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Journal of Veterinary Diagnostic

http://vdi.sagepub.com/content/13/3/252The online version of this article can be found at:

DOI: 10.1177/104063870101300312

2001 13: 252J VET Diagn InvestKrista M. D. la Perle, Fabio Del Piero, Ruthann F. Carr, Cheryl Harris and Paul C. Stromberg

Cutaneous Neosporosis in Two Adult Dogs on Chronic Immunosuppressive Therapy

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252 Brief Communications

J Vet Diagn Invest 13:252255 (2001)

Cutaneous neosporosis in two adult dogs on chronic immunosuppressive therapy

Krista M. D. La Perle, Fabio Del Piero, Ruthann F. Carr, Cheryl Harris, Paul C. Stromberg

Abstract. Antemortem diagnosis of generalized ulcerative and pyogranulomatous dermatitis with numerousintralesional tachyzoites was made from skin biopsy specimens from 2 adult dogs on chronic immunosuppres-

sive therapy. A 9-year-old Italian Greyhound was on long-term corticosteroid therapy for the treatment of a

lupus-like systemic autoimmune disorder, and a 7-year-old Labrador Retriever had received several months of

chemotherapy for lymphosarcoma. The tachyzoites were identified as Neospora caninum by immunoperoxidase

immunohistochemistry. Both dogs were treated with clindamycin. Lesions in the Greyhound resolved; however,

the Labrador Retriever was euthanized because of evidence of neuromuscular disease, despite improvement of

the skin lesions. These 2 cases indicate that cutaneous neosporosis can occur in adult dogs on chronic immu-

nosuppressive therapy. The disease may result from reactivation of a congenital infection and/or a recently

acquired primary infection.

Neospora caninum is a recently characterized, cyst-form-

ing protozoan parasite7 that causes natural disease in multi-

ple species, including dogs, cattle, sheep, goats, horses, anddeer.12 Although its life cycle has not been completely elu-

cidated, N. caninum is reportedly transmitted through trans-

placental infection3,8,9 and dog feces.22 Although neosporosis

is a major cause of abortion in cattle,12 in dogs N. caninum

mainly elicits progressive ascending neuromuscular paralysis

caused by polymyositis, polyradiculitis, and meningoenceph-

alitis, which is most severe and more common in young

dogs.2,1012,27 Clinical signs and conditions in adult dogs with

neosporosis may include neurologic disease, polymyositis,

myocarditis, and dermatitis.2,1012,27 The original case series

of 10 dogs with neosporosis published over a decade ago

included a 15-year-old mixed-breed dog with ulcerative skin

lesions and multisystemic disease.7 Since that time, Neos-

pora dermatitis has been reported in 5 other dogs, rangingin age from 5 to 12 years, which resided in France,18 Israel,25

Italy,26 and the United States.13 This report documents ul-

cerative and pyogranulomatous dermatitis due to N. caninum

diagnosed from the skin biopsies of 2 immunosuppressed

adult dogs from Ohio.

A 9-year-old castrated Italian Greyhound (dog No. 1) was

presented to the Beechmont Pet Hospital (Cincinnati, OH)

with multiple, 0.52.5-cm-diameter, firm, circular, alopecic,

and ulcerated skin nodules of several weeks duration ran-

domly distributed over the trunk, tail, legs, and feet. Two

and one-half years prior to the onset of these skin lesions,

the dog was diagnosed with a lupus-like systemic autoim-

mune disorder characterized by perioral and periocular der-

matitis, anorexia, lethargy, weakness, severe thrombocyto-

From the Department of Veterinary Biosciences, College of Veteri-

nary Medicine, Ohio State University, 1925 Coffey Road, Columbus,

OH 43210 (La Perle, Stromberg), the Department of Pathobiology, New

Bolton Center, School of Veterinary Medicine University of Pennsyl-

vania, 382 West Street Road, Kennett Square, PA 19348 (Del Piero),

Beechmont Pet Hospital, 1627 Burney Lane, Cincinnati, OH 45230

(Carr), Veterinary Internal Medicine Clinic, 931 Route 28, Milford, OH

45150 (Harris), and Veterinary Diagnostics, 5747 Cleveland Avenue,

Suite B, Columbus, OH 43231 (Stromberg).

