intern. Med. 119(8): 771-778.
- CCAC (1998). CCAC guidelines on: choosing an appropriate endpoint in experiments using animals for research, teaching, and testing. Canadian Council on Animal Care, 315 - 350 Albert Street, Ottawa, Ontario K1R 1B1.
- CCAC (1989). Ethics of Animal Investigation. Canadian Council on Animal Care, 315 - 350 Albert Street, Ottawa, Ontario K1R 1B1.
- Clemmer, T. P., Fisher, Jr., C. J., Bone, R. C., et al. (1992). Hypothermia in the sepsis syndrome and clinical outcome. Crit. Care Med. 20(10): 1395-1401.
- Conner, J. G., Eckersall, P. D., Doherty, M., et al. (1986). Acute phase response and mastitis in the cow. Res. Vet. Sci. 41: 126-128.
- Conner, J. G., Eckersall, P. D., Wiseman, A., et al. (1988). Bovine acute phase response following turpentine injection. Res. Vet. Sci. 44: 82-88.
- Connor, J. G., Eckersall, P. D., Wiseman, A., et al. (1989). Acute Phase Response in calves following infection with Pasteurella hemolytica, Ostertagia ostertagi, and endotoxin administration. Res. Vet. Sci. 47: 203-207.
- Damas, P., Ledoux, D., Nys, M., et al. (1992). Cytokine serum level during severe sepsis in human; IL-6 as a marker of severity. Ann. Surg. 215: 356-362.
- Dray, A. (1995). Inflammatory mediators of pain. Brit. J. Anaesth. 75: 125-131.
- Eckersall, P. D. (1992). Meat Inspection - The potential of acute phase protein assay. Meat Focus International October: 279-283.
- Eckersall, P. D. (1995). Acute phase proteins as markers of inflammatory lesions. Comp. Hematol. Int. 5: 93-97.
- Eckersall, P. D. and J. G. Connor (1988). Bovine and canine acute phase proteins. Vet. Res. Commun. 12: 169-178.
- Faulkner, D. B., Eurell, T., Tranquilli, W. J., et al. (1992). Performance and health of weanling bulls after butorphanol and xylazine administration at castration. J. Anim. Sci. 70: 2970-2974.
- Friedland, J. S., Porter, J. C., Daryanani, S., et al. (1996). Plasma proinflammatory cytokine concentrations, Acute Physiology and Chronic Health Evaluation (APACHE) III scores and survival in patients in an intensive care unit. Crit. Care. Med. 24(11): 1775-1781.
- Girardin, E., Lombard-Roux, P., Grau, G. E., et al. (1992). Imbalance between tumour necrosis factor alpha and soluble TNF receptor concentrations in severe meningococcaemia. Immunol. 76: 20-23.
- Godson, D. L., Baca-Estrada, M. E., Van Kessel, A. G., et al. (1995). Regulation of bovine acute phase responses by recombinant Interleukin-1b. Can. J. Vet. Res. 59: 249-255.
- Godson, D. L., Campos, M., Attah-Poku, S. K., et al. (1996). Serum haptoglobin as an indicator of the acute phase response in bovine respiratory disease. Vet. Immunol. Immunopath. 51: 277-292.
- Godson, D. L., Baca-Estrada, M. E. and L. A. Babiuk (1997). Chapter 1. Applications of bovine cytokines. In Cytokines in Veterinary Medicine. (Eds. Schijns, V. E. C. J. and Horzinek, M. C.) CAB Int., Wallingford, UK pp 3-13.
- Gregory, N. G. (1998). Physiological mechanisms causing sickness behaviour and suffering in diseased animals. Anim. Welf. 7: 293-305.
- Hack, C. E., De Groot, E. R., Felt-Bersma, R. J., et al. (1989). Increased plasma levels of interleukin-6 in sepsis. Blood 74: 1704-1710.
- Hirvonen, J., Pyorala, S. and H. Jousimies Somer (1996). Acute phase response in heifers with experimentally induced mastitis. J. Dairy Res. 63: 351-360.
- Hofner, M. C., Fosbery, M.W., Eckersall, P. D., et al. (1994). Haptoglobin response of cattle infected with foot-and-mouth disease virus. Res. Vet. Sci. 57: 125-128.
- Horadagoda, A., Eckersall, P. D., Hodgson, J. C., et al. (1994). Immediate responses in serum TNFa and acute phase protein concentrations to infection with Pasteurella haemolytica A1 in calves. Res. Vet. Sci. 57: 129-132.
- Kent, J. (1992). Acute phase proteins: their use in veterinary diagnosis. Brit. Vet. J. 148: 279-282.
