PRIMATES,12(1): 81-90, March 1971

REPORTS Isoniazid Therapy of Tuberculosis in Baboons MACKIEA. ALLGOODand G. TOWNLEYPRICE FAA Aeronautical Center

ABSTRACT. A local epizootic of tuberculosis in a laboratory colony of baboons was controlled by 40--80 mg/kg daily dose of Isoniazid. There was definite regression of disease in all animals examined at autopsy, after three weeks and three months of therapy, following their use for unrelated acute experiments. One animal survives in apparent health with a healthy infant that was born after 102 days of therapy.

INTRODUCTION Discovery of tuberculosis in a primate colony is always of great concern because most of these animals are highly susceptible to the disease (HILL, 1969; URBAIN, 1938). Tubercular rhesus monkeys have been captured (RucH, 1959) while no tubercular baboons were found in a sampling of wild animals (KIM, EUGSTER,& KALTER,1968). Monkeys often acquire tuberculosis from humans or milk prior to importation (HILL, 1969). Although squirrel monkeys were once considered to be highly resistant to tuberculosis, they (CHRISP et al., 1968; HESSLER& MORELAND, 1968) and other wild mammals have been found to be susceptible (URBAIN, 1938). Most monkeys are easily infected and transmit the organisms to one another long before symptoms are evident (CALMETTE, 1923; CLARKE,& SCHMIDT, 1969; RUCH 1959; SCHMIDT,HOFFMANN,& JOLLY, 1955). They rapidly develop extensive tuberculosis and die (ALLEN & KINARD, 1958; HABEL, 1947; RUCH, 1959). In this chemotherapeutic and nonepidemic era, much care must be exercised in extrapolating tuberculosis symptoms and progress of the disease from humans to highly susceptible animals. Those working with rhesus monkeys are most familiar with the disease as described by CALMETTE(1923) in humans and animals during the peak of the pandemic in the late 19th and early 20th centuries. The hosts were highly susceptible and the bacteria were highly virulent. Ingestion of organisms led to pulmonary as well as other types of tuberculosis. Regardless of portal of entry the bacilli were found in blood and some were excreted in the bile before local foci were found. Remaining bacilli localized in various organs and multiplied if conditions were favorable. As resistance factors in the host were stimulated, tubercles were formed. In a susceptible host, not previously exposed to tuberculosis, generalized disseminated tuberculosis developed. This rapidly progressive disease is found today in baboons exposed to tuberculosis following their capture.

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M.A. ALLGOOD& G. T. PRICE

Difficulties with tuberculin skin testing in rhesus monkeys and baboons have been reported using both KocH's old tuberculin (KOT) and purified protein derivative (PPD) (HESSLER & MORELAND, 1968; KEELING, FROEHLICH, & EDIGER, 1969; SIBINORIC, 1969; VICE et al., 1968). In our experience with baboons t~ (Papio cynocephalus) K O T gave results verifiable by necropsy while PPD false negatives (skin test negativenecropsy positive) allowed a tubercular baboon(s) to be introduced into the colony and gave us the opportunity to conduct this study. Necropsy of a baboon used in an acute experiment in mid December 1967 at the Civil Aeromedical Institute (CAMI) revealed one tubercule in the lung containing acid-fast bacilli. So limited an infection indicated an early stage of the disease and a source currently in or with the colony, but there was no clinically-ill animal in the colony and none had reacted to PPD (Table 1). Two weeks later the source revealed itself. Another baboon from the same shipment was found dead in her cage with extensive tuberculosis involving lungs, spleen, kidneys, and regional lymph nodes. All animals were re-tested using K O T (Table 1) revealing two reactors, neither of which had responded to PPD. These were in the same shipment as the two PPD negative, necropsy positive animals that alerted us to the problem. One of the reactors to K O T (No.93) was necropsied 4 January 1968 to verify the skin test. Cultures from two necropsies were identified by the National Communicable Disease Center, Atlanta, Georgia, as Myeobaeterium tuberculosis var boris. With the scarcity of bovine tuberculosis in the United States, it is highly probable that the disease was contracted outside the country. Since bovine and human strains react equally to both antigens, there is no chance of missing one by skin testing for the other (CArMETTE, 1923). Isoniazid therapy of tuberculosis in monkeys is well documented (CnRISP et al.,

TaMe 1.

