Inflammatory and Immune-Mediated Myopathies, What Do We Know?

Inflammatory myopathies or myositis encompass diseases characterized by the presence of inflammatory cellular infiltrates, mainly polymorphonuclear cells and/or lymphocytes, in muscle. This is in contrast to most forms of muscle disease characterized by myodegeneration that results in macrophage infiltration. Inflammatory myopathies could have infectious or noninfectious causes. Noninfectious causes consist of primary (genetic, autoimmune) or acquired immune-mediated disease. Focal, multifocal or diffuse, acute or recurrent forms of disease can occur. This article will mainly review immune-mediated myopathies in horses. Myositis directly caused by infection such as Clostridium spp and others will not be discussed here.

Polyautoimmunity manifest as inflammatory myopathy, uveitis, and progressive cutaneous depigmentation in a mixed breed dog: a case report.

BACKGROUND: Polyautoimmunity is the expression of more than one autoimmune disease in a single patient. This report documents polyautoimmunity in a mixed breed dog with concurrent uveitis, cutaneous depigmentation, and inflammatory myopathy. CASE PRESENTATION: A 1-year-old male neutered mixed breed dog was presented for progressive generalized leukotrichia and leukoderma, bilateral panuveitis, and masticatory muscle atrophy. The latter progressed to myositis of lingual, pharyngeal, and masticatory muscles confirmed by biopsy. Temporalis muscle was completely replaced by adipose and fibrous tissue, and necrotic myofibers with extensive infiltration of mononuclear cells indicated active myositis of lingual muscle. Skin biopsies showed severe melanin clumping in epidermis, hair follicles, and hair shafts, and perifollicular pigmentary incontinence. Uveitis, depigmentation, and myositis affecting the masticatory, pharyngeal, and tongue muscles were diagnosed based on clinical, histological, and laboratory findings. CONCLUSIONS: To the authors' knowledge, this is the first report of concurrent uveitis, progressive cutaneous depigmentation, and inflammatory myopathy in a dog.

Muscular Sarcocystosis in Sheep Associated With Lymphoplasmacytic Myositis and Expression of Major Histocompatibility Complex Class I and II.

Sarcocystosis is a protozoal disease affecting a wide range of animals. The aims of this study were to characterize the following in sheep: (1) the muscle pathology in Sarcocystis infection, (2) the inflammatory infiltrate and its relationship to severity of infection, and (3) immune markers expressed by parasitized muscle fibers and parasitic cysts. Skeletal muscle samples from 78 sheep slaughtered in southern Italy were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Polymerase chain reaction (PCR) and sequencing were used for Sarcocystis species identification. All 40 muscle samples tested were PCR-positive for Sarcocystis tenella. Histologically, cysts were identified in 76/78 cases (97%), associated with an endomysial infiltrate of lymphocytes and plasma cells. The T cells were predominantly CD8+, with fewer CD4+ or CD79α+ cells. Eosinophils were absent. Notably, sarcolemmal immunopositivity for major histocompatibility complex (MHC) I and II was found in 76/78 cases (97%) and 75/78 cases (96%), respectively, both in samples with and in those without evident inflammatory infiltrate. The number of cysts was positively correlated with inflammation. In addition, MHC I was detected in 55/78 cyst walls (72%), and occasionally co-localized with the membrane-associated protein dystrophin. The findings suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II. The possible role of MHC I and II in the inflammatory response and on the cyst wall is also discussed.

Outbreaks of severe myositis in cultured white sturgeon (Acipenser transmontanus L.) associated with Streptococcus iniae.

Outbreaks of an infectious disease affecting cultured white sturgeon (Acipenser transmontanus) were investigated. Clinical signs included erratic swimming, arching of the back and mortality. Necropsy findings included poorly demarcated yellow to dark-red and friable lesions in the epaxial muscle, ulcerative skin lesions and haemorrhages in the swim bladder and coelomic wall. Histological evaluation revealed areas of necrotizing and heterophilic myositis with aggregates of bacterial cocci. The lumen of blood vessels in the dermis, under ulcerated areas, and in the posterior kidney, was occluded by fibrin thrombi. Aggregates of Gram-positive cocci were observed in the muscle lesions and within the fibrin thrombi in the dermis and kidney. Genetically homogeneous Streptococcus iniae strains were recovered from affected fish from different outbreaks. The isolates shared high degree of similarity at gene locus (gyrB) with previously characterized S. iniae from cultured fish in California, confirming the emergence of this particular strain of S. iniae in US aquaculture.

