CCAC guidelines on: choosing an appropriate endpoint in experiments using animals for research, teaching and testing

APPENDIX C: EXAMPLES OF OBSERVATIONAL CHECKLISTS USED TO DETERMINE ENDPOINTS

1. CHECKLIST SAMPLE #1: ACUTE RESPIRATORY INFECTIOUS DISEASE IN CATTLE - A VACCINE/CHALLENGE STUDY



1. Clinical Evaluation
   
   Calves will be clinically examined each morning (8:00 a.m. - 12:00 p.m.) from day 0 to day 10 by the research veterinarian. In the afternoon, they will be examined in the pen by an animal health technician. Calves with a sickness score of >3.0, as determined by the research veterinarian, will be euthanized and necropsied that day.
   
   During the vaccination and evaluation period of the study, no concurrent medication will be administered except in the unlikely event of an anaphylactic type reaction to vaccination. Administration of antimicrobials during the study would render the trial useless since the outcomes of vaccine efficacy are sickness, weight change, death and severity of post-mortem lesions.



2. Response Variables and Scoring Method
   
   A clinical examination on each calf will be conducted daily (day 0 to 10) by the research veterinarian, using the following standardized parameters to gauge the degree of clinical illness. Scores are described in increments of 0 through 4.
   
   
   1. Weight. The weight is recorded daily for each animal. Fluctuations in weight are possible depending on when the animal has eaten. Weight loss over a few days is typical in animals which show other clinical signs.
      
      
   2. Temperature. Rectal temperature is taken with the animal restrained in a chute. Temperatures will be taken and recorded daily in all animals. Individual animal temperatures as well as mean group temperatures will be evaluated. Caution must be exercised in the evaluation process since severely ill animals on the verge of death usually have subnormal temperatures which will artificially lower the mean group temperatures. In such cases, if an animal has an elevated temperature followed by a declining temperature on the day of or day preceding death, that temperature may be excluded from calculating the daily mean group temperature for those respective days.
      
      
   3. Rhinitis or nasal score.
      
      

      0	Normal (mucosa pale pink, no visible nasal discharge).
      1	Mild Rhinitis (mild serous rhinitis with focal mucosal necrosis, nostrils moist, secretions transparent, mucosa mildly hyperemic, focal mucosal vesicles, small white circular necrotic plaques < 2mm diameter, mild serous nasal discharge).
      2	Moderate Rhinitis (moderately severe serous rhinitis with confluent areas of mucosal necrosis, mixture of focal plaques and confluent areas of necrosis, nostrils not occluded by exudates, mucosa intensely hyperemic, large confluent mucosal plaques >2mm diameter, serous nasal discharge with occasional globules or small strands of mucopurulent exudate).
      3	Severe Rhinitis (necrotizing rhinitis, nasal discharge partially occluding nostrils, necrotic plaques cover large portion of nasal mucosa, intense mucosal hyperemia, may have halitosis, nostrils partially occluded by exudates, nasal secretion a mixture of serous and mucopurulent exudates, large confluent mucosal plaques, necrotic tissues forming diphtheritic pseudomembranes that peel from septum).
      4	Very Severe Rhinitis (severe mucopurulent rhinitis with advanced mucosal necrosis, mucosal erosions bleed easily, halitosis, profuse nasal discharge, exudate hanging from nostrils, mouth breathing, head extended, thick coat of purulent of catarrhal exudate on nasal mucosa, exudates almost completely occlude nostrils).

      
      
   4. Depression score.
      
      

      0	Normal (bright, alert, eyes bright, ears erect, chews cud, curious, attentive, stays with the group, stretches back muscles, hind legs when stands, licks nostrils frequently, usually within ten seconds of release from headgate).
      1	Mildly Depressed (ears droop slightly, rarely stands alone, licks nostrils occasionally, tries to stay with the group, difficult to corner or nearly impossible to catch in corral).
      2	Moderately Depressed (walks slowly, lethargic, stands alone for prolonged periods, sometimes stands with head low, easy to corner, but would be difficult to catch).
      3	Severely Depressed (uninterested in environment, very lethargic, apathetic, stands with head lowered most of the time, a "straggler", lies in sternal recumbency frequently, often recumbent, reluctant to stand, but can stand when encouraged, easy for one person to catch).
      4	Moribund (near death, no attempt to clean nostrils, recumbent almost continuously, rarely stands up, oblivious to surroundings, unable, unwilling, or very reluctant to stand, sternal or lateral recumbency).

      
      
   5. Strength.
      
      

      0	Normal (healthy, strong, stands square, runs fast, well-coordinated, impossible to catch, lays with head and legs positioned normally, curled under, easily keeps up with group).
      1	Mild Weakness (unsteady gait, knuckles occasionally when walking, hind end wobbles, but calf does not stumble, walks slowly, but can trot and gallop when chased).
      2	Moderate Weakness (staggers noticeably, may fall down when struggling, but rises again without delay, can walk and trot, but cannot gallop, rests head on ground when laying down, fairly easy to push off balance, could be laid on side with moderate difficulty).
      3	Severe Weakness (rises with difficulty, knuckles frequently, stumbles occasionally, runs slowly, easily caught, has difficulty standing up, very easy to push off balance, often falls down when struggling).
      4	Advanced Deterioration (very weak, unable to stand, emaciated, dehydrated, sunken eyes, skin tenting).