- Kort, W. J., Hekking-Weijma, J. M., TenKate, M. T., et al. (1998). A microchip implant system as a method to determine body temperature of terminally ill rats and mice. Lab. Anim. 32: 260-269.
- Kwiatkowski, D., Hill, A. V., Sambou, I., et al. (1990). TNF concentration in fatal cerebral, non-fatal cerebral and uncomplicated Plasmodium falciparum malaria. Lancet 336: 1201-1204.
- Makimura, S. and N. Suzuki (1982). Quantitative determination of bovine serum haptoglobin and its elevation in some disease conditions. Jpn. J. Vet. Sci. 44: 15-21.
- McNair, J., Elliott, C. T. and D. P. Mackie (1995). Development of a sensitive and specific time resolved fluorimetric immunoassay for the bovine acute phase protein haptoglobin (HP). J. Immunol. Meth. 184: 199-206.
- Morimatsu, M., Sarikaputi, M., Syuto, B., et al. (1992). Bovine haptoglobin: single radial immunodiffusion assay of its polymeric forms and dramatic rise in acute-phase sera. Vet. Immunol. Immunopathol. 33: 365-372.
- Morris, D. D. and J. N. Moore (1991). Tumor necrosis factor activity in serum from neonatal foals with presumed septicemia. J. Amer. Vet. Med. Assoc. 199: 1584-1589.
- Morris, D. D., Moore, J. N. and N. Crowe (1991). Serum tumor necrosis factor activity in horses with colic attributable to gastrointestinal tract disease. Amer. J. Vet. Res. 52: 1565-1569.
- Morton, D. B. and P. H. M. Griffiths (1985). Guidelines on the recognition of pain and discomfort in experimental animals and an hypothesis for assessment. Vet. Rec. 116: 431-436.
- Morton, D. B. and P. Townsend (1995). Chapter 13: Dealing with Adverse Effects and Suffering During Animal Research. In Laboratory Animals - An Introduction for Experimenters, 2nd Ed. (Ed. Tuffery, A. A.) EdWiley & Sons Ltd. England, pp 215-231.
- Morton, D. B. (1997). Chapter 20. Socio-economic and ethical aspects. Part 4. Ethical and refinement aspects of animal experimentation. In Veterinary Vaccinology. (Eds. Pastoret , P.-P., Blancou, J., Vannier, P. and Verschueren, C.) Elsevier Science, New York. pp 763-785.
- Murata, H. and T. Miyamoto (1993). Bovine haptoglobin as a possible immunomodulator in the sera of transported calves. Brit. Vet. J. 149: 277-283.
- Nakajima, Y., Mikami, O., Yoshioka, M., et al. (1997). Elevated levels of tumor necrosis factor-alpha and interleukin-6 activities in the sera and milk of cows with naturally occurring coliform mastitis. Res. Vet. Sci. 62: 297-298.
- Nakajima, Y., Yoshioka, M., Mikami, O., et al. (1997). Association of interleukin-6 in the cerebrospinal fluid during crisis of calf with ammoniated feed syndrome. Vet. Immunol. Immunopathol. 57(1-2): 79-85.
- Saini, P. K. and D. W. Webert (1991). Application of acute phase reactants during antemortem and postmortem meat inspection. J. Amer. Vet. Med. Assoc. 198(11): 1898-1901.
- Salonen, M., Hirvonen, J., Pyorala, S., et al. (1996). Quantitative determination of bovine serum haptoglobin in experimentally induced Escherichia coli mastitis. Res. Vet. Sci. 60: 88-91.
- Sawada, T., Hirohata, S., Inoue, T., et al. (1991). Correlation between rheumatoid factor and IL-6 activity in synovial fluids from patients with rheumatoid arthritis. Clin. Exp. Rheumatol. 9: 363-368.
- Schijns, V. E. C. J. and M. C. Horzinek (1997). Introduction. In Cytokines in Veterinary Medicine. (Eds. Schijns, V. E. C. J. and Horzinek, M. C.). CAB Int., Wallingford, UK. pp xix-xxiv.
- Sehgal, P. B. (1996). Interleukin-6-type cytokines in vivo: regulated bioavailability. Proc. Soc. Exp. Biol. Med. 213(3): 238-47.
- Siems, J. J. and S. D. Allen (1989). Early euthanasia as a alternative to death in chronic infectious disease studies using a systemic Candida albicans model. Abstr. 89th Annual Meeting of the American Society for Microbiology.
- Skinner, J. G., Brown, R. A. and L. Roberts (1991). Bovine haptoglobin response in clinically defined field conditions. Vet. Rec. 128: 147-149.