Results of intradermal inoculation of the eyelid with tuberculin. PPD

KOT

KOT

No.

Arrival

Sept. 19, '67

Dec. 28, '67

85 86 88 89 93 94 95 96 97

Sept. 16, '67

Neg. Pos. Neg. Pos. Neg. Pos. Neg. Pos. Pos. Died Used Dec. 13 Pos. * Doubtful

Pos.

60 28

Nov. 28, '66 Aug. 9, '66

Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Oct. 10, '67 Neg. Neg.

Neg. Neg.

Pos. Pos.

" " " " " " "

" Feb. 5, '68

KOT

Pos. Pos.

Feb. 12, '68 Pos. Pos. Pos. Pos.

*Not tested. 1) The animals used for these experiments were lawfully acquired and treated in accordance with the Principles of Laboratory Animal Care issued by the Animal Facilities Standards Committee of the Animal Care Panel, U.S. Department of Health, Education and Welfare, Public Health Service, March 1963.

Isoniazid Therapy in Baboons

83

1968; HABEL, 1947; HESSLER& MORELAND, 1968; SCHMIDT et al., 1953, 1955) and poorly trusted. Early use of small dosage produced only a bacteriostatic effect (SCHMIDT et al., 1953). Most of the dosage recommendations are based on work with rhesus monkeys (FREMMING et al., 1957; SCHMIDT, HOFFMANN, & HU6HES, 1953; SCHMIDT, HOFFMANN, & JOLLY, 1955). RUCH (1959) recommends using isoniazid to save animals for experiments that will not be affected by tuberculosis.

METHODS

Skin test reactors were removed from the holding colony some 20 miles distant to our facility where they were housed in individual cages in the same isolation room. The room and cages were washed with a tuberculocide disinfectant-detergent mixture daily, and fecal material was rinsed down the drain at frequent intervals. Animals were maintained on a diet of Purina Monkey Chow supplemented with fruit containing Isoniazid ('Isoniazid' from Eli Lilly & Co.). Isoniazid dosage for monkeys is, at best, an approximation since it is included in food. Monkeys housed separately find their access to food unchallenged and soon become expert food examiners, rejecting anything strange. By recording our observations of INH ingested, we learned that offering 80 mg/kg/day in divided doses assured ingestion from 40-80 mg/kg/day. As the animals were used for other experiments, careful necropsies were conducted and the extent and character of the disease was noted.

RESULTS Two weeks after therapy was begun, all animals appeared well-nourished and had good appetities except for one KOT-positive animal (No.60) that died of pneumococcal pneumonia with no tuberculosis detected on gross or microscopic examination. The related autopsy reports of the other animals follow and are summarized in Table 2. Fatal No.94 Gross The lungs were totally adherent to the thoracic wall and were fibrinopurulent in nature. The lungs were solid throughout and presented extensive, confluent masses of exudative consolidation. There were tubercles visible throughout the spleen, kidneys, and area lymph nodes. Micro Lung: Confluent caseous tubercles, some invading bronchioles. There was almost no peripheral cellular reaction. Giant cells were few. Spleen: Areas of caseous necrosis with slight peripheral cellular reaction. There were a few atypical giant cells and isolated islands of epithelioid proliferation not associated with the other tubercles.

