DOI: 10.7589/2012-10-265 Journal of Wildlife Diseases, 49(3), 2013, pp. 641–645# Wildlife Disease Association 2013

Leptospirosis in Fox Squirrels (Sciurus niger) of Larimer County,

Colorado, USA

Katherine Dirsmith,1 Kaci VanDalen,2 Tricia Fry,2 Brad Charles,1 Kurt VerCauteren,2 and Colleen Duncan1,3

1College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 1601 Campus Delivery, FortCollins, Colorado, USA 80523; 2US Department of Agriculture, Animal and Plant Health Inspection Service,National Wildlife Research Center, 4101 LaPorte Ave., Fort Collins, Colorado, USA 80521; 3Correspondingauthor (email: colleen.duncan@colostate.edu)

ABSTRACT: Leptospirosis is a zoonotic diseasecaused by the bacterium Leptospira interro-gans. The organism is typically maintainedwithin a geographic region by colonizing renaltubules of carrier animals and shed into theenvironment in urine. We assessed whetherL. interrogans was present in fox squirrels(Sciurus niger) in Larimer County, Colorado,USA, and whether it is associated with disease.Twenty-two squirrels were trapped from 29November 2011 to 15 December 2011 for usein an unrelated study. The squirrels wereindividually housed for 33–65 days and eutha-nized; no clinical disease was observed. Ongross examination, significant renal lesions wereobserved in 6 of 22 animals (27%). Histologi-cally, affected animals had severe neutrophilictubulitis with interstitial nephritis. Immunohis-tochemistry was conducted on the kidneys of allanimals and 10 of 22 (45%) were positive for L.interrogans, with varying severity of infection.The same 10 squirrels were serologicallypositive for antibodies specific to L. interro-gans. These results suggest that L. interrogansis present in fox squirrels in Larimer County,Colorado, USA, and may be associated withvarying degrees of renal disease. Furtherinvestigation into the role of wildlife in theecology of leptospirosis within the region iswarranted.

Key words: Colorado, Leptospira interro-gans, leptospirosis, squirrel, wildlife.

Leptospirosis is a disease caused bygram-negative bacteria of the genus Lep-tospira. The pathogen is maintained in awide range of wild and domestic animalhosts, such as dogs (Canis lupus famil-iaris), livestock, and rodents (Rodentia;Lau et al., 2009). In geographically distinctpopulations, a single host species maycarry several serovars (Bharti et al., 2003).Leptospira spp. are typically maintainedby colonization of renal tubules in carrieranimals. Although carrier animals mayexhibit few or no symptoms, leptospires

can be shed in urine (Monahan et al.,2009). Susceptible animals can be infecteddirectly by contact with carrier animals orindirectly by contact with traces of con-taminated urine in water sources (Gautamet al., 2010). In the past several decades,Leptospira interrogans has been deemed aglobally important zoonotic pathogen(Bharti et al., 2003), occurring in manymammalian species, including humans.Historically, leptospirosis was regarded asan uncommon disease in Larimer County,Colorado, USA, but leptospires haveincreasingly been identified in domesticcanines and raccoons (Procyon lotor; Veir2009; Duncan et al., 2012). We assessedthe prevalence of L. interrogans in anoth-er peridomestic species, the fox squirrel(Sciurus niger), trapped around homes inFort Collins, Larimer County, Colorado,USA.

We used Tomahawk live traps (483

15315 cm; Tomahawk Live Trap Co.,Hazelhurst, Wisconsin, USA) baited withpeanut butter and oats to trap 22 foxsquirrels, 11 males (M) and 11 females (F)from 29 November 2011 to 15 December2011, for use in an unrelated vaccineresearch study (Fry, pers. comm.). Whenaccessioned, fox squirrels were dusted forectoparasites (DrioneH, Bayer, Montvale,New Jersey, USA) and housed individuallyin stainless-steel cages (50361342 cm) atthe National Wildlife Research Center(NWRC), Animal Research Building (FortCollins, Colorado, USA). Fox squirrelswere fed a diet comprising Purina RodentChow (Purina, St. Louis, Missouri, USA),apples, and whole mixed nuts. Water wasprovided ad libitum. The NWRC’s insti-tutional animal care and use committee

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approved all animal trapping, handling,and housing procedures.