Septicemic pasteurellosis causing peracute death and necrotizing myositis in a beef heifer calf (Bos taurus) in Alberta, Canada.

Septicemic pasteurellosis is an acute and fatal bacterial disease of cattle and wild ungulates caused by certain serotypes of Pasteurella multocida. Here we report a single case of septicemic pasteurellosis in a 6-month-old, Red Angus heifer from a cow-calf operation in Alberta, Canada. Postmortem examination revealed necrotizing and hemorrhagic myositis, fibrinous pericarditis and multisystemic bacterial emboli. Pasteurella multocida was isolated from muscle in pure culture, and the capsular antigen group was identified as serogroup B using polymerase chain reaction. To the best of our knowledge, this is the first reported case of septicemic pasteurellosis in beef cattle in Canada. Key clinical message: Veterinary practitioners and diagnosticians should include septicemic pasteurellosis on their list of differential diagnoses when they encounter similar presentations of peracute death and severe necrotizing myositis in cattle in Canada.

Pasteurellose septicémique causant la mort suraiguë et une myosite nécrosante d'une génisse d'embouche ( Bos taurus ) en Alberta, Canada. La pasteurellose septicémique est une maladie bactérienne aiguë et fatale des bovins et des ongulés sauvages causée par certains sérotypes de Pasteurella multocida. Nous rapportons ici un cas unique de pasteurellose septicémique chez une génisse Red Angus âgée de 6 mois provenant d'un élevage vache-veau en Alberta, Canada. L'examen post-mortem a révélé une myosite nécrosante et hémorragique, une péricardite fibrineuse et des embolies bactériennes multi-systémiques. Pasteurella multocida fut isolé du muscle en culture pure, et l'antigène de groupe capsulaire fut identifié comme étant le sérogroupe B à l'aide de la réaction d'amplification en chaîne utilisant la polymérase. À notre connaissance ceci représente le premier cas rapporté de pasteurelle septicémique chez des bovins d'embouche au Canada.Message clinique clé:Les vétérinaires praticiens et les diagnosticiens devraient inclure la pasteurellose septicémique sur leur liste de diagnostic différentiel lorsqu'ils rencontrent des présentations similaires de mortalité suraiguë et de myosite nécrosante sévère chez des bovins au Canada.(Traduit par Dr Serge Messier).

Polyphasic Rhabdomyositis in California Sea Lions (Zalophus Californianus): Pathology and Potential Causes.

A myositis syndrome has been recognized for more than a decade in California sea lions (CSLs; Zalophus californianus) but a detailed description of the lesions and potential causes of this condition is lacking. The tissues of 136 stranded CSLs with rhabdomyositis were examined. Rhabdomyositis was considered incidental in 67% (91/136) of the CSLs, and a factor contributing to the animal stranding (significant rhabdomyositis) in 33% (45/136). Of the 91 cases with incidental rhabdomyositis, lesions consisted of a few small foci of lymphohistiocytic inflammation. Of the 45 cases with significant rhabdomyositis, 28 (62%) also presented with major comorbidities such as leptospirosis (2 animals) and domoic acid toxicosis (6 animals), whereas 17 (38%) had severe polyphasic rhabdomyositis as the only major disease process associated with mortality. In these animals, most striated muscles had multiple white streaks and diffuse atrophy. Microscopically, there was myofiber necrosis surrounded by lymphocytes and histiocytes admixed with areas of myofiber regeneration, and/or moderate to severe rhabdomyocyte atrophy usually adjacent to intact Sarcocystis neurona cysts. At the interface of affected and normal muscle, occasional T lymphocytes infiltrated the sarcoplasm of intact myocytes, and occasional myofibers expressed MHCII proteins in the sarcoplasm. S. neurona antibody titers and cyst burden were higher in animals with significant polymyositis antibody titers of (26125 ± 2164, 4.5 ± 1.2 cysts per section) and active myonecrosis than animals with incidental rhabdomyositis antibody titers of (7612 ± 1042, 1.7 ± 0.82 cysts per section). The presented findings suggest that S. neurona infection and immune-mediated mechanisms could be associated with significant polyphasic rhabdomyositis in CSLs.

