J Vet Diagn Invest 9:320-323 (1997)

An outbreak of listeriosis in a breeding colony of chinchillas

Melinda J. Wilkerson, Amy Melendy, Eric Stauber

Listeria monocytogenes is a facultatively anaerobic shortgram-positive rod-shaped bacterium that infects a wide rangeof animals, including ruminants, monogastric animals, birds,fish, rodents, and humans.7 Infection with this obligate in-tracellular pathogen causes 3 distinct clinical entities, septi-cemia, encephalitis, and abortion, which rarely occur si-multaneously in the same animal. The septicemic form af-fects the viscera with or without meningoencephalitis and iscommon in monogastric animals, whereas encephalitis andabortion occur principally in adult ruminants. Chinchillasare considered one of the species more susceptible to viscerallisteriosis,6,7,11 especially when reared in confinement.6 Lis-teriosis in chinchillas has been described in many areas ofthe United States during a period (1949-1955) when chin-chilla pelts were prized commodities in the fur industry.9,11,12

More recently, chinchillas have become a widely used animalmodel for studies of noise-induced hearing loss.4

An outbreak of disseminated visceral listeriosis occurredin a breeding colony of 130 chinchillas located in northernIdaho. The chinchillas in this operation were raised for pelts.Polygamous breeding was practiced; 1 male had access to 8females. The feed, open sacks of commercially formulatedchinchilla pellets and a bale of hay, was stored on a woodpallet. To promote maintenance of their hair coats, a dustbath pan was provided for the animals. During December1995, there was an unusually large influx of mice into thechinchilla building, and on several occasions the owners not-ed mouse droppings in the feed. By January 1996, the colonyexperienced a 23% mortality (30 of 130) in breeding animalsof various ages. Approximately 4 days prior to death, theanimals were anorectic and hunched, and some had torti-collis. Many animals were found dead without premonitoryclinical signs. Four chinchillas (2 dead males, 1 dead female,1 moribund female) were submitted to the Washington An-imal Disease Diagnostic Laboratory for necropsy.

All 4 chinchillas were emaciated and lacked subcutaneousand retroperitoneal fat. Consistent gross lesions in the 3 deadchinchillas included multifocal 0.1-0.3-cm-diameter white-tan foci affecting the capsular surfaces and parenchyma ofthe liver, mesenteric lymph nodes, and the serosa of the smalland large intestines, particularly the cecum and colon. In-dividual differences in postmortem lesions are listed in Table1. Animal A, a dead female, had a 2-cm rectal prolapse andcolonic intussusception in addition to the visceral foci. Theintussusception consisted of a 3-cm segment of dark red prox-

From the Washington Animal Disease Diagnostic Laboratory(Wilkerson, Melendy) and The Department of Veterinary ClinicalSciences (Stauber), College of Veterinary Medicine, Washington StateUniversity, Pullman, WA 991652037. Current addresses: Depart-ment of Diagnostic Medicine/Pathobiology, College of VeterinaryMedicine, Kansas State University Manhattan, KS 66506 (Wilker-son), and 127 Elongrove Ave.#9, Providence, RI 02906 (Melendy).

Received for publication July 5, 1996.

imal colon telescoped into distal colon. Animal B, a deadmale, had fibrin tags adhered to the visceral and parietalpleura, pericardium, and diaphragm and a 1-cm-diameterabscess located on the apex of the heart. The moribund fe-male, animal D, presented with a slight head tilt and yellow-ish-tan mucoid vaginal discharge. Following euthanasia andnecropsy, the vaginal discharge was noted to extend into theuterus (mucometra). No other visceral involvement was not-ed in this animal.

Based upon postmortem findings, the following differentialetiologic agents were considered: L. monocytogenes, Sal-monella sp., and Yersina pseudotuberculosis. Samples of brain,lung, spleen, and thoracic fluid from all animals and a uterineswab from animal D were collected at necropsy for bacte-riologic cultures. Brain, lung, spleen, and thoracic fluid wereinoculated onto sheep blood agar and serum tellurite agar.a

Intestinal contents were cultured on MacConkey’s medium,enriched for Salmonella with selenite broth, followed by sub-culture to brilliant green agar and lysine iron agar.a The uter-ine swab was cultured on blood agar and MacConkey plates.a

A complete set of tissues was collected and fixed in 10%neutral buffered formalin for histopathologic examination.The tissues were embedded in paraffin, sectioned at 3 µm,and stained with hematoxylin and eosin.