As part of the original vaccine study, weobserved fox squirrels daily and collectedblood samples on day 0 of the study(approximately 22–37 days in captivity)and preceding euthanasia (4 or 28 dayslater). Fox squirrels were anesthetizedusing isoflurane before being humanelyeuthanized via intracardiac injection of anoverdose of barbiturates (BeuthanasiaH,Intervet Inc., Merck Animal Health, Sum-mit, New Jersey, USA). Complete post-mortem examinations were conducted andmultiple tissues were collected for histo-logic examination. Tissues were fixed in10% neutral-buffered formalin, paraffinembedded, cut at 5 mm, and examined bylight microscopy. A Warthin-Starry silverstain and leptospiral immunohistochemis-try (IHC) were performed on renal tissuefrom each animal. The IHC was conductedusing a primary antibody composed of acocktail of six antisera: Canicola–HondUtrecht IV, Grippotyphosa-Andaman,Hardjo-Hardojoprajtino, Copenhageni-M20A, Pomona-Pomona, and Bratislava–Jez Bratislava (US Department of Agricul-ture, Animal and Plant Inspection Service,National Veterinary Services Laboratory[NVSL], Ames, Iowa, USA). Sera from allfox squirrels were also tested for antibodiesspecific to five L. interrogans serovars:Canicola, Grippotyphosa, Hardjo, Ictero-haemorrhagiae, and Pomona, via a micro-agglutination test at the NVSL.

Gross examination revealed significantrenal lesions in six of 22 animals, (27%; 5 F,1 M). Bilaterally, the kidneys were mottledwith multifocal to coalescing areas of pallor1–4 mm diameter. On the cut surface,affected areas extended into the parenchy-ma. All other organs were grossly withinnormal limits. Histologically, renal lesionswere observed in 10 animals (19%; 6 F,4 M). In the six squirrels in which grosschanges were identified, much of the renalparenchyma was effaced by inflammation(Fig. 1A). The interstitial space was ex-panded by large aggregates of lymphocytes

and plasma cells; within affected regions,many tubules were dilated, and tubularlumens contained aggregates of viable anddegenerate neutrophils (Fig. 1B) withvarying amounts of tubular degenerationand necrosis. In four other animals (8%;1 F, 3 M), there were only mild, patchyaggregates of interstitial lymphocytes andplasma cells, devoid of the tubulitis ob-served in the more severely affectedanimals. Additional tissues reviewed histo-logically included lung, liver, and multiplesections of intestinal tract. There was mild-to-moderate, bronchiole-associated hyper-plasia of the lymphoid tissue in the lung offive animals; two animals had patchy areasof pulmonary inflammation. Very mildperiportal, lymphoplasmacytic inflamma-tion was observed in the liver of 14squirrels, but the cellular infiltrate did notdistort the hepatic parenchyma. Two ani-mals had both enteritis and colitis, whichwas moderate and mononuclear in nature.

Warthin-Starry silver stain highlightedfilamentous organisms on the luminalsurface of tubular epithelial cells of fouranimals with severe tubulitis (Fig. 1C). Inthe six severely affected animals, there wasstrong immunostaining of the tubularepithelial cells, both on the luminalmembrane, but also cytoplasmically, andpatchy staining of leukocytes within theinterstitium (Fig. 1D). Minimal immuno-staining was observed in four additionalanimals; mild-to-moderate interstitial in-flammation had been observed histologi-cally in these four animals, but none hadsevere tubulitis.

The six severely affected animals hadhigh titers of circulating antibodies to L.interrogans; highest titers were observedagainst serovars Grippotyphosa, Hardjo,and Icterohaemorrhagiae (Table 1). Serafrom four fox squirrels with histologicevidence of a mild infection were alsopositive for antibodies specific to L.interrogans. Two of these fox squirrelswere sampled 28 days apart with little orno increase in antibody titers. This mildinfection paired with stable antibody titers

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may be indicative of a ‘‘carrier’’ state oranimals recovering from infection. Threeindividuals with minimal or no histologicevidence of renal disease showed lowreactivity (titer#200) to L. interrogansserovars Hardjo or Icterohaemorrhagiaeon the last blood collection only. Theselow and inconsistent titers may indicate apossible exposure while in captivity, re-covery from infection with waning anti-body titers, or false results.

