Comprehensive immunohistochemical studies on canine necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE), and granulomatous meningoencephalomyelitis (GME)

In dogs, there are several idiopathic meningoencephalitides, such as necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE), and granulomatous meningoencephalomyelitis (GME). Although they are often assumed to be immune mediated, the etiology of these diseases remains elusive. In this study, the histopathology of the lesions caused by these conditions and the inflammatory cell populations produced in response to them were examined among dogs affected with GME, NME, or NLE to understand their pathogeneses. The brain tissues of dogs with NME (n = 25), NLE (n = 5), or GME (n = 9) were used. The inflammatory cells were identified by immunohistochemistry using antibodies against CD3, IgG, CD20, CD79acy, and CD163. In NME and NLE, malacic changes were located in the cerebral cortex, as well as the cerebral white matter and thalamus, respectively. The distribution of the brain lesions in NME and NLE was breed specific. In GME, granulomatous lesions that were mostly composed of epithelioid macrophages were observed in the cerebral white matter, cerebellum, and brainstem. Although the proportions of IgG-, CD20-, and CD79acy-positive cells (B cells) were not significantly different among the GME, NME, and NLE lesions, that of CD3-positive cells (T cells) was increased in GME. In NME and NLE, CD163-positive cells (macrophages) had diffusely infiltrated the cerebral cortex and white matter, respectively. However, in GME, CD163-positive cells accumulated around the blood vessels in the cerebral and cerebellar white matter. The distributions of these lesions were quite different among GME, NME, and NLE, whereas there were no marked differences in the proportions of inflammatory cells.     

Transglutaminase 2: a novel autoantigen in canine idiopathic central nervous system inflammatory diseases

Necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE) and granulomatous meningoencephalomyelitis (GME) are common idiopathic inflammatory central nervous system (CNS) diseases with unknown etiology in dogs. We previously showed that IgG autoantibodies in the cerebrospinal fluid (CSF) of NME cases reacted to unknown brain proteins as well as to glial fibrillary acidic protein (GFAP). In the present report, we evaluated the autoantibodies against transglutaminase2 (TG2) in the canine CNS diseases. CSF samples obtained from dogs with NME (n=19), NLE (n=7), GME (n=11) and miscellaneous CNS diseases (n=12) were subjected. CSFs from 20 healthy dogs were used as controls. Indirect fluorescent antibody test on the canine cerebrum revealed astrocyte-binding IgG in the CSF of NME. After absorption of the CSF with bovine GFAP, the CSF still possessed the reactivity to astrocytes. Double-color staining showed clear colocalization of the autoantibodies and anti-human TG2 rabbit polyclonal IgG. An immunoblot assay against human recombinant TG2 revealed anti-TG2 IgG in the CSF from dogs with NME, NLE and GME. The CSF of canine idiopathic encephalitis cases, notably of NME, tended to show high ELISA OD values against human recombinant TG2 compared to healthy controls. The presence of anti-TG2 autoantibodies in the CSF may contribute to the elucidation of the etiology of canine NME, NLE and GME.     

Heritability and transmission analysis of necrotizing meningoencephalitis in the Pug

Necrotizing meningoencephalitis (NME) in the Pug is an invariably fatal disease with an early age of onset whose cause remains unknown. Breed predilection strongly suggests genetic component(s), and viral etiology proves negative in studied cases. The current study was undertaken as the first analysis of the heritable component(s) involved in NME in the Pug. Complete medical records, individual characteristics, and pedigree information were collected for 58 affected dogs with data pertaining to 4698 dogs analyzed. A high inbreeding coefficient with differences across gender and significant differences across coat color classes and variable expression was evident. Median onset age was 19 months and median survival time 23 days. Screening for herpes-, adeno-, and parvoviruses was negative. The data demonstrate a strong familial inheritance of NME in the Pug. This investigation provides parameters of disease from the largest Pug NME cohort analyzed to date and offers evidence of previously unrecognized familial inheritance.     