Histologically, all animals had random multifocal areas ofhepatocellular necrosis and acute to subacute inflammation.Cellular debris, pyknotic nuclei, and neutrophils were locatedcentrally, with few lymphocytes and histiocytes at the pe-riphery of the necrotic foci (Fig. 1A). Transmural inflam-mation effaced the Peyer’s patches of the small intestine andmultifocal areas of cecum and colon (Fig. 2A). Mesentericlymph nodes had large necrotic foci containing degenerateneutrophils surrounded by macrophages, lymphocytes, andplasma cells. Splenic sections had multifocal areas of necrosisand fibrin deposition. Animal B had acute to subacute pleu-ritis and pericarditis consisting of fibrin, neutrophils, mac-rophages, and proliferative fibroblasts. The pericardium andepicardium were replaced by alternating zones of prolifera-tive fibroblasts, fibrin, and degenerate neutrophils. The uter-us of animal D contained hyperplastic endometrial glandsand mild metritis with moderate numbers of macrophagesand lymphocytes. Only animal D had multifocal to coalescingmicroabscessation and rarefaction of the caudal cerebrumand brain stem. Bacteria were not seen in replicate Brownand Hopps-stained sections of these tissues.

To identify listerial antigens in the tissues with inflam-matory lesions, selected formalin-fixed, paraffin-embeddedsections were immunostained with commercially availablerabbit polyclonal antiserum against L. monocytogenesb (1:2,000 dilution). A modification of a published avidin-biotinimmunoperoxidase procedure14 was performed with a stain-ing kit,c a biotinylated goat anti-rabbit immunoglobulin asthe secondary antibody,c and 3-amino-9-ethyl-carbazole(AEC)d as the chromogen. Preceding immunolabeling, the

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Table 1. Laboratory findings for 4 chinchillas (A-D).

tissue sections were deparaffinized in Pro-par,e dehydratedin 100% ethanol, blocked for endogenous peroxidases with3% hydrogen peroxide in methanol, rehydrated in gradedalcohols and water, blocked for nonspecific binding of pri-mary antibody with 3% goat serum, and digested with 0.1%Pronasef for 10 minutes at 37 C. The positive control tissueconsisted of formalin-fixed fetal bovine liver with hepaticnecrosis from which L. rnouocytogenes had been isolated inpure culture prior to fixation. Negative control for the testwas normal (nonimmune) rabbit serum applied to the Lis-teria-infected bovine fetal liver section and to the chinchillatissues. Using this technique, listerial antigen-positive debriswas identified as principally intracellular (in neutrophils andmacrophages) deep-red staining of short rods and clusters oforganisms within microabscesses and necrotic foci in thebrain stem of animal D, in pericardial abscess in animal B,and in liver and intestine of animals A, B, and C (Figs. 1B,2B). Rare extracellular listerial antigens also were highlightedby this method.

Within 3 days of submission, numerous bacteria were iso-lated from cultures of lung and thoracic fluid (animal B),spleen (animal C), and brain samples (animals A, D) andidentified as L. monocytogenes based on the following cri-teria: gram-positive stain, beta hemolysis on sheep bloodagar, a positive catalase reaction, positive CAMP test withStaphylococcus auerus, negative D-xylose fermentation, andmotility at 20 C in a mannitol agar stab. Confirmation wasperformed with 1 isolate using the API 20 STREP identifi-cation system.g Listeria were recovered from 2 of 4 brains,including animal D, with brain stem microabscesses, andanimal A, which had no macroscopic or microscopic lesions(Table 1). The Listeria isolates in all animals were susceptibleto various antibiotics, including penicillin, tetracycline, tri-methaprine-sulfadiazine, and chloramphenicol. Mixed bac-terial growth was cultured from the brains of animal A andC and from thoracic fluid of animal C, indicating possiblecontamination or postmortem overgrowth. Many S. aureus

were recovered from the uterine swab of chinchilla D. NoSalmonella or Campylobacter were isolated from the intes-tinal samples cultured as described previously. No Campy-lobacter was detected with Victoria blue staining. Althoughthe intestinal samples were not cultured specifically for Lis-teria, infection of the intestinal tract was demonstrated im-munohistochemically in animals A, B, and C (Table 1).