Granulomatous Necrotizing Myositis in Swine Affected by Porcine Circovirus Disease.

Porcine circovirus type 2 (PCV2) is associated with multiple clinical syndromes in pigs, known as porcine circovirus diseases. This work describes an outbreak of porcine circovirus diseases with severe lesions affecting the skeletal muscle. Ninety-two pigs had apathy, weight loss, and diarrhea over a clinical course of 7 to 10 days. Approximately 30 of the pigs had stiff gait, muscle weakness, hind limb paresis, and recumbency. Twelve of the 92 pigs were necropsied, and 4 had pale discoloration of skeletal muscles with microscopic lesions of granulomatous necrotizing myositis. Immunohistochemistry of skeletal muscle showed that PCV2 antigen was located primarily in the cytoplasm and nuclei of macrophages, lymphocytes, and multinucleated giant cells, with a lower amount in the cytoplasm of endothelial cells, necrotic fibers, and satellite cells. Affected muscle samples were polymerase chain reaction-positive for PCV2 and the amplicon exhibited 99% identity with sequences belonging to the PCV2b genotype. Locomotor clinical signs and granulomatous necrotizing myositis should be considered as another expression of PCV2 infection in pigs.

Inflammatory Myopathy in Horses With Chronic Piroplasmosis.

Horses affected by chronic piroplasmosis may develop poor performance and muscle atrophy. Here we investigate the pathological and immunopathological aspects of myopathy occurring in chronic equine piroplasmosis. The study included 16 horses serologically positive for equine piroplasms presenting with clinical signs and supporting serum biochemical evidence of a myopathy. Skeletal muscle was evaluated by histopathology, immunohistochemistry, indirect immunofluorescence, and molecular detection of piroplasms and inflammatory cytokines in skeletal muscle. Histologic lesions included muscle fiber atrophy (100% of cases), degenerative changes (13/16, 81%), and perivascular perimysial and endomysial lymphocytic infiltrates (81% of cases). In 15 cases (94%), muscle fibers had strong immunostaining for major histocompatibility complex classes I and II. T lymphocyte populations were mainly CD3+, CD8+, and CD4+ in equal proportions, with a lower number of CD79α+ cells. The serum from affected horses was tested by indirect immunofluorescence for binding of IgG, IgM, or IgA to sections of normal equine muscle to detect circulating autoantibodies against muscle antigen(s). In all cases, distinct sarcolemmal staining was detected in sections incubated with serum from affected horses, in contrast to sections incubated with phosphate-buffered saline or equine control sera. Reverse transcription polymerase chain reaction (RT-PCR) testing of muscles from affected animals revealed a significant increase of interferon-γ, interleukin-12, and tumor necrosis factor-α gene expression compared to healthy controls. Theileria equi or Babesia caballi was not detected in samples of affected muscle by RT-PCR. Thus, inflammatory myopathy associated with equine piroplasmosis may involve an autoimmune pathogenesis with upregulation of inflammatory cytokines that may cause myofiber atrophy and degeneration.

High prevalence of chigger mite infection in a forest-specialist frog with evidence of parasite-related granulomatous myositis.

Amphibians are hosts for a wide variety of micro- and macro-parasites. Chigger mites from the Hannemania genus are known to infect a wide variety of amphibian species across the Americas. In Chile, three species (H. pattoni, H. gonzaleacunae and H. ortizi) have been described infecting native anurans; however, neither impacts nor the microscopic lesions associated with these parasites have been described. Here, we document 70% prevalence of chigger mite infection in Eupsophus roseus and absence of infection in Rhinoderma darwinii in the Nahuelbuta Range, Chile. Additionally, we describe the macroscopic and microscopic lesions produced by H. ortizi in one of these species, documenting previously undescribed lesions (granulomatous myositis) within the host's musculature. These findings highlight that further research to better understand the impacts of chigger mite infection on amphibians is urgently required in Chile and elsewhere.

