The otter belongs to the mammalian order Carnivora, family Mustelidae (7). Otters feed on mussels, shellfish, crabs, and frogs living in rivers, creeks, and along coastlines. There are 13 extant species of otters distributed worldwide, five species; Eurasian otter (Lutra lutra), Smooth-coated otter (Lutrogale perspicillata), Hairy-nosed otter (Lutra sumatrana), Asian small-clawed otter (Aonyx cinereus) and Sea otter (Enhydra lutris) occur in various freshwater, coastal and marine ecosystems of Asia and Asia Pacific regions (7,16). The Asian small-clawed otter is the smallest otter with a total body length of around 65.2 to 93.9 cm and native to South and Southeast Asia (7).

Pasteurella (P.) multocida is Gram-negative, non-motile pleomorphic coccobacilli with bipolar staining character. P. multocida is normally maintained as commensal of the oropharynx of mammal but it usually causes respiratory disease and septicemia in pigs and cattle (13). There are several reports of P. multocida infection in otter characterized by respiratory infection, septicemia and endocarditis with a very high mortality rate (2). However, peritonitis associated P. multocida infection is rarely reported in domestic animals (3). In this study, we report peritonitis and necrotic hepatitis associated with P. multocida infections in Asian small-clawed otter.

A 12-year-old female Asian small-clawed otter was raised in the Aquaplanet Jeju had a week history of anorexia, chill, hypothermia, and abrupt abdominal distention. The otter was found dead in the cages on the morning of January 2013. At that time, there were four otters in the exhibits of aquarium. Among them, the oldest individual died. According to the history taking from aquarists and veterinarian, the dead otter often fought with two other otters, but got along well with the other one relatively without fighting. After presenting clinical signs, the dead otter was being reared at the same cage with the other well-off otter. No additional otter deaths were seen. Necropsy was performed at the Pathology Laboratory of the College of Veterinary Medicine, Jeju National University.

Grossly, the otter showed severe abdominal distension. Many black spots with 1-2 mm in diameter were scattered in both surface and parenchyma of the lungs (Fig. 1B). Yellowish-brown turbid fluids accumulated in abdominal cavity of the otter, and yellowish thread-like fibrinous materials were found on the surface of abdominal organs (Fig. 1A). Also, the liver and spleen were enlarged with dark red to yellowish in color (Fig. 1A). Several variable sized yellowish-white crystalloids were scattered on the medullary to cortex of kidneys (Fig. 1C). Collected visceral organs were fixed with 10% neutral buffered formalin, routine processed and prepared into paraffin sections and stained with hematoxylin & eosin and Gram staining for light microscopy.

Figure 1. Gross findings. (A) Note enlarged liver and spleen with blunt margin. Yellowish fibrinous materials (arrow) and yellowish-brown turbid fluid were occupied in abdominal cavity. (B) Many black spots (white arrow) were scattered in the lungs. (C) Several variable sized yellowish-white crystalloids (white arrows) were scattered in the cut surface of kidney.

Histologically, multifocal anthracosis and focal granulomatous inflammation with intra-lesional cholesterol clefts were observed in lungs (Fig. 2A). Multifocal necrosis, hemorrhage, infiltration of macrophage, and brown pigments were presented in the liver (Fig. 2B). Many megakaryocytes and reticular cell hyperplasia were founded in the spleen (Fig. 2C). Diffuse serositis (peritonitis) characterized by the fibrinous exudates, thickened serosal capsule and the swelling of mesothelial cells were observed in the serosa of liver, spleen, stomach, and intestine (Fig. 2D). Cystic endometrial hyperplasia with inflammation was noted in the uterus. Gram-negative coccobacilli were observed in the serosa of small intestine (Fig. 2D insert) using Gram staining for internal organs.

Figure 2. Histopathologic findings. (A) The lungs. Note anthracosis (black arrow) and focal granulomatous inflammation with cholesterol clefts (white arrow) in lung (Bar = 100 μm). H&E. (B) The liver. Note focal necrotic hepatitis in the liver (Bar = 50 μm). H&E. (C) The spleen. Note some megakaryocytes (black arrows) and reticular cell (white arrow) hyperplasia in the spleen (Bar = 20 μm). H&E. (D) The serosa of the small intestine. Serositis characterized by fibrinous exudates (black arrow) and swelling of mesothelial cells (white arrow) (Bar = 100 μm). H&E. Insert of (D) is higher magnification of Gram negative coccobacilli (open arrows) in fibrinous exudates of the serosa (Bar = 10 μm). Gram stain.