Received for publication April 11, 2000.

penia, mild neutrophilia, and anemia. The dog was initially

treated with immunosuppressive doses of prednisone. As a

result of the onset of hyperadrenocorticism-like symptoms,prednisone treatment was subsequently reduced to daily anti-

inflammatory doses 6 months prior to the dogs presentation

for nodular skin lesions. Fine-needle aspirates of the ulcer-

ated nodules revealed many segmented neutrophils with

some monocytes. After 1 week of antimicrobial therapy with

cephalexin, all lesions were exudative and enlarged to 1

cm. Punch biopsies from the dorsal neck were obtained,

fixed in 10% neutral buffered formalin, routinely processed,

and embedded in paraffin.

Histologic examination of 5-m sections stained with he-

matoxylin and eosin (HE) revealed severe, diffuse pyogran-

ulomatous dermatitis with nodular aggregates of lympho-

cytes and plasma cells in the deep dermis, multifocal epi-

dermal ulceration, and seropurulent crusts. Numerous ovoidintracellular tachyzoites compatible with N. caninum were

present within endothelial cells, macrophages, and kerati-

nocytes (Fig. 1).

The identification of N. caninum tachyzoites was con-

firmed by immunoperoxidase immunohistochemical staining

using the murine monoclonal antibody MAB-6G7, IgG2a is-

otypea (1:1,200), which recognizes low-molecular-mass epi-

topes (3197.4 kD) located on bradyzoites, tachyzoites, and

cysts of N. caninum (Fig. 2).5 Positive controls consisted of

sections of bovine cerebrum containing N. caninum zoites

and cysts and spun formalin-fixed tissue culture cells con-

taining numerous intra- and extracellular N. caninum zoites.

Tissue sections containing Toxoplasma gondii served as con-

trols to evaluate the specificity of the monoclonal antibody.

Three weeks of cephalexin therapy produced mild im-

provement. Cephalexin was discontinued and clindamycin

was initiated upon definitive diagnosis of Neospora derma-

titis. The dog had not exhibited signs of neurologic or other

nondermatologic disease, and lesions completely resolved

with no evidence of recurrence.

Dog No. 2, a 6-year-old spayed female Labrador Retriever,

presented to the Veterinary Internal Medicine Clinic (Milford,

OH) with a history of anorexia, exercise intolerance, polyuria/

polydipsia, hind limb weakness, and generalized lymphadenop-

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253Brief Communications

Figure 1. Skin from the dorsal neck of dog No. 1. Note the pyogranulomatous dermatitis with numerous clusters of tightly packed

tachyzoites within macrophages and endothelial cells. HE. Bar 18.25 m.

Figure 2. Skin from dog No. 1. Clusters of tachyzoites within dermal macrophages and endothelial cells stain intensely positive with

a monoclonal antibody specific for Neospora caninum. Avidinbiotin complex immunoperoxidase immunohistochemistry, hematoxylin

counterstain. Bar 18.25 m.

athy. Other than mild conscious proprioceptive deficits, there

were no other neurologic abnormalities. The hind limb weak-

ness was attributed to mild hypercalcemia (12.1 mg/dl; refer-

ence range, 8.810.5 mg/dl). Fine-needle lymph node aspirates

were consistent with a diagnosis of lymphosarcoma. The hind

limb weakness and hypercalcemia resolved within 1 week, and

complete remission was obtained within 6 months with a com-

bination chemotherapy protocol using vincristine, L-asparagi-

nase, prednisone, and chlorambucil. A maintenance protocol of

prednisone and chlorambucil was instituted and continued until

relapse occurred 4 months later. Chemotherapy with actino-

mycin-D and cytarabine failed, but remission was again ob-

tained with lomustine.

Seven months later, the dog presented again with fever,

diarrhea, hind limb weakness, and multiple erythematous

skin lesions that were ulcerative on the head and exudative

on the thorax and abdomen. These clinical signs were at-

tributable to a secondary problem; the lymphosarcoma was

believed to be in continued remission due to the absence of

palpable lymph nodes and hypercalcemia. Fine-needle as-



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254 Brief Communications

pirates of the skin lesions demonstrated purulent inflamma-

tion with numerous tachyzoites. Histologic findings from a

skin biopsy were similar to those for dog No. 1, and N.caninum tachyzoites were confirmed with immunohisto-chemistry using the same protocol. Although the skin lesions

and hind limb weakness resolved with clindamycin therapy,

the dog became febrile, nonresponsive, and laterally recum-

bent 2 months later, at which time euthanasia was elected.No necropsy was performed.