An epidemiological investigation of an idiopathic myopathy in hunting dogs in New Zealand.

AIM: To conduct an epidemiological investigation of an idiopathic myopathy, known as "Go Slow" (GSM), which was initially recognised in dogs used for pig hunting. A secondary aim was to describe the hunting activities, diet and health of dogs used for pig hunting in New Zealand. METHODS: A retrospective cohort study was conducted between June 2014-June 2017. Cases of GSM in dogs were diagnosed by veterinarians using a combination of clinical history, physical examination findings, serum biochemistry and/or skeletal muscle histology. A telephone interview was conducted with the owner or primary veterinarian to provide information regarding the dog's diet and exercise over the 7 days preceding the onset of clinical signs. In August 2015, a separate online survey of owners of dogs used for pig hunting was conducted to characterise the normal hunting activities, diet and health of these dogs. RESULTS: A total of 86 cases of GSM were recruited, of which 58 (67%) were pig hunting dogs, 16 (19%) pet dogs and 12 (14%) working farm dogs. Cases were most commonly reported in the upper North Island, and 65 (76 (95% CI=67-85)%) were from the Northland region. Processed commercial dog food had been fed to 93 (95% CI=88-98)% of affected dogs. Ingestion of raw, frozen or cooked wild pig in the preceding week was reported for 76 (88 (95% CI=82-95)%) dogs with the myopathy. In the survey of owners of healthy pig hunting dogs, 203 eligible responses were received; pig hunting was reported to most commonly occur in Northland (20.2%), Waikato (22.3%) and Bay of Plenty (23.2%) regions. Commercial dog food was fed to 172 (85 (95% CI=80-90)%) of the dogs included in this survey, and 55 (27 (95% CI=20-33)%) had eaten wild pig in the preceding week. The most common reported health problem in pig hunting dogs was traumatic wounds. CONCLUSIONS: Cases of GSM were most commonly recognised in dogs used for pig hunting, but also occurred in pet and working farm dogs. The disease was most frequently reported in the upper North Island of New Zealand and ingestion of wild pig was a consistent feature in cases of this myopathy. CLINICAL RELEVANCE: To minimise the risk of dogs developing this myopathy, it would seem prudent to avoid feeding any tissues from wild pigs to dogs in areas where the disease is known to occur.

MASTICATORY MUSCLE MYOSITIS IN A GRAY WOLF (CANIS LUPUS).

A 10-yr-old male, neutered gray wolf ( Canis lupus ) was presented for atrophy of the temporalis and masseter muscles. Clinical signs and magnetic resonance imaging were consistent with a myopathy. Positive serology for antibody titers directed against Type 2M myofibers, and the observation of a mixed mononuclear inflammatory cell infiltrate along with eosinophils and neutrophils within the temporalis muscle, were diagnostic for masticatory muscle myositis. Importantly, protozoal myositis was excluded based on other clinicopathologic data. The case highlights the potential for immune-mediated polymyositis in canids other than the domesticated dog ( Canis lupus familaris). Additionally, awareness of a diet in which raw meat is used should prompt a thorough investigation for an underlying infectious myositis in the gray wolf.

Sarcocystis caninum and Sarcocystis svanai n. spp. (Apicomplexa: Sarcocystidae) Associated with Severe Myositis and Hepatitis in the Domestic Dog (Canis familiaris).