Ascites and fibrinous materials within serosal surface of liver and spleen were aseptically collected for bacterial culture and were inoculated on sheep blood and MacConkey agar and aerobically incubated for 48 hr at 37°C. Isolated bacteria were white, smooth and convex with characteristic mousy odor on blood agar plate. However, no typical bacteria were grown in MacConkey agar plate. P. multocida was confirmed by VITEK 2 system (Biomerieux, USA). To differentiate and confirm the causative bacteria, polymerase chain reaction (PCR) were applied to the isolated bacterial colonies. Bacterial colonies from sheep blood agar were dissolved in DNase-free distilled water (Invitrogen, USA), and centrifuged at 14,000 × g for 10 min to discard the supernatant. The pellets were then re-suspended in 300 µL of water to incubate for 10 min at 100°C and kept at –20°C. After defrosting at room temperature, samples were centrifuged at 14,000 × g for 10 min and supernatants were used for the template DNA. PCR analysis was performed to differentiate the capsule types of P. multocida and the oligonucleotide primer sets in Table 1 were used (18). PCR amplification was an initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 sec, annealing at 55°C for 30 sec, 72°C for 30 sec, and finished with a final extension at 72°C for 5 min and stored at 4°C (18). The amplified products were visualized by electrophoresis on a 1.2% agarose gel. Therefore, these bacteria were confirmed as P. multocida type A by PCR analysis (Fig. 3).

Table 1 . Primer sets used for the detection of capsular types of Pasteurella multocida .

Serogroup	Primer	Sequences (5` to 3`)	Size of products (bp)
All	KMT1T7	ATC CGC TAT TTA CCC AGT GG	460
	KMT1SP6	GCT GTA AAC GAA CTC GCC AC
A	CAPA-FWD	TGC CAA AAT CGC AGT CAG	1,044
	CAPA-REV	TTG CCA TCA TTG TCA GTG
D	CAPD-FWD	TTA CAA AAG AAA GAC TAG GAG CCC	657
	CAPD-REV	CAT CTA CCC ACT CAA CCA TAT CAG

Figure 3. Result of polymerase chain reaction (PCR) for the capsule types of P. multocida. PCR products of P. multocida. N, negative control; PC, P. multocida all type (460 bp), type A (1,044 bp) and type D (657 bp) positive control; M, 100 bp ladder; lane 1 and 2, isolated bacterial colony from ascites and the serosa of abdominal organs including liver and spleen.

Nephrolithiasis and urolithiasis are a common finding in otters both in the wild and in captive individuals (2,14). As otters mainly represent a piscivorous feeding (a rich source of purine) type and also excrete a lot of uric acid, their purine metabolism might partly explain the etiology of renal calculi. Kidney calculi may cause severe back pain and bleeding. In captive Asian small-clawed otters, the calculi are mostly composed of calcium oxalate, but in Eurasian otters, the great majority of urinary calculi are composed of ammonium urate (2,14). In the present study, the otter did not show any significant clinical signs associated with renal calculi. Unfortunately, we could not perform the analysis for the urinary calculi.

P. multocida is capable of causing various disease in a wide range of animals including birds (13). The strains of these bacteria are classified into 5 serogroups (A, B, D, E and F) based on capsule antigens (6). P. multocida serogroup A strains are typically associated with fowl cholera, types B and E strains cause the haemorrhagic septicemia in cattle, and type D strains induce atrophic rhinitis in pigs (6). In a recent literature, there have been reported about fatal peritonitis caused by P. multocida type F in 2 weeks old calves in Germany (3). The virulence of P. multocida type A is the most prevalent capsular type among P. multocida isolates from cattle, swine, poultry and rabbits (13). In both clinical and histopathologic characteristics in this case had similarity with fowl cholera in avian, such as chill, anorexia, hypothermia, necrotic hepatitis and serositis (6). In acutely affected birds, general hyperemia and increased amounts of pericardial and peritoneal fluids usually occurs like a septicemia of sudden onset with high morbidity and mortality. Petechial and ecchymotic hemorrhages are usually found in internal organs and the livers are swollen, and usually contain multifocal coagulative necrosis and infiltration of heterophils. Similarly to birds, diffuse peritonitis and necrotic hepatitis were observed in this otter case associated with P. multoicida infection. In wild animals, septicemia associated with P. multoicida infection was also describe in a stoat, a kind of small mustelids (15). The lungs of stoat showed multiple irregular hemorrhage, consolidation and emphysema. In addition, this is a well-known cause of death in small wild mammals, bats, and small birds submitted to wildlife hospitals.

In the case of Salmonella infection in animals, there are tiny yellowish foci (referred to as paratyphoid nodules) in the affected liver and necrotic or ulcerative enteritis in ileum and large intestine (8). Histopathologically, these paratyphoid nodules were characterized by lytic necrosis of hepatocytes and reactive histiocytic granulomas (9). Salmonella infections in marine mammals have been associated with enteritis, cholecystitis, abscesses, pneumonia and septicemia (1,10). Although Salmonella species were isolated from an otter with hepatitis (2), infection of this bacteria was uncommon in sea otter (<1.0%) compared to the frequency of detection in sympatric terrestrial and marine vertebrates (10). Recently experimental infection of P. multoicida infection demonstrated hepatic necrosis in chickens and fibrinous peritonitis and multifocal necrotizing hepatitis in pigs, respectively (5,12). According to this paper (5), hepatic lesions were characterized by coagulative necrosis with fibrin thrombosis. In the present otter case, hepatic necrotic inflammation is similar with coagulative necrosis rather than lytic necrosis. In addition, this otter had no clinical signs and pathologic lesions related with enteric disease. Hence, it is estimated that hepatic lesion of this otter is more likely to be related to the P. multoicida infection than to the Salmonella infection.

