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APPENDIX I Suggested Standard Procedures for Detection of Tuberculosis ILAR Subcommittee on Laboratory Animal Quality Standards L. R. Christensen (Chairman), R. J. Flynn, W. G. Hoag, R. Holdenried, D. L. Huxsoll, H. M. Kaplan, L. M. Kraft, J. B. Nelson, and M. Pollard PREFACE The objective of the Institute of Laboratory Animal Resources' Subcom- mittee on Laboratory Animal Quality Standards is to recommend sampling and testing procedures for the detection of infectious agents in laboratory animal colonies. In developing the report on tuberculosis, the group con- sidered the advice of several primatologists. Although there is not complete agreement on universally applicable testing procedures for tuberculosis in primates, or on the species and families within the order Primates that should be routinely tested, the subcommittee members feel that the procedure as presented gives the best possible assurance that an individual or colony is tuberculosis-free. The report is presented as "Suggested Standard Procedures" to encourage further comment by specialists in primate medicine. AGENT Mycobacterium tuberculosis ANIMALS All nonhuman primates 44
45 INTRODUCTION Tuberculin-testing is the single most practical and reliable method for de- tection of tuberculosis in primates. Necropsy and x-ray examination are valuable adjuncts. Available evidence indicates that Old World monkeys are more suscep- tible to tuberculosis than New World monkeys and prosimian primates. Nevertheless, tuberculosis can occur in all nonhuman primates. These standards, therefore, shall apply to all nonhuman primate colonies. Most primate colonies will fall within one of two categories. Transient colonies are those in which animals are held for a limited time, usually before delivery to a using laboratory. Typical of such colonies are those maintained by importers and dealers holding animals for sale, and quaran- tine, isolation, and conditioning colonies maintained by both dealers and research institutions to prepare animals for use in research. Because of their continually changing populations, such colonies can never be termed tuber- culosis-free, but animals leaving such colonies can be termed tuberculosis-free if they meet the criteria specified below under A(l-3). Maintenance colonies represent the usual laboratory situation. Animals are maintained for longer periods and the populations are relatively stable. Only tuberculosis-free primates should be admitted to such a colony. SAMPLING PROCEDURE A. Transient Colony 1. Each animal shall be examined by chest x-ray on arrival to detect advanced cases of tuberculosis which may be tuberculin-negative. 2. Each animal in an incoming group* shall be tuberculin-tested at 2- week intervals! until all animals in the group have passed three consecutive negative tests. If a positive reaction occurs, the reactor shall be necropsied and appro- *"Group" refers to a population with a similar history and environment. For example, a shipment of animals received at one time and kept physically isolated from older and newer arrivals would constitute a group. All animals housed in one room in a research facility would constitute a group, assuming effective separation of these from primates in other rooms. f There is a considerable difference of opinion regarding the proper interval between tuberculin tests, due largely to inadequate data on the length of time required for an infected animal to become tuberculin-positive. It is the opinion of the committee that the intervals given will be adequate if the testing programs for transient and maintenance colonies are rigorously adhered to.
46 priate tests for confirmation shall be performed. Such tests shall include histopathological examination, acid-fast stains, and cultures for the pur- pose of determining the type of organism involved. All remaining animals in the reactor's group shall be retested at 2-week intervals until all animals in the group have passed three consecutive negative tests. 3. Each animal shall receive a final tuberculin test not more than 2 weeks prior to delivery to a maintenance colony. The final negative test in A(2) shall satisfy this requirement if delivery is made not more than 2 weeks sub- sequent to it. B. Maintenance Colony 1. All additions to the colony shall meet the requirements outlined in A(l-3). 2. Each animal shall be tuberculin-tested at 3-month intervals. All ani- mals with a positive tuberculin test shall be necropsied and appropriate con- firmatory tests shall be performed as outlined in A(2). 3. All animals dying of unexplained causes shall be necropsied and appro- priate confirmatory tests for tuberculosis shall be performed. 4. In the event that a positive tuberculin reactor appears in the colony or tuberculosis is discovered at necropsy, all animals in the group shall be tested at 2-week intervals until all have passed three consecutive negative tests, as in A(2). TESTING PROCEDURE 1. 0.1 ml of Koch's Old Tuberculin (KOT),* containing 15 mg of KOT, shall be injected into the upper eyelid with a 25-gauge needle. Injury to the eye- lid must be avoided. Proper restraint or drug immobilization of the animal is essential. Intradermal injections in other sites may be used (e.g., near the umbilicus), but the palpebral site is preferred because of the ease of reading the test and the dramatic response in animals with a positive reaction. If other sites are used, and removal of hair is required, injury to the skin must be avoided. 2. Injected animals shall be examined daily for three successive days. *Nonhuman primates require larger doses of tuberculin to elicit a response than does man. While smaller doses of KOT than recommended (15 mg in 0.1 ml) are frequently used, there appear to be no adverse effects from the higher dose, and there is reason to believe that the latter may be more effective in eliciting a response. Other tuberculins (e.g., PPD) have been used. However, the committee recommends the use of KOT until adequate evidence is obtained that other preparations are equally effective.
