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Journal of Veterinary Diagnostic Investigation Cutaneous Neosporosis in Two Adult Dogs on Chronic Immunosuppressive Therapy

Krista M. D. la Perle, Fabio Del Piero, Ruthann F. Carr, Cheryl Harris and Paul C. Stromberg J VET Diagn Invest 2001 13: 252 DOI: 10.1177/104063870101300312 The online version of this article can be found at:

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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 numerous intralesional tachyzoites was made from skin biopsy specimens from 2 adult dogs on chronic immunosuppressive 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 identied 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 immunosuppressive 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-forming protozoan parasite7 that causes natural disease in multiple species, including dogs, cattle, sheep, goats, horses, and deer.12 Although its life cycle has not been completely elucidated, N. caninum is reportedly transmitted through transplacental 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 meningoencephalitis, 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, Neospora dermatitis has been reported in 5 other dogs, ranging in age from 5 to 12 years, which resided in France,18 Israel,25 Italy,26 and the United States.13 This report documents ulcerative 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, rm, circular, alopecic, and ulcerated skin nodules of several weeks duration randomly 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 autoimmune disorder characterized by perioral and periocular dermatitis, anorexia, lethargy, weakness, severe thrombocytoFrom the Department of Veterinary Biosciences, College of Veterinary 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 Pennsylvania, 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 antiinammatory doses 6 months prior to the dogs presentation for nodular skin lesions. Fine-needle aspirates of the ulcerated 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, xed in 10% neutral buffered formalin, routinely processed, and embedded in parafn. Histologic examination of 5- m sections stained with hematoxylin and eosin (HE) revealed severe, diffuse pyogranulomatous dermatitis with nodular aggregates of lymphocytes and plasma cells in the deep dermis, multifocal epidermal ulceration, and seropurulent crusts. Numerous ovoid intracellular tachyzoites compatible with N. caninum were present within endothelial cells, macrophages, and keratinocytes (Fig. 1). The identication of N. caninum tachyzoites was conrmed by immunoperoxidase immunohistochemical staining using the murine monoclonal antibody MAB-6G7, IgG2a isotypea (1:1,200), which recognizes low-molecular-mass epitopes (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-xed tissue culture cells containing numerous intra- and extracellular N. caninum zoites. Tissue sections containing Toxoplasma gondii served as controls to evaluate the specicity of the monoclonal antibody. Three weeks of cephalexin therapy produced mild improvement. Cephalexin was discontinued and clindamycin was initiated upon denitive diagnosis of Neospora dermatitis. 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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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.

athy. Other than mild conscious proprioceptive decits, there were no other neurologic abnormalities. The hind limb weakness was attributed to mild hypercalcemia (12.1 mg/dl; reference 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 combination chemotherapy protocol using vincristine, L-asparaginase, prednisone, and chlorambucil. A maintenance protocol of prednisone and chlorambucil was instituted and continued until

relapse occurred 4 months later. Chemotherapy with actinomycin-D and cytarabine failed, but remission was again obtained 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 attributable 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-

Figure 2. Skin from dog No. 1. Clusters of tachyzoites within dermal macrophages and endothelial cells stain intensely positive with a monoclonal antibody specic for Neospora caninum. Avidinbiotin complex immunoperoxidase immunohistochemistry, hematoxylin counterstain. Bar 18.25 m.

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pirates of the skin lesions demonstrated purulent inammation with numerous tachyzoites. Histologic ndings from a skin biopsy were similar to those for dog No. 1, and N. caninum tachyzoites were conrmed with immunohistochemistry using the same protocol. Although the skin lesions and hind limb weakness resolved with clindamycin therapy, the dog became febrile, nonresponsive, and laterally recumbent 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. caninum, Sarcocystis canis, and T. gondii. Conrmed cases of dermatitis have been associated with Caryospora sp.6 and S. canis14 in young immunosuppressed dogs. Distinguishing features include the presence of caryocysts in cases of Caryospora infection and the presence of schizonts and division by endopolygeny in cases of Sarcocystis infection. Although leishmaniasis 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), Kansas (1982), Ohio (1988), Michigan (1989), Texas and Alabama (1991), and New York (2000) (P. M. Schantz, Centers for Disease Control, personal communication).23 Amastigotes containing kinetoplasts are pathognomonic for Leishmania. Toxoplasma gondii can infect virtually all body tissues; however, there are no reported cases of cutaneous toxoplasmosis in dogs.13 Although it is structurally similar to Neospora in cytologic smears and HE-stained sections, Toxoplasma can be differentiated by means of serology, electron microscopy, and immunohistochemistry. Although canine neosporosis typically presents as progressive 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 rst described in a 15-year-old mixed-breed dog from the United States with ulcerative and stulous cutaneous lesions that began ventrolateral to the anus.7 Postmortem lesions included interstitial pneumonia, hepatitis, leptomeningitis, myositis, interstitial nephritis, cyclitis, and granulocytic hyperplasia of the bone marrow. A 12-year-old Golden Retriever, 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 pseudotumoral 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 immunodeciency 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 It is not known whether the disease in adult dogs is due to reactivation of a congenital infection or is a recently acquired 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 cellular immunity. IL-12 stimulates the production of IFN by T cells and natural killer cells. Lymphocytes and phagocytic cells also secrete IL-10, which inhibits IL-12 and potentiates TH2 or humoral responses. This downregulation of the cellular 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 prednisone and various cytotoxic drugs. Reactivation of dormant neosporosis has been demonstrated experimentally with administration of glucocorticoids,10 which inhibit TH1 and enhance TH2 cytokine secretion.15 T cells are exquisitely more sensitive than B cells to the cytotoxic effects of drugs such as L-asparaginase, chlorambucil, and cytarabine.19,24 Therefore, immunosuppressive therapy in both of these dogs most likely preferentially suppressed cellular immunity, enabling the establishment of clinically apparent N. caninum infection. Neospora caninum should be included on the list of differential diagnoses for ulcerative and pyogranulomatous dermatitis, 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 conrmed with immunohistochemistry. Acknowledgements. We thank Dr. D. S. Lindsay (Department of Biomedical Sciences and Pathobiology, VirginiaMaryland Regional College of Veterinary Medicine) and Dr. E. J. Dubovi (New York State Diagnostic Laboratory, Cornell 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 Weinstock (Pennsylvania State University) and Dr. Evelyn W. Polack (Department of Pathology, Cornell University). We are also 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 Pathology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, and Dr. E. J. Dubovi, New York State Diagnostic Laboratory, Cornell University, Ithaca, NY.