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M.A. ALLGOODt~ G. T. PRICE

UNTREATED

No.95 Gross A firm nodule was detected on palpation of the apex, left lung. The hilar lymph nodes were enlarged and firm. Pus escaped on section. The capsular surface of the spleen was smooth except where it presented several small tubercles. Micro Lung: Tubercle not available, used for culture. Hilar lymph node showed caseous necrosis, relatively few epithelioid cells, very few macrophages and plasma cells. Spleen: Occasional aggregations of epithelioid and giant cells were present with necrosis. Very few plasma cells and macrophages were detected. No.93 Gross The apex of the right lung presented numerous, confluent tubercles. Section of this revealed a large cavity of caseous necrosis. The left lung presented numerous small tubercles. Hilar lymph nodes contained caseous necrotic material. Several small tubercles were present subcapsularly and on cut surfaces of the spleen. Micro Spleen: Large numbers of irregular, foreign-body type giant cells. LANGHANS'types absent. Epithelioid cells relatively few. Macrophages and plasma cells were not detected. There was a portion of one large caseous nodule and a much smaller nodule composed of numerous giant cells and epithelioid elements. Lung: Confluent tubercles, early caseation. Numerous irregular giant cells. One huge cell with very numerous (50÷) nuclei (Fig. 1). A few giant cells approximately the size and appearance of LANGHANS'types. Epithelioid cells were numerous. No increased peripheral vascularization. Very few identifiable macrophages and plasma cells. No.96 Gross Palpation disclosed two firm nodules, 1 cm in diameter in apex of each lung. The hilar lymph nodes were firm and on section revealed necrotic areas. The spleen was normal except for the presence of one small tubercle. Micro Lung: Confluent tubercles, early caseation. Moderate numbers of giant cells. Many were irregular morphologically. Moderate epithelioid, very few macrophages and plasma cells were identified. One preparation presented a large caseous nodule. Few giant cells were present. Spleen: Large tubercles, moderate number of LANGHANS'type giant cells. Numerous epithelioid cells. TREATED

No.88 Gross The left lung was totally collapsed (due to surgery). The right lung presented numerous tubercles on palpation. The hilar lymph nodes were enlarged and firm. The spleen presented several small subcapsular tubercles. A few tubercles were exposed on the cut surface. Micro Lung: Early easeation. Confluent tubercles. Moderate numbers of irregular giant

Isoniazid Therapy in Baboons

85

cells. Moderate expanses of epithelioid cells. No increased peripheral vascularization. Slight numbers of macrophages and plasma cells were identified. A few LANGHANS' type giant cells were detected. Spleen: Confluent tubercles. Early caseation. Moderate numbers of irregular giant cells. Moderate expanse of epithelioid cells. No.86 Gross On palpation a few minute, discrete, nodules were detected; the hilar lymph nodes were normal. There were no tubercles detected in the spleen. Micro Lung: One slide. A tubercle was present consisting of a center of inflammatory cells and epithelioid cells. The latter appeared to be in strands rather than in sheets. No giant cells were detected. Peripherally epithelioid cells form a thin mantle. The impression is that the epithelioid cells were fewer in number than usually seen in a tubercle. Macrophages and plasma cells were identified among the inflammatory elements. There was no particular increase of peripheral vascularization. Epithelioid cells appeared in some prominent follicles. No.28 Gross Neither the lungs nor the spleen presented any indication of tubercles. Micro Lung: Negative. Spleen: Negative. No.85 Gross There were no palpable nodules in the lungs or spleen. The hilar lymph nodes appear normal. Micro Lung: Negative. Spleen: Negative. No.97 Gross Palpation revealed numerous small nodules scattered throughout both lungs. The hilar lymph nodes were normal. The spleen appeared normal. Micro Lung: Confluent tubercles. Atypical foreign-body type giant cells. Epithelioid cells did not appear to form broad expanses or strands. They appeared to be depleted. There was no peripheral vascularization; no encapsulation; very few plasma cells. Fibrocytes were numerous as were monocytes. Spleen: Numerous small foci of epithelioid cells were observed.