The differential diagnoses for protozoal dermatitis in the dog

include infection with Caryospora sp., Leishmania sp., N. can-inum, Sarcocystis canis, and T. gondii. Confirmed cases of der-matitis have been associated with Caryospora sp.6 and S.canis14 in young immunosuppressed dogs. Distinguishing fea-tures include the presence of caryocysts in cases ofCaryosporainfection and the presence of schizonts and division by endo-

polygeny in cases of Sarcocystis infection. Although leishman-iasis primarily occurs in dogs that reside in or have visited

Mediterranean countries and Northern Europe,17 cases have

been reported in the United States1 in Oklahoma (1980), Kan-

sas (1982), Ohio (1988), Michigan (1989), Texas and Alabama

(1991), and New York (2000) (P. M. Schantz, Centers for Dis-

ease Control, personal communication).23 Amastigotes contain-

ing kinetoplasts are pathognomonic for Leishmania. Toxoplas-ma gondii can infect virtually all body tissues; however, thereare no reported cases of cutaneous toxoplasmosis in dogs.13

Although it is structurally similar to Neospora in cytologicsmears and HE-stained sections, Toxoplasma can be differen-tiated by means of serology, electron microscopy, and immu-

nohistochemistry.

Although canine neosporosis typically presents as progres-

sive ascending paralysis in young dogs, affected animals have

ranged in age from several weeks to 15 years, with involvement

of virtually any organ system.2,1012,27 All cases of cutaneous

infection have been in middle-aged or older dogs. Cutaneous

neosporosis was first described in a 15-year-old mixed-breed

dog from the United States with ulcerative and fistulous cuta-neous lesions that began ventrolateral to the anus.7 Postmortem

lesions included interstitial pneumonia, hepatitis, leptomenin-

gitis, myositis, interstitial nephritis, cyclitis, and granulocytic

hyperplasia of the bone marrow. A 12-year-old Golden Retriev-

er, also from the United States, had draining nodules on the

head and thorax.13 Affected dogs from other countries include

a 6-year-old female Siberian Husky from France with pseu-

dotumoral nodules on the face and front legs,18 an 11-year-old

male Boxer from Israel with ulcerative lesions on the thorax

and abdomen,25 and a 5-year-old Bernese Cattle Dog from Italy

with nodular dermatitis on the tarsus.26 The clinical history in

each of these cases was unremarkable, and it was suggested

that age-related immunodeficiency might have played a role in

inducing or provoking dermal neosporosis in these dogs.13,25

Unlike T. gondii, which is considered an opportunistic

pathogen, N. caninum is regarded as a primary pathogen.7 Itis not known whether the disease in adult dogs is due to

reactivation of a congenital infection or is a recently ac-

quired primary infection. The immune response elicited by

the host in response to N. caninum has been characterized

primarily through studies in cattle and mice.4,16,20,21 These

studies implicate cellular and humoral immune responses in

which interleukin (IL)-12, interferon- (IFN), and IL-10 are

the key cytokines. It is believed that N. caninum, like T.

gondii, induces phagocytic cells to produce IL-12, which

stimulates the differentiation of uncommitted T helper (TH)cells toward the TH1 phenotype and therefore promotes cel-

lular immunity. IL-12 stimulates the production of IFN byT cells and natural killer cells. Lymphocytes and phagocyticcells also secrete IL-10, which inhibits IL-12 and potentiates

TH2 or humoral responses. This downregulation of the cel-lular immune response facilitates survival of the parasite and

the host.The clinical histories for the 2 dogs described here were

extensive and included chronic immunosuppression by pred-

nisone and various cytotoxic drugs. Reactivation of dormantneosporosis has been demonstrated experimentally with admin-istration of glucocorticoids,10 which inhibit TH1 and enhance

TH2 cytokine secretion.15 T cells are exquisitely more sensitivethan B cells to the cytotoxic effects of drugs such as L-aspa-

raginase, chlorambucil, and cytarabine.19,24 Therefore, immu-

nosuppressive therapy in both of these dogs most likely pref-

erentially suppressed cellular immunity, enabling the establish-

ment of clinically apparent N. caninum infection. Neospora caninum should be included on the list of dif-

ferential diagnoses for ulcerative and pyogranulomatous der-

matitis, particularly in immunosuppressed dogs and in

breeds exhibiting increased incidences of neosporosis, such

as Basset Hounds, Boxers, Golden and Labrador retrievers,

and Greyhounds.12 Tachyzoites, which are usually abundant,

are readily observed by routine histopathology and can be

easily confirmed with immunohistochemistry.