Evaluation of Brain Tissue or Cerebrospinal Fluid with Broadly Reactive Polymerase Chain Reaction for Ehrlichia,Anaplasma, Spotted Fever Group Rickettsia,Bartonella, and Borrelia Species in Canine Neurological Diseases (109 Cases)

Background: Vector‐transmitted microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, Bartonella, and Borrelia are commonly suspected in dogs with meningoencephalomyelitis (MEM), but the prevalence of these pathogens in brain tissue and cerebrospinal fluid (CSF) of dogs with MEM is unknown. Hypothesis/Objectives: To determine if DNA from these genera is present in brain tissue and CSF of dogs with MEM, including those with meningoencephalitis of unknown etiology (MUE) and histopathologically confirmed cases of granulomatous (GME) and necrotizing meningoencephalomyelitis (NME). Animals: Hundred and nine dogs examined for neurological signs at 3 university referral hospitals. Methods: Brain tissue and CSF were collected prospectively from dogs with neurological disease and evaluated by broadly reactive polymerase chain reaction (PCR) for Ehrlichia, Anaplasma, Spotted Fever Group Rickettsia, Bartonella, and Borrelia species. Medical records were evaluated retrospectively to identify MEM and control cases. Results: Seventy‐five cases of MUE, GME, or NME, including brain tissue from 31 and CSF from 44 cases, were evaluated. Brain tissue from 4 cases and inflammatory CSF from 30 cases with infectious, neoplastic, compressive, vascular, or malformative disease were evaluated as controls. Pathogen nucleic acids were detected in 1 of 109 cases evaluated. Specifically, Bartonella vinsonii subsp. berkhoffii DNA was amplified from 1/6 dogs with histopathologically confirmed GME. Conclusion and Clinical Importance: The results of this investigation suggest that microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, and Borrelia are unlikely to be directly associated with canine MEM in the geographic regions evaluated. The role of Bartonella in the pathogenesis of GME warrants further investigation.     

Correlation between fluorodeoxyglucose positron emission tomography and magnetic resonance imaging findings of non-suppurative meningoencephalitis in 5 dogs

This study characterized the [(18)F]2-deoxy-2-fluoro-D-glucose positron emission tomography (FDG-PET) findings of encephalitis in dogs and assessed the role of FDG-PET in the diagnosis of meningoencephalitis. The medical records, magnetic resonance (MR), and FDG-PET images of 3 dogs with necrotizing meningoencephalitis (NME), 1 dog with granulomatous meningoencephalitis (GME), and 1 dog with meningoencephalitis of unknown etiology (MUE) were reviewed. On the FDG-PET, glucose hypometabolism was identified in the dog with NME, whereas hypermetabolism was noted in the dog with GME. The T2-weighted images (WI) and fluid attenuated inversion recovery (FLAIR) images were characterized by hyperintensity, whereas the signal intensity of the lesions on the T1-WI images was variable. The metabolic changes on the brain FDG-PET corresponded well to the hyper- and hypointense lesions seen on the MR imaging. This type of tomography (FDG-PET) aided in the differentiation of different types of inflammatory meningoencephalitis when the metabolic data was combined with clinical and MR findings.     

Autoantibodies against glial fibrillary acidic protein (GFAP) in cerebrospinal fluids from Pug dogs with necrotizing meningoencephalitis

Cerebrospinal fluids (CSFs) from 9 Pug dogs with necrotizing meningoencephalitis (NME: Pug dog encephalitis) were examined to identify the antigens for anti-astrocyte autoantibodies. Each CSF exhibited a positive reaction to the cytoplasm of cultured canine astrocytes by an indirect fluorescent antibody test. In an immunoblotting analysis on normal canine brain proteins, eight of 9 CSFs showed a common band of 52 kDa, corresponding to glial fibrillary acidic protein (GFAP), and all of 9 CSFs reacted with purified bovine GFAP. From these results, GFAP is one of the common autoantigens in Pug dogs with NME. On the other hand, the reactivity of CSFs to chymotrypsin-digested bovine GFAP fragments were variable among dogs, indicating that the antibodies in the CSFs recognized different epitopes on GFAP.     

