A Novel Presentation of Tuberculosis with Intestinal Perforation in a Free-Ranging Australian Sea Lion (Neophoca cinerea) Open Access

Tuberculosis in pinnipeds is typically a pulmonary, pleural, or multicentric granulomatous disease resulting from infection with Mycobacterium pinnipedii, one of 11 highly genetically related and variably human- or animal-adapted members of the Mycobacterium tuberculosis complex (Cousins et al. 1993; Silva-Pereira et al. 2019). Infection with M. pinnipedii has been reported sporadically in free-ranging fur seals and sea lions (otariids) of the southern hemisphere, with increasingly frequent documentation of cases in captive pinnipeds worldwide. This disease in free-ranging animals has been reported in Australasia in the Australian sea lion (Neophoca cinerea), Australian fur seal (Arctocephalus pusillus doriferus; Cousins et al. 1993; Woods et al. 1995), New Zealand sea lion (Phocarctos hookeri), and New Zealand fur seal (Arctocephalus forsteri; Roe et al. 2006, 2019) and in South America in the South American sea lion (Otaria flavescens) and South American fur seal (Arctocephalus australis; Romano et al. 1995; Bernardelli et al. 1996). Disease has also been reported in the subantarctic fur seal (Arctocephalus tropicalis; Bastida et al. 1999).

Reporting of tuberculosis in captive Australian sea lions is limited to that of four deaths in 1985–86 described in a case series in a mixed pinniped population housed at a Western Australian marine park (Forshaw and Phelps 1991). Three animals had been collected from the Archipelago of Recherche in southern Western Australia (34°39′S, 122°27′E) and the fourth from closer to the marine park (31°33′S, 115°41′E). To date, bacteriologically confirmed tuberculosis in free-ranging Australian sea lions involves two subadult males found dead on beaches on the south coast of Western Australia (Albany: 35°01′S, 117°53′E; Bremer Bay: 34°24′S, 119°23′E) in 1990–91 (Cousins et al. 1993). Reports of tuberculosis in free-ranging pinnipeds in South Australian waters is limited to that of a subadult male Australian fur seal found dead at Beachport (37°29′S, 140°01′E; Boardman et al. 2014).

The Australian sea lion is endemic to Australian waters and ranges from the Houtman Abrolhos on the west coast of Western Australia to the Pages Islands in South Australia (Goldsworthy et al. 2009). The species is listed as endangered with a decreasing population trend by the International Union for Conservation of Nature (Goldsworthy 2015). Common to other pinnipeds of small population size, the Australian sea lion is at increased risk of significant population decline from epizootic disease (Lynch et al. 2011). Furthermore, population recovery from the effects of disease would be limited by the species' low fecundity (including a unique 18-mo breeding cycle) and extreme female natal site fidelity dispersed across 76 asynchronous breeding sites (Goldsworthy et al. 2009). The majority (86%) of pup births occur in South Australia, with just five large breeding colonies, including Seal Bay, Kangaroo Island, contributing more than half of the state's pup production (Goldsworthy et al. 2009; Goldsworthy 2015).

On 1 February 2017, a juvenile male Australian sea lion died shortly after hauling out at a public beach at Kingscote (35°39′S, 137°38′E), Kangaroo Island, South Australia. Preliminary assessment was of an animal in fair to thin body condition with no external wounds. Microchip scanning confirmed its birth was at Seal Bay Conservation Park (35°58′S, 137°21′E), Kangaroo Island, and provided a known age of 3 yr. The cadaver was preserved by freezing at –20 C within hours of death for later postmortem examination.

Standard morphometric postmortem data were a weight of 33.3 kg, a standard length (nose to tail) of 122.3 cm, and an axillary girth of 78.0 cm. External examination confirmed the earlier assessment of body condition and found below average muscling and moderate abdominal distension. Internal examination revealed a large volume (exceeding 2 L) of serosanguinous and slightly turbid peritoneal effusion and the absence of subcutaneous blubber or visceral adipose tissue. Mid-jejunum, the intestine was completely encircled by a 120-mm-diameter spherical mesenteric mass with fibrous adhesions to the stomach and spleen (Fig. 1). The 0.60-kg mass was smooth surfaced with fibrin tags, and its cut surface was fibrous, friable, and patchy red-green to yellow. The entrapped jejunal lumen remained patent but nonexpansile and constricted to 25% of the adjacent organ diameter. A 12-mm-diameter wall perforation communicated with a shallow 30-mm-diameter cavity within the encircling mass.