There are several reports of Sarcocystis sarcocysts in muscles of dogs, but these species have not been named. Additionally, there are two reports of Sarcocystis neurona in dogs. Here, we propose two new names, Sarcocystis caninum, and Sarcocystis svanai for sarcocysts associated with clinical muscular sarcocystosis in four domestic dogs (Canis familiaris), one each from Montana and Colorado in the USA, and two from British Columbia, Canada. Only the sarcocyst stage was identified. Most of the sarcocysts identified were S. caninum. Sarcocysts were studied using light microscopy, transmission electron microscopy (TEM), and polymerase chain reaction. Based on collective results two new species, S. caninum and S. svanai were designated. Sarcocystis caninum and S. svanai were structurally distinct. Sarcocystis caninum sarcocysts were up to 1.2 mm long and up to 75 µm wide. By light microscopy, the sarcocyst wall was relatively thin and smooth. By TEM, the sarcocyst wall was "type 9", 1-2 µm thick, and contained villar protrusions that lacked microtubules. Bradyzoites in sections were 7-9 µm long. Sarcocysts of S. svanai were few and were identified by TEM. Sarcocystis svanai sarcocysts were "type 1", thin walled (< 0.5 µm), and the wall lacked villar protrusions but had tiny blebs that did not invaginate. DNA was extracted either from infected frozen muscle biopsies or formalin-fixed paraffin-embedded sections. Dogs were either singly infected with S. caninum or multiply co-infected with S. caninum and S. svanai (the result of a mixed infection) based on multilocus DNA sequencing and morphology. BLASTn analysis established that the sarcocysts identified in these dogs were similar to, but not identical to Sarcocystis canis or Sarcocystis arctosi, parasites found to infect polar bears (Ursus maritimus) or brown bears (Ursus arctosi), respectively. However, the S. caninum sequence showed 100% identify over the 18S rRNA region sequenced to that of S. arctica, a parasite known to infect Arctic foxes (Vulpes lagopus).

Clinical features of idiopathic inflammatory polymyopathy in the Hungarian Vizsla.

BACKGROUND: A retrospective study of the clinicopathological features of presumed and confirmed cases of idiopathic inflammatory polymyopathy in the Hungarian Vizsla dog and guidelines for breeding. RESULTS: 369 medical records were reviewed (1992-2013) and 77 Hungarian Vizslas were identified with a case history consistent with idiopathic inflammatory polymyopathy. Inclusion criteria were: group 1 (confirmed diagnosis); histopathology and clinical findings compatible with an inflammatory polymyopathy and group 2 (probable diagnosis); clinical findings compatible with a polymyopathy including dysphagia, sialorrhea, temporal muscle atrophy, elevated serum creatine kinase (CK) activity, and sufficient clinical history to suggest that other neuromuscular disorders could be ruled out. Some group 2 dogs had muscle biopsy, which suggested muscle disease but did not reveal an inflammatory process. The mean age of onset was 2.4 years; male dogs were slightly overrepresented. Common presenting signs were dysphagia, sialorrhea, masticatory muscle atrophy, and regurgitation. Common muscle histopathological findings included degenerative and regenerative changes, with multifocal mononuclear cell infiltration with lymphoplasmacytic myositis of variable severity. A positive response to immunosuppressive treatment supported an immune-mediated aetiology. The mean age at death and survival time were 6.4 and 3.9 years, respectively. Recurrence of clinical signs and aspiration pneumonia were common reasons for euthanasia. CONCLUSIONS: Diagnosis of Vizsla idiopathic inflammatory polymyopathy can be challenging due to lack of specific tests, however the presence of dysphagia, regurgitation and masticatory muscle atrophy in this breed with negative serological tests for masticatory muscle myositis and myasthenia gravis, along with muscle biopsies suggesting an inflammatory process, support the diagnosis. However, there is an urgent need for a more specific diagnostic test. The average of inbreeding coefficient (CoI) of 16.3% suggests an increased expression of a Dog Leukocyte Antigen Class II haplotype, leading to an increased disease risk. The prognosis remains guarded, as treatment can only manage the disease. Recurrence of clinical signs and perceived poor quality of life are the most common reasons for humane euthanasia.

Dynamic exophthalmos and lateral strabismus in a dog caused by masticatory muscle myositis.