For almost 25 yr, no case reports orprevalence studies were published, to ourknowledge, regarding L. interrogans inanimals of Larimer County, Colorado,USA. However, between 2005 and 2008,a growing number of domestic dogs inColorado, USA, were diagnosed withleptospirosis; 61% of those were dogs

living in urban or suburban environments,including Larimer County, Colorado,USA, where there were 85 suspected and15 confirmed cases; Grippotyphosa wasthe most commonly reported serovar (Veir2009). Many peridomestic animals alsolive in these environments, includingNorway rats (Rattus norvegicus), mice(Mus musculus), raccoons, and fox squir-rels. Previous studies in Larimer County,Colorado, USA, have shown evidence ofL. interrogans infection in Norway rats,house mice, and raccoons (Roberts 1972;Al Saadi and Post 1976; Duncan et al.,2012). Although fox squirrels in Colorado,USA, have not been investigated until thisstudy, to our knowledge, a gray squirrel(Sciurus carolinensis) from Connecticut,USA, had antibodies to L. interrogans

FIGURE 1. Kidney of a fox squirrel (Sciurus niger) infected with Leptospira interrogans. (A) There ismarked inflammation affecting both the interstitium and renal tubules. H&E. 34. (B) Inflammatory cellsdilate renal tubules and are predominantly neutrophils. H&E. 320. (C) Lining renal tubules are manyargyrophilic organisms. 320 Warthin-Starry silver stain. (D) Strong L. interrogans immunostaining is presentin the tubular epithelial cells and inflammatory cells in tubular lumens. Immunohistochemistry. 320.

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serovars Grippotyphosa and Canicola(Richardson and Gauthier, 2003). Similar-ly, L. interrogans serovar Grippotyphosahas been isolated from the kidneys of fivesouthern flying squirrels (Glaucomys vo-lans) and was subsequently identified intwo human handlers, demonstrating thepotential for zoonotic transmission (Masu-zawa et al., 2006).

We provide evidence of L. interrogansinfection, with both mild and severe renaldisease, in a population of peridomestic foxsquirrels in Larimer County, Colorado,USA. The high prevalence of disease(45%) and antibody-positive animals(55%) was unexpected and suggests thatfox squirrels may be important in theepidemiology of leptospirosis in the region.Although the causative serovar cannot beunequivocally determined, the high anti-body titers suggest probable infection withserovars Grippotyphosa, Hardjo, or Icter-ohaemorrhagiae. Many species of rodentsare considered reservoirs of L. interrogans;therefore, squirrels may be important hostsin the maintenance and transmission of L.interrogans in some regions. Although thecourse of infection with L. interrogans isunknown, the squirrels in this study werecaptured in urban and suburban areas, andthe epidemiology of leptospirosis in foxsquirrels and increased incidence in dogsmay be related. The possibility of squirrel-dog transmission warrants further investi-gation to better understand the spread ofleptospirosis at the urban-wildlife interface.

We thank Daniel Gossett and the animalcare staff at the National Wildlife ResearchCenter for their help in completing this

research, and Cindy Hirota for assistancewith serology.

LITERATURE CITED

Al Saadi M, Post G. 1976. Rodent leptospirosis inColorado. J Wildl Dis 12:315–317.

Bharti AR, Nally J, Ricaldi J, Matthias M, Diaz MM,Lovett MA, Levett PN, Gilman RH, Willig MR,Gotuzzo E, et al. 2003. Leptospirosis: A zoonoticdisease of global importance. Lancet Infect Dis3:757–771.

Duncan C, Krafsur G, Podell B, Baeten LA. 2012.Leptospirosis and tularaemia in raccoons (Pro-cyon lotor) of Larimer County, Colorado.Zoonoses Public Health 59:29–34.

Gautam R, Guptill LF, Wu CC, Potter A, Moore GE.2010. Spatial and spatio-temporal clustering ofoverall and serovar-specific Leptospira micro-scopic agglutination test (MAT) seropositivityamong dogs in the United States from 2000through 2007. Prev Vet Med 96:122–131.

Lau C, Smythe L, Weinstein P. 2009. Leptospirosis:an emerging disease in travelers. Travel MedInfect Dis 8:33–39.

Masuzawa T, Okamoto Y, Une Y, Takeuchi T. 2006.Leptospirosis in squirrels imported from UnitedStates to Japan. Emerg Infect Dis 12:1153–1155.

Monahan AM, Callanan JJ, Nally JE. 2009. Reviewpaper: Host-pathogen interactions in the kidneyduring chronic leptospirosis. Vet Pathol 46:792–799.

Roberts JR. 1972. Factors influencing growth,virulence, viability, and antigenicity of Lepto-spira icterohaemorrhagiae. MS Thesis. ColoradoState University, Fort Collins, Colorado, 310 pp.

Richardson DJ, Gauthier JL. 2003. A serosurvey ofleptospirosis in Connecticut peridomestic wild-life. Vector Borne Zoonotic Dis 3:187–193.

Veir J. 2009. Take a new look at canine lepto inColorado. Colo Vet Med Assoc Voice 2. http://www.colovma.com/associations/2956/files/CVMA%20Voice%20Lepto%20story_FIN.pdf.Accessed February 2013.

Submitted for publication 24 October 2012.Accepted 12 January 2013.

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