Prevalence of autoantibody in cerebrospinal fluids from dogs with various CNS diseases

To examine the prevalence of autoantibody in canine cerebrospinal fluids (CSFs), CSFs were collected from 14 healthy controls and 88 clinical cases with various diseases in the central nervous system (CNS), and were analyzed by an indirect fluorescence antibody test on frozen sections of the cerebrum from normal Beagle dogs. An anti-astrocyte autoantibody was detected in 31 clinical cases with titers ranging from 1:1 to >/=1:100. All tested cases with necrotizing meningoencephalitis (NME: n=22) and granulomatous meningoencephalitis (GME: n=3) possessed the anti-astrocyte autoantibody, while the autoantibody was negative in most cases with other inflammatory CNS diseases. The autoantibody was also detected in 4 of 12 cases with brain tumors. Hence, examination of the autoantibody in the canine CFS would be significant for diagnosing NME and/or GME, as well as for understanding peritumoral events in cases with brain tumors.     

A comparative pathological study on canine necrotizing meningoencephalitis and granulomatous meningoencephalomyelitis

Canine necrotizing meningoencephalitis (NME) and granulomatous meningoencephalomyelitis (GME) were compared pathologically. Gross observation exhibited lateral ventricular dilation and discoloration, malacia and/or cavitation of the cerebrum in NME. On the contrary, gross changes were milder in GME, except for occasional visible granulomatous mass formation. Histopathologically, the lesions of NME were distributed predominantly in the cerebral cortex and various degrees of inflammatory and necrotic changes were observed according to clinical stages. Besides, microscopic lesions of GME were mainly distributed in the white matter of the cerebrum, cerebellum and brainstem, which are characterized by perivascular cuffing, multiple granulomas and leptomeningeal infiltrates. Although macrophages and lymphocytes were predominant in the inflammatory lesions of both disorders, macrophages in GME transformed into epithelioid cells and exhibited more massive infiltration. Although lectin RCA-1-reactive cells were numerous in both disorders, lysozyme immunoreactive cells in NME were fewer than that in GME. Parenchymal infiltration of MAC387-positive cells was common in GME and limited in NME. The number of CD3-positive lymphocytes in the GME lesions tended to be greater than in NME, though the difference was not statistically significant. Morphological and immunohistochemical differences of the lesions, in particular, the characteristics of infiltrative macrophages may reflect these different pathogeneses of the two disorders.     

Impaired expression of excitatory amino acid transporter 2 (EAAT2) and glutamate homeostasis in canine necrotizing meningoencephalitis

To clarify the involvement of excitatory and inhibitory amino acids in canine necrotizing meningoencephalitis (NME), glutamate, aspartate, taurine and gamma-aminobutylic acid (GABA) were determined in the cerebrospinal fluids (CSF) from eight NME cases and ten healthy controls. NME dogs exhibited significantly higher concentrations of glutamate and aspartate than those in controls (p<0.001 and p<0.001, respectively), while there was no difference in taurine or GABA between the two groups. When fetal canine astrocytes were cultured for 24 hr in the presence of NME-CSF, supernatant concentrations of glutamate, aspartate and taurine were significantly elevated. Simultaneously, expression of excitatory amino acid transporter 2 (EAAT2) mRNA was significantly reduced in the astrocytes without change in EAAT1 mRNA. Hence, reduced expression of EAAT2 and impaired glutamate homeostasis may contribute to the pathogenesis of NME.     

Epidemiology of necrotizing meningoencephalitis in Pug dogs

BACKGROUND: Although the histopathologic features of necrotizing meningoencephalitis (NME) have been described previously, little information is available concerning the signalment, geographic distribution, seasonal onset, treatment, and survival of affected dogs. ANIMALS: Sixty Pugs with NME and 14 contemporaneous control Pugs with other intracranial diseases (non-NME group). METHODS: Pugs that were euthanized or died because of intracranial disease were prospectively obtained. All dogs had necropsy, histopathology, and testing for various infectious diseases and were subsequently divided into NME and non-NME groups. Signalment, geographic distribution, seasonal onset, treatment, and survival were compared between groups. RESULTS: In Pugs with NME, median age at onset of clinical signs was 18 months (range, 4-113 months). A greater proportion of female dogs were present in the NME group (40/60) compared with the control group (6/14). Pugs with NME had a significantly lower mean weight (7.81 kg) than control Pugs (9.79 kg) (P= .012). Mean survival in Pugs with NME was 93 days (range, 1-680 days), with dogs receiving any form of treatment living significantly longer than those that were not treated (P= .003). Anticonvulsive drugs were the only treatment significantly associated with longer survival (P= .003). CONCLUSIONS AND CLINICAL IMPORTANCE: NME appears to be a common cause of intracranial signs in Pugs, based on the high proportion of NME dogs reported in this population. Pugs with NME are most commonly young adult female dogs. Although further investigation is needed to determine the optimal treatment of NME, anticonvulsive drugs appear to beneficially affect duration of survival.     