There was marked generalized abdominal lymphadenomegaly (gastric: 50×25 mm; mesenteric chain: 160×28 mm, 181×43 mm, and 81×25 mm); the cut surfaces of lymph nodes were pale yellow, nodular, and fibrous (Fig. 2). Extra-abdominal findings were limited to mild tracheobronchial lymphadenomegaly with normal cortical-medullary differentiation; localized grossly detectable pulmonary lesions were not apparent. Ziehl-Neelsen–stained cytologic specimens of a mesenteric lymph node reviewed at necropsy contained large numbers of acid-fast bacilli (AFBs), presumed to be Mycobacterium spp., supporting a preliminary diagnosis of tuberculosis.

Histologic interpretation (limited by substantial freezing and autolysis artifacts) included fibrosing granulomatous jejunitis surrounding the jejunal perforation and marked necrotizing and fibrosing granulomatous peritonitis in the surrounding mass. Multicentric necrotizing and fibrosing granulomatous lymphadenitis of the abdominal lymph nodes was severe. The tracheobronchial lymph node showed reactive hyperplasia. Additionally, mild multifocal nodular change, probably fibrosis and necrosis, was apparent within this lymph node and sampled lung. Microscopic pulmonary and thoracic lymph node involvement in mycobacterial disease was therefore suspected but remained inconclusive.

In histologic sections, AFBs were detected in low number in the mesenteric lymph node, as rare organisms within the mesenteric mass, and as rare organisms within chronically inflamed jejunal submucosa at and adjacent to the perforation site. Organisms were not detected in thoracic tissues.

Mycobacterium tuberculosis complex, subsequently identified as M. pinnipedii, was isolated from a mesenteric lymph node sample. The pathogen was identified by the Xpert MTB/RIF DNA assay (Cepheid, Sunnyvale, California, USA) culture was positive after 3-wk use of the Bactec MGIT 960 automated broth-based system (Becton Dickinson, Cockeysville, Maryland, USA), and the isolate grew on Lowenstein Jensen media (Biomerieux Australia Pty. Ltd., Baulkham Hills, New South Wales, Australia) containing glycerol and pyruvate (SA Pathology, Adelaide, South Australia, Australia). Nitrate reduction testing was not performed on the isolate, which was susceptible to streptomycin, isoniazid, rifampicin, ethambutol, and pyrazinamide. The isolate was positive for the MPT64 antigen by a rapid immunochromatographic identification test (SD Bioline TB Ag MPT64, Alere, Waltham, Massachusetts, USA). Molecular typing to M. pinnipedii used mycobacterial interspersed repetitive-unit–variable number tandem repeat (MIRU-VNTR) typing with 12 loci, followed by analysis on the MIRU-VNTR plus website (Allix-Beguec et al. 2008); 12-locus MIRU analysis was conducted (LabPlus, Auckland City Hospital, New Zealand) and compared with two profiles from otariids supplied from the Victorian Infectious Diseases Reference Laboratory (Melbourne, Australia).

While suspicion of microscopic pathology within the thorax existed in the present case, gross lesions were not seen, which is uncommon but not unprecedented in tuberculous disease in pinnipeds. In the Western Australian case series, nodular pulmonary and pleural disease predominated in the six culture-positive animals, although lesions were limited to the liver and its draining hepatic lymph node in one of these animals (Forshaw and Phelps 1991). In our case, detection of intralesional AFBs in the intestine, together with limited extra-abdominal disease, suggested primary alimentary infection with ingestion as the route of infection and a potential for fecal shedding, as previously asserted (Forshaw and Phelps 1991). More commonly, pulmonary disease in pinnipeds results from inhalation, with subsequent bacterial shedding in airway secretions (Cousins et al. 1993). In aquarium and rehabilitation facilities, mucosal secretions, feces, and urine have been suggested as potential sources of zoonotic infection (Thompson et al. 1993; Waltzek et al. 2012).