DISCUSSION The skin test data (Table 1) show that those KOT positive on 28 December, plus No.94 and No.95, were more than likely diseased at the time of the previous PPD. Both gross and microscopic examination (Table 2) showed that the untreated animals (Nos. 93, 95, and 96) with the exception of the fatal case (No.94) had the expected extent of disease and cellular response to it. These were all received in the same

86

M.A. ALLGOOD(~ G. T. PRICE

shipment. Their small nodules and primary complex indicate a recent acquisition of the disease and they can be considered untreated controls. That the uninterrupted progress would result in death is proven by No.94, as well as many years of observation by various workers (ALLEN& KINARD, 1958; CAL~ETTE, 1923; HABEL, 1947; RUCI-I, 1959). This animal (No.94) was infected when received and at autopsy presented a classic picture of overwhelming tuberculosis infection rarely seen today except in some monkeys. The lungs showed massive exudative tuberculous pneumonia. The spleen and kidneys and area lymph nodes were mottled with lesions. Treatment was begun on all animals within two weeks of their first positive skin test. In each instance there was a conversion of the KOT from negative or doubtful to positive. Those animals with earlier infections, the ones that had given negative responses to PPD but were positive with the first KOT used (Table 1), were used in experiments before therapy was begun, unfortunately denying us the comparison of treatment effect between animals with early infection and those with more established disease. Animal No.97 was missed by the 28 December skin test and may have converted earlier than the doubtful response on 5 February indicated. The necropsy did indicate that she had a more advanced disease than the other treated animals. There were irregular giant cells; the tubercles were small, some were confluent, and the acid-fast bacteria remaining were intracellular, poorly stained, and not well defined. Comparing these features to those described by NESET (1964) correlating the number of bacteria destroyed with the increase of macrophages, epithelioid and plasma cells, this animal had passed the peak of cell destruction but the lesions had not had time to completely regress. Animals No.85 and No.28 showed no sign of tuberculosis on either gross or microscopic examination (Table 2) although their skin test (KOT) had converted from negative to positive prior to treatment (Table 1). The term 'skin test positive- necropsy negative' indicates something other than a nonspecific false-positive skin test. The earlier negative reaction showed that these animals were not allergic to the antigen. The skin test conversion from negative to positive suggests that there was a specific immunological response. Following CALMETTE'Sdescription of the progress of tuberculosis given above, the absence of detectable lesions in No.60, who had only 5 days of therapy, and in Nos. 28 and 85 suggests that therapy was begun before visible lesions were formed. Baboon No.88 is of particular interest because of the limited time of treatment which was interrupted by extensive surgery for implantation of transducers in the aorta and the brain cavity preparatory to an acute experiment. After only three weeks of interrupted therapy extracellular acid-fast bacilli were no longer detected and there were already cellular changes including more irregular giant cells, fewer of the LANGI-IANS'type, fewer epithelioid cells and macrophages, indicating regression of the disease. Baboon No.89 was not used in an acute experiment because of pregnancy. Therapy continued on this animal and she delivered a healthy male after 102 days of INH. Therapy was continued on both animals for a total of one year after which all periodic skin tests continued to be negative. As treatment progressed, all acid-fast organisms seen were in macrophages, appeared less distinct and stained poorly. In untreated animals there were broad sheets

Isoniazid Therapy in Baboons

87

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M.A. ALLGOOD& G. T. PRICE

of epithelioid cells, numerous giant cells, and plasma cells, and, peripherally, a mantle of inflammatory cells (Fig. 2). In the early treatment animal atypical giant cells (Fig. 3) and fewer of the LAN6HANS' type were found. Little change was observed in the rest of the tubercle with plasma cells and macrophages becoming progressively fewer. Fewer giant cells, strands, instead of broad sheets, of epithelioid cells, and decreased number of macrophages (Fig. 4) were found in the late treatment group. These findings agree with those of NESET (1964) except that irregular type giant cells, which are very small when compared to LANGHANS' and foreign-body type, were found in untreated animals, while he observed them in humans only following treatment. Isoniazid therapy of tuberculosis in baboons appears to be justified for a control of the disease in a small colony, both to save a group of animals for acute experiments, as recommended by RtJcn (1959) and as a general treatment to rid the animals of disease.