- Soothill, J. S., Morton, D. B. and A. Ahmad (1992). The HID50 (hypothermia inducing dose 50): an alternative to the LD50 for the measurement of bacterial virulence. Int. J. Exp. Pathol. 75: 95-98.
- Sordillo, L. M. and J. E. Peel (1992). Effect of interferon-gamma on the production of tumor necrosis factor during acute Escherichia coli mastitis. J. Dairy Sci. 75(8): 2119-2125.
- Spooner, R. L. and J. K. Miller (1971). The measurement of haemoglobin reactive protein in ruminants as an aid to the diagnosis of acute inflammation. Vet. Rec. 88: 2-4.
- Stokka, G. L., Edwards, A. J., Spire, M. F., et al. (1994). Inflammatory response to clostridial vaccines in feedlot cattle. J. Amer. Vet. Med. Assoc. 204: 415-419.
- Sullivan, J. S., Kilpatrick, L., Costarino, T., et al. (1992). Correlation of plasma cytokine elevations with mortality rate in children with sepsis. J. Ped. 120: 510-515.
- Suputtamongkol, Y., Kwiatkowski, D., Dance, D. A. B., et al. (1992). Tumor necrosis factor in septicemic melioidosis. J. Infect. Dis. 165: 561-564.
- Suter, P. M., Suter, S., Girardin, E., et al. (1992). High bronchoalveolar levels of tumor necrosis factor and its inhibitors interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock or sepsis. Amer. Rev. Resp. Dis. 145: 1016-1022.
- Thompson, D., Milford-Ward, A. and J. T. Whicher (1992). The value of acute phase proteins in clinical practice. Ann. Clin. Bioch. 29: 123-131.
- Uchida, E., Katoh, N. and K. Takahashi (1993). Appearance of haptoglobin in serum from cows at parturition. J. Vet. Med. Sci. 55: 893-894.
- van Deuren, M., Dofferhoff, A. S. M., and J. W. M. van der Meer (1992). Cytokines and the response to infection. J. Pathol. 168: 349-356.
- Waage, A., Brandtzaeg, P., Halstensen, A., et al. (1989). The complex pattern of cytokines in serum from patients with meningococcal septic shock. J. Exp. Med. 169: 333-338.
- Waage, A., Halstensen, A., Shalaby, R., et al. (1989). Local production of tumor necrosis factor alpha, interleukin 1 and interleukin 6 in meningococcal meningitis. Relation to the inflammatory response. J. Exp. Med. 170: 1859-1867.
- Wagner, L., Gessl, A., Baumgartner Parzer, S., et al. (1996). Haptoglobin phenotyping by newly developed monoclonal antibodies. J. Immunol. 156: 1989-1996.
- Wallace, J., Sanford, J., Smith, M. W. and K. V. Spencer (1990). The assessment and control of the severity of scientific procedures on laboratory animals. Lab. Anim. 24: 97-130.
- Wittum, T. E., Young, C. R., Stanker, L. H., et al. (1996). Haptoglobin response to clinical respiratory tract disease in feedlot cattle. Amer. J. Vet. Res. 57: 646-652.
- Wong, J. P., Saravolac, E. G., Clement, J. G. and L. P. Nagata (1997). Development of a murine hypothermia model for study of respiratory tract influenza virus infection. Lab. Anim. Sci. 47(2): 143-147.
- Workman, P., Twentyman, P., Balkwill, F., et al. (1998). United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines for the welfare of animals in experimental neoplasia (Second Edition, July 1997). Brit. J. Cancer 77(1): 1-10.
- Yoshino, K., Katoh, N., Takahashi, K., et al. (1992). Purification of a protein from serum of cattle with hepatic lipidosis, and identification of the protein as haptoglobin. Amer. J. Vet. Res. 53: 951-956.
- Yoshino, K., Katoh, N., Takahashi, K., et al. (1993). Possible involvement of protein kinase C with induction of haptoglobin in cows by treatment with dexamethasone and by starvation. Amer. J. Vet. Res. 54: 689-694.
- Young, C. R., Eckersall, P. D., Saini, P. K., et al. (1995). Validation of immunoassays for bovine haptoglobin. Vet. Immunol. Immunopath. 49: 1-13.
- Young, C. R., Wittum, T. E., Stanker, L. H., et al. (1996). Serum haptoglobin concentrations in a population of feedlot cattle. Amer. J. Vet. Res. 57: 138-141.
New ALTEX: 4/2016
January 8-13, 2017
Various locations throughout Europe
Beginning April 2017
June 14-16, 2017
SAVE THE DATE:
10th World Congress on Alternatives and Animal Use in the Life Sciences
August 20-24, 2017