CASE DESCRIPTION: A 5.5-year-old neutered male mixed-breed dog was presented for evaluation of a 5-month history of deviation of the right globe upon opening the mouth and a 1-month history of conjunctivitis in the same eye. Clinical findings, diagnostic imaging results, treatment and follow-up are described. CLINICAL FINDINGS: When the mouth was opened, the right globe deviated rostrally and laterally. There was no pain or resistance to opening the mouth, or resistance to retropulsion of the globe. No other abnormalities were observed. Computed tomography was performed, revealing a contrast-enhancing region associated with the right masseter muscle impinging into the right orbit; this was more pronounced with the mouth open. Cytology revealed a small number of mildly to moderately atypical mesenchymal cells. Histopathology was consistent with polyphasic myositis, with a predominance of lymphocytes and plasma cells. No infectious agents were identified. Serum antibody titers for Toxoplasma gondii and Neospora caninum were negative. Serum titers for 2 M antibody were positive at 1:500, consistent with a diagnosis of masticatory muscle myositis. TREATMENT AND OUTCOME: Therapy with prednisone was initiated. During a follow-up period of 5 months, there was no recurrence of clinical signs, and the dose of prednisone was reduced by 25%. CLINICAL SIGNIFICANCE: To the authors' knowledge, this is the first reported case of masticatory muscle myositis causing dynamic exophthalmos and strabismus in a dog.

MANAGEMENT OF ACUTE RENAL FAILURE WITH DELAYED HYPERCALCEMIA SECONDARY TO SARCOCYSTIS NEURONA-INDUCED MYOSITIS AND RHABDOMYOLYSIS IN A CALIFORNIA SEA LION (ZALOPHUS CALIFORNIANUS).

A 3-yr-old captive-born California sea lion (Zalophus californianus) developed Sarcocystis neurona-induced myositis and rhabdomyolysis that led to acute renal failure. The sea lion was successfully managed with fluid therapy, antiprotozoals, antibiotics, anti-inflammatories, antiemetics, gastroprotectants, and diuretics, but developed severe delayed hypercalcemia, a syndrome identified in humans after traumatic or exertion-induced rhabdomyolysis. Treatment with calcitonin was added to the management, and the individual recovered fully. The case emphasizes that animals with rhabdomyolysis-induced renal failure risk developing delayed hypercalcemia, which may be life threatening, and calcium levels should be closely monitored past the resolution of renal failure.

Use of MRI for the early diagnosis of masticatory muscle myositis.

The medical records of two dogs that were diagnosed with masticatory muscle myositis (MMM) were reviewed. The reported clinical signs included intense pain when opening the mouth and restricted jaw movement. MRI detected widespread, symmetrical, and inhomogeneously hyperintense areas in the masticatory muscle. Electromyography (EMG) demonstrated severe and spontaneous pathologic activity in the temporal and masseter muscles. With early therapeutic treatment, remission of symptoms occurred within 2 mo, and no relapses were observed for the subsequent 2 yr. The gold standard for the diagnosis of MMM is the 2M antibody test, but the purpose of this study was to evaluate the use of MRI as an accurate and efficient diagnostic tool for the initiation of early therapy for the treatment of muscle myositis.

Bovine sarcocystosis: Sarcocystis species, diagnosis, prevalence, economic and public health considerations, and association of Sarcocystis species with eosinophilic myositis in cattle.

Infections by Sarcocystis in cattle are ubiquitous worldwide. There is considerable debate concerning the identity of Sarcocystis spp. in cattle. Proper diagnosis of Sarcocystis spp. is important to assess their economic and public health importance. Currently there are seven named species: Sarcocystis hirsuta, Sarcocystis cruzi, Sarcocystis hominis, Sarcocystis bovifelis, arcocystis heydorni, Sarcocystis bovini and Sarcocystis rommeli. Additionally, there are unnamed Sarcocystis spp. Two species, S. hominis and S. heydorni, are zoonotic. One out of seven species (S. hirsuta, contracted from cats) forms macroscopic cysts which can be visible during carcass inspection. Current molecular characterization is based on DNA extracted from sarcocysts from naturally infected cattle because DNA was not characterized from tissues of experimentally infected cattle or feces of experimentally infected definitive hosts. Sarcocystis cruzi (transmitted via canids) is recognized as the most pathogenic species and it causes abortion, low milk yield, poor body growth, and outbreaks of clinical sarcocystosis and death. Additionally, Sarcocystis infections have been linked to an inflammatory condition of striated muscles termed bovine eosinophilic myositis (BEM). Cattle affected by BEM appear clinically normal. Diagnosis of BEM at slaughter occurs when inspecting the carcass surface, or once the carcass has been divided into prime cuts or quarters. Sex and breed have no apparent influence on prevalence of BEM. The condition evidently occurs with equal frequency in steers, cows, and heifers. Virtually all striated muscles can be affected including skeletal muscles, the muscles of the eye, larynx, and the heart. In the USA, regulations require condemnation of BEM-affected parts, or (in severe cases) the entire carcass. These aesthetic considerations result in economic losses. Cattle experimentally infected with Sarcocystis did not have BEM at slaughter. Here, we review the status of Sarcocystis spp. and BEM in cattle including prevalence, lesions, epidemiology, and association of BEM with different species of Sarcocystis.