Polymerase chain reaction analysis for viruses in paraffin-embedded myocardium from dogs with dilated cardiomyopathy or myocarditis

OBJECTIVE: To perform polymerase chain reaction (PCR) analysis on paraffin-embedded myocardium from dogs with dilated cardiomyopathy (DCM) and dogs with myocarditis to screen for canine parvovirus, adenovirus types 1 and 2, and herpesvirus. SAMPLE POPULATION: Myocardial specimens from 18 dogs with an antemortem diagnosis of DCM and 9 dogs with a histopathologic diagnosis of myocarditis were evaluated. PROCEDURE: Paraffin-embedded myocardial specimens were screened for viral genome by PCR analysis. Positive-control specimens were developed from cell cultures as well as paraffin-embedded tissue specimens from dogs with clinical and histopathologic diagnoses of viral infection with canine parvovirus, adenovirus types 1 and 2, and herpesvirus. The histologic characteristics of all myocardial specimens were classified regarding extent, location, and type of inflammation and fibrosis. RESULTS: Canine adenovirus type 1 was amplified from 1 specimen from a dog with DCM. Canine parvovirus, adenovirus type 2, and herpesvirus were not amplified from any myocardial specimens. Histologic analysis of specimens from dogs with DCM revealed variable amounts of fibrosis; myocardial inflammation was observed in 1 affected dog. Histopathologic analysis of specimens from dogs with myocarditis disclosed variable degrees of inflammation and fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE: Viral agents canine parvovirus, adenovirus types 1 and 2, and herpesvirus are not commonly associated with DCM or active myocarditis in dogs. Additional studies evaluating for nucleic acid from viruses that less commonly affect dogs or different types of infectious agents may be warranted to gain insight into the cause of DCM and myocarditis in dogs.     

A comparison of combination therapy (cyclosporine plus prednisolone) with sole prednisolone therapy in 7 dogs with necrotizing meningoencephalitis

Administration of immunosuppressive doses of glucocorticosteroids is the traditional primary treatment in necrotizing meningoencephalitis (NME) in dogs. However, response is variable and clinical signs often recur quickly with tapering dosage. Prognosis is poor and long-term therapy causes many complications. In the present study, we compared the long-term effects of combination (cyclosporine plus prednisolone) therapy with sole prednisolone therapy in management in dogs with NME. All NME cases in this study were examined with magnetic resonance imaging and cerebrospinal fluid analysis, and confirmed by histopathologic examination. The mean survival time of combination therapy group was 305.7 +/- 94.7 days. The mean survival time of sole prednisolone therapy group was 58.3 +/- 30.5 days. This case report demonstrates that combination treatment of cyclosporine with prednisolone is more effective in survival time than administration of only prednisolone in NME cases.     

Polymerase chain reaction (PCR) amplification of parvoviral DNA from the brains of dogs and cats with cerebellar hypoplasia

Cerebellar hypoplasia in cats is caused most commonly by an in utero or perinatal infection with feline panleukopenia virus (parvovirus). Cerebellar hypoplasia has been reported infrequently in dogs, but no viral etiology has been identified to date. DNA was extracted from archival, paraffin-embedded, cerebellar tissue from 8 cats and from 2 canine littermates with cerebellar hypoplasia, 2 canine littermates with cerebellar cortical abiotrophy, 6 dogs with congenital cerebellar vermal defects, 1 dog with congenital hydranencephaly, and 15 dogs and cats with various encephalitdes. The DNA extracted from each cerebellum was subject to polymerase chain reaction (PCR) amplification by 3 primer pairs specific for parvovirus DNA. Sequence analysis of PCR products from each of the 8 cats and 2 dogs with cerebellar hypoplasia confirmed their identity with parvoviral DNA. The 6 dogs with cerebellar vermal defects, 2 dogs with cortical abiotrophy, 1 dog with congenital hydranencephaly, and all control samples were PCR negative for parvovirus. Parvoviral structural proteins were not identified by immunohistochemistry in either dog with cerebellar hypoplasia. This study shows that parvoviral DNA can be amplified from feline and canine archival brain tissue and that cerebellar hypoplasia in dogs might be associated with in utero parvovirus infection.     