REFERENCES

ALLEN, A. M. & R. G. KINARD, 1958. Primary cutaneous inoculation tuberculosis in the Macaea mulatta monkey. Amer. J. Path., 34: 337-345. CALMETTE,A., 1923. Tubercle Bacillus Infection and Tuberculosis in Man and Animals'. Williams & Wilkins Company, Baltimore. C~RISP, C. E., B. J. COHEN,D.H. RINGLER,& G. D. ABRAMS, 1968. Tuberculosis in a squirrel monkey (Saimiri sciureus). J. amer. vet. reed. Assoc., 153: 918-922. CLAR~:E, G. L. & J. P. SCHMIDT, 1969. Effect of prophylactic isoniazid on early developing experimental tuberculosis in Macaca mulatta. Am. Rev. resp. Dis., 100: 224-227. FREMMING, B. D., R. E. BENSON,R. J. YOUNG, & M. D. HARRIS,JR., 1957. Antituberculosis therapy in Macaca mulatta monkeys. Amer. Rev. Tuberc., 76: 225-231. HABEL, K., 1947. Tuberculosis in a laboratory monkey colony: Its spread and control. A mer. Rev. Tuberc., 55: 77-92. HESSLER, J. R. & A. F. MORELAND, 1968. Pulmonary tuberculosis in a squirrel monkey (Saimiri sciureus). J. amer. vet. reed. Assoc., 153 : 923-927. HILL, W. C. O., 1969. The use of primates in biomedical studies: A review of suitable species. Ann. N. Y. Acad. Set., 162: 7-14. KEELING, N. E., R. E. FROEHLICH,& R. D. EDIGER, 1969. An epizootic of tuberculosis in a rhesus monkey conditioning colony. Laboratory Animal Care, 19: 629-634. KIM, C. S., A. K. EUGSTER,& S. S. KALTER, 1968. Pathologic study of the African baboon (Papio sp.) in his native habitat. Primates, 9 : 93-104. NESET, G., 1964. Specific drug therapy in pulmonary tuberculosis. Acta chit. scand. Suppl., 335: 1-94. RUCH, T. C., 1959. Dieseases o f Laboratory Primates. W. B. Saunders Company, Philadelphia. pp. 199-253. SCHMIDT, L. H., R. HOFFMANN, & H. B. HUGHES, 1953. The toxicity of isoniazid for the rhesus monkey. Amer. Rev. Tuberc., 67: 798-807. , & N. JOLLY,1955. Induced pulmonary tuberculosis in the rhesus monkey: Its usefulness in evaluating chemotherapeutic agents. Trans. Conf. on the Chemotherapy o f Tuberc., 14: 226-231. SmINORIC, S., 1969. Tuberculin testing in monkeys (Macaca rnulatta) with naturally occurring tuberculosis. Laboratory Animal Care, 19: 621-623. URnAIN, A., 1938. L'Infection tuberculeuse spontanGe chez les mammifGres sauvages en captivitG. Annales de l'Institut Pasteur, 61 : 705-730.

lsoniazid Therapy in Baboons

Fig. Fig. Fig. Fig.

1. 2. 3. 4.

Irregular giant cell with numerous nuclei. Giant cells, plasma and epithelioid cells surrounding zone of necrosis. Atypical giant ceils, few plasma cells. Calcification, epithelioid cells in strands and poorly stained.

*All slides stained with Hematoxylin and Eosin, magnified 125 times.

89

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M . A . ALLGOOD & G. T. PRICE

VICE, T. E., M. E. PINKERTON,F.A. FEAR, & S. S. KALTER,1968. An outbreak of tuberculosis in a group of experimental baboons. Primates, 9 : 105-122.

---Received August 19, 1970; Accepted December 12, 1970. Authors' Address: MACKIE A. ALLGOOD and G. TOWNL~¥ PgIc~, AC-119, Civil Aeromedieal Institute, FAA Aeronautical Center, P.O. Box 25082, Oklahoma City, Oklahoma 73125, U.S.A.