Identification of Bartonella henselae in 2 cats with pyogranulomatous myocarditis and diaphragmatic myositis.

Most cats infected with Bartonella henselae remain outwardly healthy carriers for years; however, self-limiting fever, transient anemia, neurologic dysfunction, lymphadenopathy, reproductive disorders, aortic valvular endocarditis, and neutrophilic myocarditis have been described in experimentally or naturally infected cats. Two cats in a North Carolina shelter died with pyogranulomatous myocarditis and diaphragmatic myositis. Bacteria were visualized in the lesions by Warthin-Starry silver impregnation and by B. henselae immunohistochemistry. B. henselae DNA was amplified and sequenced from the heart of 1 cat and from multiple tissue samples, including heart and diaphragm, from the second cat. This study supports a potential association between B. henselae and what has been historically described as "transmissible myocarditis and diaphragmitis" of undetermined cause in cats.

Granulomatous myositis associated with a novel alveolate pathogen in an adult southern leopard frog (Lithobates sphenocephalus).

Since 1999, infections with an incompletely characterized alveolate protozoan variously reported as a Dermocystidium-like organism, a Perkinsus-like agent, and Dermomycoides sp. have been associated with mortality events in tadpoles of ranid frogs from the USA. However, disease or mortality events due to this organism have not been described in post-metamorphic animals. We describe infection with a morphologically similar protozoan presenting itself as a leg mass in a free-ranging adult southern leopard frog Lithobates sphenocephalus. Using histological examination, we found a mass within skeletal muscle; this mass was composed of macrophages with intracytoplasmic, thick-walled, 4 to 6 µm in diameter, spherical basophilic protozoal organisms that exhibited green autofluorescence with epiflorescence illumination. Using transmission electron microscopy, organism cell walls were found to have electron-dense plates that, when viewed by scanning electron microscopy, were reminiscent of the thecal plates of dinoflagellates. Additional morphologic and molecular phylogenetic research is needed to resolve the taxonomic status of this organism.

Myonecrosis by Clostridium septicum in a dog, diagnosed by a new multiplex-PCR.

Clostridial myositis is an acute, generally fatal toxemia that is considered to be rare in pet animals. The present report describes an unusual canine clostridial myositis that was diagnosed by a new multiplex-PCR (mPCR) designed for simultaneous identification of Clostridium sordellii, Clostridium septicum, Clostridium perfringens type A, Clostridium chauvoei, and Clostridium novyi type A. A ten-month-old male Rottweiler dog, that had displayed lameness and swelling of the left limb for 12 h, was admitted to a veterinary hospital. The animal was weak, dyspneic and hyperthermic, and a clinical examination indicated the presence of gas and edema in the limb. Despite emergency treatment, the animal died in only a few minutes. Samples of muscular tissue from the necrotic area were aseptically collected and plated onto defibrinated sheep blood agar (5%) in anaerobic conditions. Colonies suggestive of Clostridium spp. were submitted to testing by multiplex-PCR. Impression smears of the tissues, visualized with Gram and also with panoptic stains, revealed long rod-shaped organisms, and specimens also tested positive using the fluorescent antibody technique (FAT). The FAT and mPCR tests enabled a diagnosis of C. septicum myonecrosis in the dog.