Histopathologic and Bacteriologic Testing and Polymerase Chain Reaction Amplification

Various organs and tissues from seven banded mongooses and a suricate were fixed in 10% buffered Formol-saline, embedded in paraffin wax, cut into 4-µm sections, and stained with hematoxylin and eosin, as well as Kinyoun’s method for acid-fast bacteria (AFIP modification) (7).

Pooled organ specimens from one banded mongoose (No. 2075) and one suricate (No. VLF005) and liver samples from another banded mongoose (No. 2344) were homogenized, and equal parts were decontaminated with either 2% HCl or 4% NaOH. After neutralization, sediments were placed on two slants of Löwenstein-Jensen (LJ) medium containing 0.5% pyruvate and one slant containing glycerine. The cultures were incubated at 37°C and checked weekly for growth. Acid-fast culture isolates were subjected to polymerase chain reaction (PCR) specific for M. tuberculosis-complex organisms (8) and subcultured onto the same type of medium for identification by growth characteristics and standard biochemical tests. PCR-positive Mycobacterium isolates were subjected to a second PCR test to differentiate M. bovis and M. tuberculosis (9).

Epidemiologic Features

In the suricates, the epizootic occurred from October 1998 to December 1999, after an unknown infected male joined a study group consisting of 5 adult suricates and 15 pups. One human case of TB was known to have occurred in the study area in the human population living near the suricate burrows. Suricates were not observed feeding in garbage pits; however, they were seen foraging around roads and investigating human sputum.

In the banded mongooses, the epizootic spread quickly in six troops living along the Chobe River Front. New clinical cases were identified from the initial outbreak site at an average rate of 0.109 km/day into Chobe National Park and the township at an average rate of 0.282 km/day. Although the last case was observed on September 15, monitoring continued until January 21, 2001; no new cases were identified. Garbage pits and a known human TB case were in close proximity to initial outbreak points (Figure). Banded mongooses were observed feeding regularly at these garbage pits and would therefore be exposed to human excretions and any infectious material from TB-infected humans.

Clinical Signs and Gross Pathologic Findings

The first suricate noted to have clinical signs had enlarged cervical lymph nodes when it emigrated to the study site. The lymph nodes ruptured within a month and continued to discharge pus for 2 months (November–December 1998). By January 1999 the lesion had become a persistent nonhealing wound. The animal became progressively more debilitated and cachectic until he disappeared from the study site in April 1999. In June, clinical signs first appeared in other animals and then spread through the entire troop by December 1999. The signs included emaciation, weakness, and dyspnea with variable enlargement of the lymph nodes of the head, neck, and axilla. All troop members were euthanized, died, or disappeared and were presumed dead. Gross postmortem examination of one suricate showed abscesses filled with yellow fluid in the left parotid (3 cm in diameter), right axilla (1 cm), lung, liver, and mediastinal lymph nodes (5 mm), and pancreas (8 mm). The lungs were congested and edematous.

The mongoose troop size ranged from 8 to 35; in each troop, 2 to 4 animals had clinical signs. Affected animals were often found separated from the troop during periods of foraging. In some cases, although alert, they showed a pronounced lack of fear response to humans. Affected animals that were not euthanized disappeared within a few days after onset of illness and were presumed dead. Clinical signs included progressive cachexia, ataxia, and weakness. No other external abnormalities were noted. Necropsies of seven animals from five troops showed numerous miliary grayish white nodular masses (0.5 cm–2 cm in diameter) scattered over the liver and spleen surfaces, causing massive enlargement of the two organs; numerous grayish white infiltrative masses in the lung and kidney; enlargement of the mesenteric lymph nodes, which had necrotic gritty centers with chalky material; and occasional grayish white foci scattered throughout the length of the intestines.

Histopathologic Findings

Granulomas of varying size, predominantly consisting of aggregated epithelioid macrophages, were found in most of the organs and tissues examined and were consistently present in the liver, spleen, lymph nodes, and lungs. Such granulomas were occasionally noted in the adrenal gland, kidney, myocardium, pancreas, epididymus, pleura, intestine, peritoneum, and skin. Lesions were absent from the brain, skeletal muscle, urinary bladder, and testis. The smaller granulomas consisted purely of macrophages, while large ones showed central necrosis and sometimes contained small aggregates of lymphocytes and plasma cells. Giant cells were rare, and no calcification was seen. Acid-fast rods, typical of Mycobacterium species, were noted in the cytoplasm of macrophages in all eight mongooses but varied in numbers from scarce to abundant.

Bacteriologic and PCR Amplification Results

Acid-fast bacteria were detected on Ziehl-Neelsen–stained impression smears, and Mycobacterium species were isolated from both banded mongooses and the suricate specimens. For material from animal No. 2075, growth first appeared after 2 weeks on LJ slants with and without pyruvate. LJ-pyruvate cultures of the specimens from mongoose No. 2344 and suricate No. VLF005 yielded very few acid-fast colonies after a 5- to 6-week incubation period. Following PCR amplification, the Mycobacterium sp. isolated from mongoose No. 2344 and the suricate produced a 372-bp DNA product. Both isolates showed a 336-bp DNA product, characteristic for M. tuberculosis when amplified by the protocol of de Wit et al. (9). This method had been found useful in differentiating isolates of M. bovis and M. tuberculosis (A. Michel, unpub. data). The isolates produced positive results in both the niacin production and nitrate reduction tests, confirming them as M. tuberculosis. A subculture of an isolate from banded mongoose No. 2075 was classified as a fast-growing Mycobacterium species after it had shown growth at 27°C and 37°C at 7 and 5 days, respectively. No growth was observed at 45°C.