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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

Introduction

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.

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