Transcranial Doppler sonographic findings in granulomatous meningoencephalitis in small breed dogs

Granulomatous meningoencephalitis (GME) is an acute, progressive, and often fatal inflammatory disease of the central nervous system, affecting mainly small and toy dog breeds. A definitive diagnosis of GME can only be achieved through histopathologic examination of samples collected after death. This retrospective study describes transcranial Doppler ultrasonography (TDS) findings in dogs with confirmed clinical histopathology of GME. Eleven dogs were selected for this study. Sonographic findings in B-mode demonstrated diffuse decreased brain parenchyma echogenicity in 9 dogs, ventriculomegaly in 8 dogs, brain atrophy in 4 dogs, and hyperechoic focal lesions in 6 dogs. Color Doppler imaging revealed more obvious vessels of the arterial circle in 10 dogs. Spectral Doppler examination was performed in 10 dogs to detect the 6 major cerebral arteries of interest. The examination showed normal and high resistive index (RI) values in the outlined arteries. The TDS findings were consistent with pathology found on postmortem examination.     

Glial fibrillary acidic protein (GFAP) and anti-GFAP autoantibody in canine necrotising meningoencephalitis

To establish clinical markers for canine necrotising meningoencephalitis (NME) and to elucidate its pathogenesis, glial fibrillary acidic protein (GFAP) and anti-GFAP autoantibodies were measured in the cerebrospinal fluid (CSF) of 32 dogs with NME, 23 dogs with other inflammatory central nervous system (CNS) diseases, 27 dogs with miscellaneous CNS diseases and 25 healthy dogs, including five pugs. The dogs with NME had the highest levels of anti-GFAP autoantibodies. The diagnostic sensitivity and specificity of anti-GFAP autoantibodies for NME were 91 per cent and 73 per cent, respectively. Some of the dogs with NME and the healthy pugs, had high CSF concentrations of GFAP, suggesting a breed-specific fragility of astrocytes. The leakage of GFAP and the development of autoimmunity may be key to understanding the pathogenesis of NME.     

Necrotizing meningoencephalitis in five Chihuahua dogs

An acute to chronic idiopathic necrotizing meningoencephalitis was diagnosed in 5 Chihuahua dogs aged between 1.5 and 10 years. Presenting neurologic signs included seizures, blindness, mentation changes, and postural deficits occurring from 5 days to 5.5 months prior to presentation. Cerebrospinal fluid analyses from 2 of 3 dogs sampled were consistent with an inflammatory disease. Magnetic resonance imaging of the brain of 2 dogs demonstrated multifocal loss or collapse of cortical gray/white matter demarcation hypointense on T1-weighted images, with T2-weighted hyperintensity and slight postcontrast enhancement. Multifocal asymmetrical areas of necrosis or collapse in both gray and white matter of the cerebral hemispheres was seen grossly in 4 brains. Microscopically in all dogs, there was a severe, asymmetrical, intensely cellular, nonsuppurative meningoencephalitis usually with cystic necrosis in subcortical white matter. There were no lesions in the mesencephalon or metencephalon except in 1 dog. Immunophenotyping defined populations of CD3, CD11d, CD18, CD20, CD45, CD45 RA, and CD79a immunoreactive inflammatory cells varying in density and location but common to acute and chronic lesions. In fresh frozen lesions, both CD1b,c and CD11c immunoreactive dendritic antigen-presenting cells were also identified. Immunoreactivity for canine distemper viral (CDV) antigen was negative in all dogs. The clinical signs, distribution pattern, and histologic type of lesions bear close similarities to necrotizing meningoencephalitis as described in series of both Pug and Maltese breed dogs and less commonly in other breeds.     