The most common manifestation of listeriosis in chin-chillas is microabscessation of the liver, mesenteric lymphnodes, and small and large intestines.3,10 Intestinal intussus-ception and rectal prolapse has been reported as complica-tions of enteritis.5 These lesions may be confused with thoseproduced by Y. pseudotuberculosis and Salmonella in chin-chillas.6 The bacteriologic isolation and immunohistochem-ical techniques confirmed the presence of Listeria in thisbreeding colony of chinchillas. Although Listeria is mostfrequently isolated from the liver, intestine, colon, and spleenin chinchillas, the organism also has been isolated from thelung, heart, and brain .5,10,11 Torticollis is a commonly de-scribed symptom in chinchillas with Listeria-induced me-ningoencephalitis .6,11 Chinchilla D had brain stem enceph-alitis without evidence of septicemia or digestive tract in-fection. Although the pathogen in chinchilla D could haveaccessed the brain via the oral cavity, as is speculated ofListeria invasion into the ruminant brain in which entry viaaxons and trigeminal nerves follows penetration of oral mu-cosa and the dental pulp when animals cut or lose teeth,2 nooral lesions were observed in this animal. However, mac-roscopic oral wounds are not essential to the developmentof meningoencephalitis; it can be reproduced in mice andrabbits by microscopic inoculation of L. monocytogenes inthe lip.1 Because microscopic examination of trigeminal nerveand its branches to the oral cavity was not performed in thesechinchillas, such a route of infection could not be confirmed.A hematogenous route of infection was not considered likelybecause of the lack of a septicemic process.

The predominance of digestive tract infection in chin-

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Figure 1. Immunoperoxidase stain of a focus of hepatic necrosis in chinchilla liver with (A) negative control serum and (B) antiserumagainst Listena monocytogenes, revealing few antigen-positive debris and short rods. AEC chromogen, counterstained with hematoxylin.Bar = 25 µm.

Figure 2. Immunoperoxidase stain of a transmural microabscess with necrosis in chinchilla cecum with (A) negative control serumand (B) antiserum against Listeria monocytogenes, depicting a myriad of intracellular and extracellular bacteria. AEC chromogen, coun-terstained with hematoxylin. Bar = 70 µm.

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chillas A, B, and C is suggestive of oral entry via contami-nated feed. Hay contaminated with rodent, chicken, or ru-minant feces has been implicated in outbreaks of chinchillalisteriosis, and the removal of such contaminated feed oftenstops the development of new infections.3,5 Moreover, thevisceral syndrome has been reproduced experimentally inadult nonpregnant chinchillas and white mice by oral inoc-ulation with L. monocytogenes.7,9 Although the pelleted feedand hay was not cultured during this outbreak, they werestrongly suspected to be contaminated with mouse droppingsor ruminant feces. This pathogen also could have been trans-mitted easily among animals by coprophagia because animalsdefecated in the dust pan during dust baths and this dust panwas transferred from cage to cage. Recommendations to breakthe cycle of oral transmission included removal of contam-inated feed, euthanasia and disposal of all moribund animals,and disinfection of cages, water bottles, and dust baths. Theowners discontinued feeding hay, stored pelleted feed in acovered metal container, provided individual dust baths, eu-thanatized 30 sick animals, and removed mice from thepremises. No problems were noted in the subsequent 6months.

Listeria monocytogenes was isolated from tissues and iden-tified readily within 72 hours by bacterial culture withoutcold enrichment. Listerial antigens could be detected in chin-chilla tissues immunohistochemically utilizing commerciallyavailable rabbit polyclonal antiserum, which verified thepresence of intracellular bacteria in microabscesses and ne-crotic foci even though a tissue Gram stain did not. Failureof Gram stains to detect Listeria has been reported for bovinebrain sections with compatible histologic lesions and cere-brospinal fluid cells from human patients.8,13 The rapid im-munohistochemical test used for these chinchillas could beutilized to verify Listeria organisms if tissues are unavailablefor culture. Lack of Gram staining is likely due to the obligateintracellular life cycle of Listeria. This particular organismis capable of utilizing the host cell’s actin machinery to propelitself through the cytoplasm of 1 cell into another cell withoutexposure to the extracellular environment.13

Acknowledgements. We thank the Washington AnimalDisease Diagnostic Laboratory Faculty and Staff for theircritical review of the manuscript and technical expertise inperforming diagnostic tests.

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Sources and manufacturers

Becton Dickinson, Cockeysville, MD.Listeria O antiserum poly, serotypes 1 and 4, Difco, Detroit,MI.Vector Laboratories Burlingame, CA.BioGenex, San Ramon, CA.Anatech, Battlecreek, MN.Protease Type XIV, Sigma Chemical Co., St. Louis, MO.Biomerieux, Hazelwood, MO.

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