      
      
   6. Respiratory distress.
      
      

      0	Normal (normal nasal breathing, mouth closed, lips dry).
      1	Mild Respiratory Distress (intermittent mouth breathing, lips and jaw moist from salivation, but not observed holding mouth open, mucous membranes pink unless stressed).
      2	Moderate Respiratory Distress (stands with head extended, salivates profusely, muzzle dripping wet).
      3	Severe Respiratory Distress (mouth breathes when stressed, opens mouth frequently, occasionally extends tongue, breathes through mouth when disturbed, labored breathing, becomes cyanotic when stressed minimally).
      4	Very Severe Respiratory Distress (cyanotic, stands with head lowered, neck extended, mouth open and tongue extended, severe drooling, anxious expression).

      
      
   7. Sickness score. Subjective - meant to emulate decision to select animals for treatment on the farm or at feedlot. This score is made after other scores recorded and clinician is informed of the animal's temperature. Since animals are not treated with antibiotics, this scoring system is used to determine the endpoint where an animal is euthanized rather than being allowed to die.
      
      

      0	Healthy Animal (normal healthy animal, no treatment required, no fever, no clinical symptoms).
      0.5	Suspicion of Disease (animal would not be treated, slight clinical signs and no fever, fever 39.5oC-39.9oC and no clinical signs, single chronic unresolved clinical sign and no fever, animal not depressed and still eating well).
      1	Mildly Sick Animal (animal would be treated, febrile (40.0oC) and/or clinical signs, disease progressed to where it would be selected to initiate therapy on the farm or feedlot, recovery would be expected with appropriate therapy).
      2	Obviously Sick Animal (animal would be treated, obvious clinical signs usually febrile, disease is now serious - animal should have been treated before disease progressed to this point, recovery even with appropriate therapy would not be certain). These animals are watched to see if they progress to the next sickness score.
      3	Very Sick Animal (animal would be treated in a clinical setting, severe clinical signs, including depression - fever may be present or temperature dropping, disease is very serious, recovery unlikely even with appropriate therapy). These animals are euthanized.
      4	Moribund (near death, treatment would be futile; animal would be euthanized in a clinical or experimental setting).

      
      
      
2. CHECKLIST SAMPLE #2: CHRONIC INFECTIOUS DISEASE IN MICE - LEISHMANIASIS
   
   
   1. General
      
      
      1. The laboratory animal veterinarians are to be notified at the beginning of each experiment involving foot inoculation - date of inoculation, number of animals involved, etc.
         
         
      2. Any negative effects with respect to animal condition are to be recorded on a checklist by the research staff or animal technicians, and reported to the laboratory animal veterinarian.
         
         
      3. Animals on experiment are to be weighed weekly.
         
         
      4. Records relating to animal observations and endpoint measurements will be kept by the principal investigator. Such records to be kept in the animal room, accessible to both technical animal care and veterinary staff.
         
         
      5. All unexpected negative effects on an animal's health and well-being are to be reported to the laboratory animal veterinarian immediately.
         
         
   2. Response Variables and Scoring System
      
      
      1. Body weight changes.
         
         

         0	Normal
         1	< 10% Weight loss
         2	10-15% Weight loss
         3	> 20% Weight loss.

         
         
      2. Physical appearance.
         
         

         0	Normal
         1	Lack of grooming
         2	Rough coat, nasal/ocular discharge
         3	Very rough coat, abnormal posture.

         
         
      3. Behavior.
         
         

         0	Normal
         1	Minor changes; limping, favoring inoculated limb
         2	Abnormal; reduced mobility, inactive
         3	Unsolicited vocalizations, self-mutilation, either very restless or immobile.

         
         
      Endpoint scoring: When a total score of three or more is reached, based on above checklist, the laboratory animal veterinarian must be notified.
      
      
   3. Specific
      
      
      1. Foot inoculation. Foot swelling will be measured with a caliper. Measurements of foot swelling will be taken weekly. When the foot swelling is 4mm the animals will be humanely killed. In the event that this endpoint is reached and the principal investigator is unavailable for action, the decision regarding the immediate fate of the animals will be made by the veterinarian.
         
         
      2. Rump skin inoculation. The diameter of the skin lesion will be measured weekly. When the skin lesion reaches 8mm diameter, the animals will be humanely killed. In the event that this endpoint is reached and the principal investigator is unavailable for action, the decision regarding the immediate fate of the animals will be made by the veterinarian.
         
3. CHECKLIST SAMPLE #3: CHEMICALLY INDUCED (AZOXYMETHANE) COLON CANCER IN RATS
   
   In this model, using Sprague Dawley rats, colonic tumors develop six to eight months after initiation of the carcinogenesis protocol, and grow relatively slowly after that. Routine animal care and routine monitoring is done for the first four months. Four months after initiation, the animals are examined three times weekly for signs of tumor burden. One animal is killed at this time to assess the progression of the tumor in each particular experiment. The examinations include palpation of the abdomen for presence of tumors or ascitic fluid, examination of the eyes for signs of anemia, testing the feces for blood, and examination of the general appearance and behavior of the animal.
   
   
   1. Response Variables and Scoring System
      
      
      1. Body weight changes.
      
      
      

      0	Normal
      1	< 10% Weight loss
      2	10-15% Weight loss
      3	> 20% Weight loss.

      
      
      2. Physical appearance.
      
      
      

      0	Normal
      1	Anemia (pale eye color)
      2	Anemia, and blood in feces, diarrhea and/or small fecal pellets
      3	Above signs, plus abdominal swelling
      4	Ruffled fur coat, lack of grooming, abnormal posture.

      
      
      Endpoint scoring: If weight loss exceeds 15% compared to control animals and the animals are not euthanized immediately, they will receive analgesics until the end of the experiment. Any animal reaching a total score of four or higher using the above checklist, will be euthanized and a necropsy done for tumor load evaluation and tissue sampling.