According to previous large scaled survey for 560 necropsies of southern sea otter in USA, high mortality due to shark bite, cardiomyopathy, toxoplasmosis, sarcocystosis, acanthocephalan peritonitis and coccidiodomycosis was confirmed in the sea otters (11). Among them, bacterial infection (6%, 33/558) was a primary cause of death and sequelae to acanthocephalan peritonitis, shark bite, mating trauma and fight trauma. Bacteria related with fatal lesions in sea otter included Streptococcus spp., Erysipelothrix sp. (e.g., E. rhusiopathiae), Klebsiella pneumoniae, P. multoicida, Salmonella sp., hemolytic Escherichia coli, and others. In other previous survey for mortality trends in northern sea otters collected from the coasts of Washington and Oregon, infectious diseases were the primary cause of death (56%, n = 52) for 93 necropsied cases. The primary infectious disease was protozoal encephalitis (30%, n = 28) due to Sarcocystis neurona. Streptococcus (S.) phocae septicemia (7.5%, n = 7) was second most common disease in northern sea otters. Associated suppurative lesions in these cases included subcutaneous, intramuscular, or tooth root abscesses, embolic-septic pneumonia, suppurative arthritis, suppurative meningitis, infectious endocarditis, suppurative splenitis, or suppurative peritonitis. P. multoicida was also isolated from the same suppurative lesions and was considered as a synergistic pathogen in the septic process in two otters with S. phocae septicemia. (19). Similar to this study, P. multoicida was isolated from two otters that died from pleuropneumonia and fibrinopurulent pleuritic and was suspected the cause of death in three other otters with fibrinopurulent pleuritis. Infections with Pasteurella species generally often occur due to bite wounds or the intake of contaminated food. Infection with this bacteria can lead to respiratory infection, septicemia and endocarditis with a very high mortality rate (2). In the present study, a clear route of infection of P. multoicida in the otter could not be determined. However, based on the epidemiology of frequent fights with two other co-breeding otters, it seems very likely that the oldest otter developed traumatic skin wounds while fighting with other individuals, and P. multoicida in the upper respiratory system of other individuals may entered this wound site and then penetrated into the body.

Most Pasteurellla species are commensals on the mucosa of the upper respiratory system of animals (6,13). These bacteria may invade the tissues of immunosuppressed or immunocompromised animals. Exogenous transmission can also occur either direct contact or through aerosols. Factors of importance in the development of disease include adhesion of the bacteria to the mucosa of hosts and the avoidance of phagocytosis. There has been little progress in understanding exactly how P. multoicida invades mucosal surfaces to gain access to the blood stream and how the host responds to this infection. The pathogenesis by which P. multoicida can invade the mucosa, evade innate immunity and cause systemic disease including septicemia are slowly being elucidated. Key virulence factors identified to date include capsule and lipopolysaccharide of these bacteria. The capsule is clearly involved in bacterial avoidance of phagocytosis by macrophages and resistance to complement-mediated lysis, while complete lipopolysaccharide is critical for bacterial survival in the host (6).

P. multoicida is a bacterial pathogen of pet and agricultural animals. This bacteria can be a facultative pathogen of the upper respiratory tracts and can be transmitted via bite wounds (1). Although relatively uncommon, human infections of P. multoicida have been observed in a range of sites, commonly following cat or dog bites (6). Recently, a case of P. multoicida infection was reported in a child with peritonitis caused by close contact of the domestic cat to the dialysis machine and tubes, without any evidence of bites, scratches or leaks (17). In addition, over 25 human cases of P. multoicida peritonitis in association with peritoneal dialysis have been reported. Most cases were indeed closely associated with the presence of dogs or cats in the household, especially when contact with, or even puncture of, dialysis tubing is present. Animal bites are common in human. Land mammals are responsible for most bites, whereas marine mammals are an infrequently reported cause of animal bites (4). Bacterial infections are an important complication of animal bites. The pathogenic organisms may originate from the environment, the victim’s skin flora or the animal’s oral flora. The otter’s oral flora may have included Gram-negative organisms such as Escherichia coli, Salmonella sp. and Pasteurella multocida (1,4).

The Asian small-clawed otters is one of the most popular animals raised for the purpose of exhibition at zoos of wild animals, aquariums, and theme parks. And these animals are traditionally kept as pets not only in the Asian countries but also in many western countries (7). Therefore, great efforts should be warrant to prevent infection through biting accidents from wild animals including otter in field veterinarian, aquarists, and pets’ owners. Appropriate wound care after an animal bite is essential for the patients. In addition to antimicrobial chemoprophylaxis, consideration must be given to administering tetanus toxoid and rabies vaccine (4).

Based on the gross examinations, pathologic findings and bacterial experiments, this otter was diagnosed as severe peritonitis and necrotic hepatitis associated with P. multocida type A. In our best knowledge this is the first pathologic study for peritonitis and necrotic hepatitis caused by P. multocida type A infection in a captive Asian small-clawed otter in Korea.

This research was supported by the 2023 scientific promotion program funded by Jeju National University.

The authors have no conflicting interests.