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 neosporosis in dogs. Vet Rec 139:439443. 3. Barber JS, Trees AJ: 1998, Naturally occurring vertical transmission of Neospora caninum in dogs. Int J Parasitol 28:5764. 4. Baszler TV, Long MT, McElwain TF, Mathison BA: 1999, Interferon-gamma and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice. Int J Parasitol 29: 16351646. 5. Cole RA, Lindsay DS, Dubey JP, Blagburn BL: 1993, Detection of Neospora caninum in tissue sections using a murine monoclonal antibody. J Vet Diagn Invest 5:579584. 6. Dubey JP, Black SS, Sangster LT, et al.: 1990, Caryospora-associated 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 Assoc 192:12691285. Dubey JP, Koestner A, Piper RC: 1990, Repeated transplacental transmission of Neospora caninum in dogs. J Am Vet Med Assoc 197:857860. Dubey JP, Lindsay DS: 1989, Transplacental Neospora caninum infection in dogs. Am J Vet Res 50:15781579. Dubey JP, Lindsay DS: 1990, Neosporosis in dogs. Vet Parasitol 36:147151. Dubey JP, Lindsay DS: 1993, Neosporosis. Parasitol Today 9: 452458. Dubey JP, Lindsay DS: 1996, A review of Neospora caninum and neosporosis. Vet Parasitol 67:159. Dubey JP, Metzger FL Jr, Hattel AL, et al.: 1995, Canine cutaneous neosporosis: clinical improvement with clindamycin. Vet Dermatol 6:3743. Dubey JP, Slife LN, Speer CA, et al.: 1991, Fatal cutaneous and visceral infection in a Rottweiler dog associated with a Sarcocystis-like protozoon. J Vet Diagn Invest 3:7275. Elenkov IJ, Papanicolaou DA, Wilder RL, Chrousos GP: 1996, Modulatory effects of glucocorticoids and catecholamines on human interleukin-12 and interleukin-10 production: clinical implications. Proc Assoc Am Physicians 108:374381. Eperon S, Bronnimann K, Hemphill A, Gottstein B: 1999, Sus ceptibility of B-cell decient C57GL/6 ( MT) mice to Neospora caninum infection. Parasite Immunol 21:225236. Ferrer L, Rabanal R, Fondevila D, et al.: 1988, Skin lesions in canine leishmaniasis. J Small Anim Pract 29:381388.

18. Fritz D, George C, Dubey JP: 1997, Neospora caninum: associated nodular dermatitis in a middle-aged dog. Canine Pract 22:2124. 19. Kazmers IS, Daddona PE, Dalke AP, Kelley WN: 1983, Effect of immunosuppressive agents on human T and B lymphoblasts. Biochem Pharmacol 32:805810. 20. Khan IA, Schwartzman JD, Fonseka S, Kasper LH: 1997, Neospora 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 Neospora caninum. Parasite Immunol 20:519526. 22. McAllister MM, Dubey JP, Lindsay DS, et al.: 1998, Dogs are denitive hosts of Neospora caninum. Int J Parasitol 28:14731478. 23. Monti DJ: 2000, Hunters hounded as leishmaniasis is diagnosed in Foxhounds. J Am Vet Med Assoc 216:1887, 1890. 24. Ohnuma T, Arkin H, Holland JF: 1980, Differences in chemotherapeutic susceptibility of human T-, B-, and non-T-/non-Blymphocytes in culture. Recent Results Cancer Res 75:6167. 25. Perl S, Harrus S, Satuchne (Goldvaser) C, et al.: 1998, Cutaneous neosporosis in a dog in Israel. Vet Parasitol 79:257261. 26. Poli A, Mancianti F, Carli MA, et al.: 1998, Neospora caninum infection in a Bernese Cattle Dog from Italy. Vet Parasitol 78: 7985. 27. Ruehlmann D, Podell M, Oglesbee M, Dubey JP: 1995, Canine neosporosis: a case report and literature review. J Am Anim Hosp Assoc 31:174183.

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 ghting 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 maltophilia 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 previously been classied as Pseudomonas maltophilia and Xanthomonas maltophilia. This bacterium was transferred to the new genus Stenotrohomonas because of comparative enzymology data, results of DNAribosomal RNA hybridization studies, guanine/cytosine content, and fatty acid composition.16 Despite earlier reports that S. maltophilia had limited pathogenicity,2 the bacterium has recently gained imFrom 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 pneumonia,7,10 urocystitis,17 and wound infection.18 Human patients considered at risk for S. maltophilia infections include the severely debilitated or immunosuppressed, those receiving 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 important in the pathogenesis of infection.1,15 Although S. maltophilia is now recognized as a signicant human pathogen, the role of this bacterium in diseases of animals is less clear. In 1 report, S. maltophilia was considered to be the cause of eece rot in sheep.9 Stenotrophomonas maltophilia has been isolated from sh,6 lizards,

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