Acknowledgements. We thank Dr. D. S. Lindsay (Depart-ment of Biomedical Sciences and Pathobiology, Virginia-

Maryland Regional College of Veterinary Medicine) and Dr.

E. J. Dubovi (New York State Diagnostic Laboratory, Cor-

nell University) for providing the MAB-6G7 antibody and

sections of bovine cerebrum and tissue culture cells for use

as positive controls. We appreciate the comparative staining

using different antibodies performed by Dr. Daniel Wein-

stock (Pennsylvania State University) and Dr. Evelyn W. Po-

lack (Department of Pathology, Cornell University). We arealso grateful to Dr. Eric A. G. Blomme (Searle/Pharmacia,

Chicago) for critical review of this manuscript.

Sources and manufacturers

a. Dr. D. S. Lindsay, Department of Biomedical Sciences and Pa-

thology, Virginia-Maryland Regional College of Veterinary Med-

icine, Blacksburg, VA, and Dr. E. J. Dubovi, New York State

Diagnostic Laboratory, Cornell University, Ithaca, NY.

References

1. Anderson DC, Buckner RG, Glenn BL, MacVean DW: 1980,Endemic canine leishmaniasis. Vet Pathol 17:9496.

2. Barber JS, Trees AJ: 1996, Clinical aspects of 27 cases of neos-porosis in dogs. Vet Rec 139:439443.

3. Barber JS, Trees AJ: 1998, Naturally occurring vertical trans-mission of Neospora caninum in dogs. Int J Parasitol 28:5764.

4. Baszler TV, Long MT, McElwain TF, Mathison BA: 1999, In-terferon-gamma and interleukin-12 mediate protection to acuteNeospora caninum infection in BALB/c mice. Int J Parasitol 29:16351646.

5. Cole RA, Lindsay DS, Dubey JP, Blagburn BL: 1993, Detectionof Neospora caninum in tissue sections using a murine mono-clonal antibody. J Vet Diagn Invest 5:579584.

6. Dubey JP, Black SS, Sangster LT, et al.: 1990, Caryospora-as-sociated dermatitis in dogs. J Parasitol 76:552556.

7. Dubey JP, Carpenter JL, Speer CA, et al.: 1988, Newly recog-



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nized fatal protozoan disease of dogs. J Am Vet Med Assoc192:12691285.

8. Dubey JP, Koestner A, Piper RC: 1990, Repeated transplacentaltransmission of Neospora caninum in dogs. J Am Vet Med As-soc 197:857860.

9. Dubey JP, Lindsay DS: 1989, Transplacental Neospora caninuminfection in dogs. Am J Vet Res 50:15781579.

10. Dubey JP, Lindsay DS: 1990, Neosporosis in dogs. Vet Parasitol

36:147151.11. Dubey JP, Lindsay DS: 1993, Neosporosis. Parasitol Today 9:452458.

12. Dubey JP, Lindsay DS: 1996, A review of Neospora caninumand neosporosis. Vet Parasitol 67:159.

13. Dubey JP, Metzger FL Jr, Hattel AL, et al.: 1995, Canine cu-taneous neosporosis: clinical improvement with clindamycin.Vet Dermatol 6:3743.

14. Dubey JP, Slife LN, Speer CA, et al.: 1991, Fatal cutaneous andvisceral infection in a Rottweiler dog associated with a Sarco-cystis-like protozoon. J Vet Diagn Invest 3:7275.

15. Elenkov IJ, Papanicolaou DA, Wilder RL, Chrousos GP: 1996,Modulatory effects of glucocorticoids and catecholamines onhuman interleukin-12 and interleukin-10 production: clinicalimplications. Proc Assoc Am Physicians 108:374381.