A canine case of necrotizing meningoencephalitis for long-term observation: clinical and MRI findings

A 3-year-old female pug presented with general seizure following a partial seizure. During the remaining 48 months till death, the dog showed various neurological signs such as disturbance of consciousness, myoclonus and various types of partial seizure after each occurrence of the seizure clusters, and the dog eventually exhibited inability to stand and dementia. Magnetic resonance imaging findings revealed atrophy of the brain over the course of the disease. On histopathological analysis, the dog was diagnosed with necrotizing meningoencephalitis. This case of a canine necrotizing meningoencephalitis observed over the long term is valuable.     

Evaluation of the presence of selected viral and bacterial nucleic acids in pericardial samples from dogs with or without idiopathic pericardial effusion

Many viruses have been identified in pericardial fluid and in tissue samples from humans with pericarditis by means of molecular diagnostics. In canine idiopathic pericardial effusion there is as yet no conclusive evidence to support the involvement of an infectious agent. This study was designed to investigate a possible relationship between idiopathic pericardial effusion in dogs and viruses most commonly encountered in humans affected with viral pericarditis. Coxsackievirus B3 RNA, influenza virus type A RNA, human adenovirus type 2 DNA, human cytomegalovirus DNA, and parvovirus B19 DNA were investigated using PCR on pericardial effusion samples and pericardial tissue specimens collected from 14 dogs with idiopathic pericardial effusion. PCR was also used to test for two bacteria, Borrelia burgdorferi and Chlamydia pneumoniae. The same microorganisms were also looked for in pericardial effusions or pericardial washes from 10 dogs with neoplastic pericardial effusion, and in samples collected from 10 dogs which died of a non-cardiac disease. One pericardial effusion sample from a dog with the idiopathic form of the disease tested positive for influenza virus type A and sequencing of the amplicon confirmed the PCR result. In another dog from the same group a cytomegalovirus was detected by PCR in the effusion, but sequencing showed this to be a false-positive result. The genomes of the microorganisms investigated were not detected in neoplastic effusions or pericardial washes. The results indicate that viral and bacterial DNA/RNA of relevance for human pericarditis is rare in pericardial samples from dogs with idiopathic pericardial effusion. The finding of influenza type A viral RNA in pericardial fluid from one dog with the idiopathic form of the disease warrants further investigation.     

Greyhound meningoencephalitis: PCR-based detection methods highlight an absence of the most likely primary inducing agents

Greyhound meningoencephalitis is currently classified as a breed-associated idiopathic central nervous system inflammatory disorder. The non-suppurative inflammatory response can be distinguished from the other breed-associated disorders based on histopathology and lesion topography, however the nature of the response primarily suggests a viral infection. In the present study PCR and RT-PCR technologies were employed on frozen cerebral tissue from confirmed cases of meningoencephalitis to target specific viruses and protozoa likely to be implicated and to exclude the presence of bacterial 16SrRNA. Secondly, degenerate primers were used to detect viruses of the herpesvirus and flavivirus families. In addition cerebral tissues were probed for West Nile Virus. Viral nucleic acid sequences to Borna disease virus, to louping ill, tick borne encephalitis, West Nile and other flaviviruses were not detected. Canine distemper virus was detected in one animal with 97% homology to strain A75/15. Degenerate PCR for herpesviruses detected viral amplification products in one animal with 90% homology to canine herpesvirus DNA polymerase gene. Protozoal amplification products were only detected in a single dog with pathological confirmation of a combination of lesions of greyhound meningoencephalitis and a protozoal encephalomyelitis. Neospora was confirmed with sequence homology to Austrian strain 1. Bacterial 16SrRNA was not detected. The present study supports previous observations that many of the known microbial causes of canine meningoencephalitis are not involved. Findings could reflect that the causal agent was not specifically targeted for detection, or that the agent is at undetectable levels or has been eliminated from brain tissue. The potential roles of genetics and of molecular mimicry also cannot be discounted.     

A necrotizing meningoencephalitis of pug dogs

Clinical and pathologic features of a sporadic, necrotizing meningoencephalitis affecting adolescent and mature pug dogs are described. Many of the affected animals were closely related. Acute and chronic forms occur, with clinical signs reflecting the pathologic affinity of the disease for the cerebral hemispheres. No etiologic agent has been identified. The extensive necrosis and affinity for the cerebral hemispheres are similar to alphatype herpesvirus encephalitides of other species.