47 Positive reactions may range from a minimal reddening, usually with slight edema, through severe swelling with closure of the eyelids, to hemorrhage into the injection site and necrosis in the more severe reactions. Doubtful reactors shall be placed in isolation and retested in the oppo- site eyelid within 2 weeks. If the retest is negative or doubtful, the animal shall be retested three times at 2-week intervals. Upon completion of three consecutive negative tests, it may be returned to the colony. 3. All positive reactors shall be considered infected and infective and shall be removed from the colony immediately for necropsy and confirmatory tests as in A(2). 4. Retention of positive reactors is not justified except in the most un- usual circumstances. If compelling reasons exist for maintaining a positive reactor, it must be isolated in an area completely removed from other pri- mates and properly equipped for work with infectious agents, and investiga- tors and caretakers must take effective precautions to prevent transmission to humans or to other primates. Treatment with isoniazid is recommended.* PERSONNEL Investigators, caretakers, and others in regular or frequent contact with pri- mates in either transient or maintenance colonies shall be examined at least twice yearly for evidence of active tuberculosis. Persons with infective tuberculosis shall not be admitted to primate colonies. EVALUATION False negative tuberculin reactions may occur early in the infection before hypersensitivity develops, or late in the disease when a previously positive animal may become nonreactive (anergic). Animals with early infections will usually become tuberculin-positive within a few weeks' time, hence the recommendation for initial testing at *Isoniazid prophylaxis appears to be valuable in the prevention of tuberculosis in pri- mates, although treatment of active cases is less satisfactory. Evidence indicates that isoniazid does not always result in eradication of tubercle bacilli from infected animals and the disease may become active on cessation of therapy, not only in animals pre- viously known to be infected, but also in animals with no prior evidence of infection. In addition, the possibility of toxic drug reactions and selection of drug-fast strains must be considered. For these reasons the committee does not consider continuous drug prophylaxis a substitute for an adequate testing program.
48 2-week intervals. Probably few of these early infections (before the develop- ment of a tuberculin-positive reaction) result in the shedding of organisms, so they do not represent a great hazard. Animals with far-advanced disease who have become anergic will show lesions in the lungs demonstrable by x-ray examination. Such animals are usually prolific shedders of tubercle bacilli, so every attempt should be made to prevent their introduction into colonies. A few instances are known where positive tuberculin reactors have ap- peared in maintenance colonies 6 to 9 months after their introduction into the colony, although tuberculin tests were negative in the intervening period. In these instances no source of infection could be found, either in other pri- mates or in humans. The possibility exists, therefore, that a rare animal may develop a disease that progresses much more slowly than is usual Primates may be infected with human, bovine, or avian strains of tuber- culosis. Although the majority of infections are of the human type, isolation and determination of the infecting type of organism is important since it may provide evidence for unsuspected sources of infection. A transient colony cannot be certified as tuberculosis-free because of the constant introduction of untested animals. However, individual animals from such a colony that meet the criteria in A(l-3) may be designated tuberculosis- free. A maintenance colony meeting the criteria in B(l-3) may be designated tuberculosis-free. In the event tuberculosis is subsequently detected in such a colony, re-establishment of the tuberculosis-free status shall be dependent on successful completion of the testing program outlined in B(4). REFERENCES Dolowy, W. C., M. H. Frank, G. E. Cox, and A. L. Hesse. 1958. Detection of Pulmonary Tuberculosis in Laboratory Monkeys by Chest Radiography. Amer. J. Vet. Res. 10:225-229. Habel, K. 1947. Tuberculosis in a Laboratory Monkey Colony: Its Spread and Control. Amer. Rev. Tuberc. 55:77-92. Ruch, T. C. 1959. Diseases of Laboratory Primates. W. B. Saunders, Philadelphia, Pa., p. 598. Schmidt, L. H., R. Hoffmann, and P. N. Jolly. 1955. Induced Pulmonary Tuberculosis in the Rhesus Monkey: Its Usefulness in Evaluating Chemotherapeutic Agents. Trans. Conf. Chemotherap. Tuberc. 74:226-231.