16. Eperon S, Bronnimann K, Hemphill A, Gottstein B: 1999, Sus-ceptibility of B-cell deficient C57GL/6 (MT) mice to Neospora

caninum infection. Parasite Immunol 21:225236.17. Ferrer L, Rabanal R, Fondevila D, et al.: 1988, Skin lesions incanine leishmaniasis. J Small Anim Pract 29:381388.

18. Fritz D, George C, Dubey JP: 1997, Neospora caninum: asso-ciated nodular dermatitis in a middle-aged dog. Canine Pract22:2124.

19. Kazmers IS, Daddona PE, Dalke AP, Kelley WN: 1983, Effectof immunosuppressive agents on human T and B lymphoblasts.Biochem Pharmacol 32:805810.

20. Khan IA, Schwartzman JD, Fonseka S, Kasper LH: 1997, Neos-pora caninum: role for immune cytokines in host immunity. Exp

Parasitol 85:2434.21. Lunden A, Marks J, Maley SW, Innes EA: 1998, Cellular im-

mune responses in cattle experimentally infected with Neosporacaninum. Parasite Immunol 20:519526.

22. McAllister MM, Dubey JP, Lindsay DS, et al.: 1998, Dogs aredefinitive hosts of Neospora caninum. Int J Parasitol 28:14731478.

23. Monti DJ: 2000, Hunters hounded as leishmaniasis is diagnosedin Foxhounds. J Am Vet Med Assoc 216:1887, 1890.

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J Vet Diagn Invest 13:255258 (2001)

Septicemia associated with Stenotrophomonas maltophilia in a

West African dwarf crocodile (Osteolaemus tetraspis subsp. tetraspis)

N. Beth Harris, Douglas G. Rogers

Abstract. A 17-year-old male captive West African dwarf crocodile (Osteolaemus tetraspis subsp. tetraspis)died 1 month after fighting with a penmate. Abrasions were present on the head and mandible. Necropsy

revealed a vegetative valvular lesion of the left atrioventricular valve, miliary foci of necrosis in the endocardium

and myocardium, multiple duodenal and rectal ulcers, and serous atrophy of body fat. Stenotrophomonas mal-

tophilia was isolated in pure culture from lung, liver, and kidney. Gram-negative bacilli were seen histologically

in the valvular lesion and in foci of necrosis in the myocardium, liver, spleen, pancreas, kidney, and intestine.

Septic thrombi in multiple tissues, arteritis, and pneumonia were additional histologic lesions. Findings indicated

that the crocodile died from acute S. maltophilia septicemia, although the primary site of infection was not

determined. Stenotrophomonas maltophilia is ubiquitous in the environment and is recognized as an important

nosocomial pathogen in humans.

Stenotrophomonas maltophilia is a straight or slightly

curved nonfermentative gram-negative bacillus that has pre-

viously been classified as Pseudomonas maltophilia andXanthomonas maltophilia. This bacterium was transferred to

the new genus Stenotrohomonas because of comparative en-

zymology data, results of DNAribosomal RNA hybridiza-

tion studies, guanine/cytosine content, and fatty acid com-

position.16 Despite earlier reports that S. maltophilia had lim-ited pathogenicity,2 the bacterium has recently gained im-

From the Veterinary Diagnostic Center, Department of Veterinary

and Biomedical Sciences, University of Nebraska, Lincoln, NE

68583-0907.

Received for publication April 15, 2000.

portance as a nosocomial pathogen in humans, in which it

causes septicemia,7,10,11 endocarditis,4,12 meningitis,13 pneu-

monia,7,10

urocystitis,17

and wound infection.18

Human pa-tients considered at risk for S. maltophilia infections include

the severely debilitated or immunosuppressed, those receiv-

ing antimicrobial and/or intravenous therapy, and individuals

subjected to invasive surgical procedures.2 A protease and

elastase elaborated by S. maltophilia are believed to be im-portant in the pathogenesis of infection.1,15

Although S. maltophilia is now recognized as a significant

human pathogen, the role of this bacterium in diseases of

animals is less clear. In 1 report, S. maltophilia was consid-

ered to be the cause of fleece rot in sheep.9 Stenotropho-

monas maltophilia has been isolated from fish,6 lizards,


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