Microchipping data confirmed the subject of this report as a 3-yr-old (juvenile) Seal Bay born male sea lion; its young age was consistent with previous reporting of tuberculosis in pinnipeds. In the Western Australian cases of tuberculosis in Australian sea lions, the free-ranging animals were subadult males (approximately 7–10 yr old), with the captive animals 5–14 yr old and three of the latter four animals being 5 yr old or younger. Even for relatively gregarious pinniped species, disease transmission is more likely to occur during animal congregation observed during the breeding season. Preferential involvement of young males, as observed in reports from the Australian sea lion, has been attributed to a tendency for this age and sex cohort to congregate before maturation to breeding age (Cousins et al. 1993).

A recent report of disseminated tuberculous disease in a 3-yr-old New Zealand sea lion described multifocal granulomatous enteritis and bronchopneumonia, with AFBs detected in intestine and lung (Chatterton et al. 2020). By comparison, gross enteric disease in our case was limited to one site, speculatively beginning as focal granulomatous enteritis (supported by detection of intralesional AFBs) with subsequent perforation and mesenteric reaction contributing to the gross observations. It is also possible that the suspected small foci of pulmonary pathology represented a comparatively early stage of subsequent disease within the lung.

As with other M. tuberculosis complex members, M. pinnipedii infection can spill over into nonpinniped mammalian species, most significantly to humans working with infected marine mammals in zoologic collections and marine parks (Thompson et al. 1993). Less well considered than this occupational disease risk to individuals working with captive animals is the risk to individuals briefly or infrequent exposed to infection. At risk individuals include those involved in animal rescue or washed-up carcass disposal, those working at rehabilitation centers, and those involved in disease diagnosis. The young age of infected animals and clinical signs that can be absent, nonspecific, or difficult to detect warrant consideration of zoonotic risk when handling any pinniped (Forshaw and Phelps 1991). Reviews of antemortem testing for confirmation of tuberculosis in captive animals and in quarantine surveillance settings outline the specificity and sensitivity limitations of currently available serologic and intradermal tests (Jurczynski et al. 2012; Chatterton et al. 2020).

Individuals hauled out or washed up on the coast provide a biased sample but remain population sentinels for endemic diseases such as tuberculosis. It remains to be determined if the rare reports of tuberculosis in the Australian sea lion reflect a low level of observation and testing or a genuinely low prevalence. A review of tuberculosis in native New Zealand marine mammals found that despite an overall low prevalence (1.6%), the disease was a common cause of mortality in New Zealand sea lions on both the mainland (25%) and Enderby Island (14.6%; Roe et al. 2019). Furthermore, a comparative genomic study of M. pinnipedii in the South American sea lion has sequenced two isolates from different organs in the same individual, consistent with mixed-strain infection (Silva-Pereira et al. 2019). This was suggested to indicate an infection that is highly endemic within the population. Large scale opportunistic sampling and serologic testing is needed to define tuberculosis prevalence in the Australian sea lion, similar to that previously conducted in the Australian fur seal (Lynch et al. 2011).

Our report documented a novel presentation for M. pinnipedii infection in a marine mammal. It confirmed the presence of this disease in a juvenile male Australian sea lion of known age in South Australian waters and added to the limited reporting of disease in free-ranging animals of this and other otariid species. We recommend increased serologic surveillance as a means of determining population risk from this and other endemic diseases. Furthermore, it highlights the need for constant vigilance of zoonotic disease risk by any person in close contact with free-ranging pinnipeds of juvenile age or older, independent of presenting signs.

The authors thank Melanie Stonnill, Anthony Maguire, and Michelle Nairn (Department of Environment and Water, South Australia) and David Stemmer (South Australian Museum, South Australia) for collection, storage, and transport of the cadaver. We greatly appreciate the description of laboratory methodology provided by Lisa Shephard from the Mycobacterium Reference Laboratory, SA Pathology.