Role of FcγRIII in the nasal cavity of BALB/c mice in the primary amebic meningoencephalitis protection model.

Different mechanisms of the host immune response against the primary amebic meningoencephalitis (PAM) in the mouse protection model have been described. It has been proposed that antibodies opsonize Naegleria fowleri trophozoites; subsequently, the polymorphonuclear cells (PMNs) surround the trophozoites to avoid the infection. FcγRs activate signaling pathways of adapter proteins such as Syk and Hck on PMNs to promote different effector cell functions which are induced by the Fc portion of the antibody-antigen complexes. In this work, we analyzed the activation of PMNs, epithelial cells, and nasal passage cells via the expression of Syk and Hck genes. Our results showed an increment of the FcγRIII and IgG subclasses in the nasal cavity from immunized mice as well as Syk and Hck expression was increased, whereas in the in vitro assay, we observed that when the trophozoites of N. fowleri were opsonized with IgG anti-N. fowleri and interacted with PMN, the expression of Syk and Hck was also increased. We suggest that PMNs are activated via their FcγRIII, which leads to the elimination of the trophozoites in vitro, while in the nasal cavity, the adhesion and consequently infection are avoided.

Role of cathepsin B of Naegleria fowleri during primary amebic meningoencephalitis.

Naegleria fowleri causes primary amoebic meningoencephalitis in humans and experimental animals. It has been suggested that cysteine proteases of parasites play key roles in metabolism, nutrient uptake, host tissue invasion, and immune evasion. The aim of this work was to evaluate the presence, expression, and role of cathepsin B from N. fowleri in vitro and during PAM. Rabbit-specific polyclonal antibodies against cathepsin B were obtained from rabbit immunization with a synthetic peptide obtained by bioinformatic design. In addition, a probe was designed from mRNA for N. fowleri cathepsin B. Both protein and messenger were detected in fixed trophozoites, trophozoites interacted with polymorphonuclear and histological sections of infected mice. The main cathepsin B distribution was observed in cytoplasm or membrane mainly pseudopods and food-cups while messenger was in nucleus and cytoplasm. Surprisingly, both the messenger and enzyme were observed in extracellular medium. To determine cathepsin B release, we used trophozoites supernatant recovered from nasal passages or brain of infected mice. We observed the highest release in supernatant from recovered brain amoebae, and when we analyzed molecular weight of secreted proteins by immunoblot, we found 30 and 37 kDa bands which were highly immunogenic. Finally, role of cathepsin B during N. fowleri infection was determined; we preincubated trophozoites with E-64, pHMB or antibodies with which we obtained 60%, 100%, and 60% of survival, respectively, in infected mice. These results suggest that cathepsin B plays a role during pathogenesis caused by N. fowleri mainly in adhesion and contributes to nervous tissue damage.

Heme oxygenase-1 modulates brain inflammation and apoptosis in mice with angiostrongyliasis.

The rat nematode lungworm Angiostrongylus cantonensis undergoes obligatory intracerebral migration in its hosts and causes eosinophilic meningitis or meningoencephalitis. Heme oxygenase 1 (HO-1) has several cytoprotective properties such as anti-oxidative, anti-inflammatory, and anti-apoptotic effects. HO-1 in brain tissues was induced in A. cantonensis-infected group and showed positive modulation in cobalt protoporphyrin (CoPP)-treated groups. Assay methods for the therapeutic effect include western blot analysis, enzyme-linked immunosorbent assay, gelatin zymography, blood-brain barrier permeability evaluation and eosinophil count in cerebrospinal fluid. The combination of albendazole (ABZ) and CoPP significantly decreased pro-inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-5, and IL-33 but significantly increased anti-inflammatory cytokines IL-10 and transforming growth factor-ß. In addition, worm recovery, matrix metalloproteinase-9, BBB permeability, and eosinophil counts were decreased in the ABZ and CoPP co-treated groups. Induction of HO-1 with CoPP strongly inhibited the protein levels of caspase-3 and increased the induction of annexin-V and B-cell leukemia 2. Thus, co-treatment with ABZ and CoPP prevented A. cantonensis-induced eosinophilic meningoencephalitis and its anti-apoptotic effect by promoting HO-1 signaling prior to BBB dysfunction. HO-1 induction might be a therapeutic modality for eosinophilic meningoencephalitis.

Neurocysticercosis in Children.

Neurocysticercosis is one of the most common parasitic infections in the central nervous system in children. The usual clinical manifestation is new-onset focal seizure. However, there are other multiple clinical manifestations, such as increased intracranial pressure, meningoencephalitis, spinal cord syndrome, and blindness. The diagnosis needs high index of suspicion with clinical history, physical examination, neuroimaging, and immunologic studies. Recent advances in neuroimaging and serology facilitate the accurate diagnosis. Management of neurocysticercosis should focus on critical symptoms first, such as the use of antiepileptic drugs and medical or surgical therapy for increased intracranial pressure.

Peroxisome-Proliferator Activator Receptor γ in Mouse Model with Meningoencephalitis Caused by Angiostrongylus cantonensis.

Peroxisome-proliferator activator receptor γ (PPARγ) has an anti-inflammatory role that inhibits the nuclear factor-κB (NF-κB) pathway and regulates the expressions of pro-inflammatory proteins, whereas its role in parasitic meningoencephalitis remains unknown. In this study we investigated the role of PPARγ and related mechanisms in eosinophilic meningoencephalitis caused by the rat lungworm Angiostrongylus cantonensis. We observed increased protein NF-κB expression in mouse brain tissue using GW9662, which is the specific antagonist of PPARγ, in a mouse model of angiostrongyliasis. Then we investigated NF-κB-related downstream proteins, such as COX-2, NOSs, and IL-1ß, with Western blot or enzyme-linked immunosorbent assay and found that the protein expression was upregulated. The results of gelatin zymography also showed that the MMP-9 activities were upregulated. Treatment with GW9662 increased the permeability of the blood-brain barrier and the number of eosinophils in cerebrospinal fluid. These results suggested that in angiostrongyliasis, PPARγ may play an anti-inflammation role in many inflammatory mediators, including NOS-related oxidative stress, cytokines, and matrix metalloproteinase cascade by decreasing the NF-κB action.

Magnetic resonance imaging in dogs with neuroangiostrongyliasis (rat lungworm disease).

The magnetic resonance imaging (MRI) appearance of the brain and spinal cord in humans with neuroangiostrongyliasis (NA) due to Angiostrongylus cantonensis infection has been well reported. Equivalent studies in animals are lacking. This case series describes clinical and MRI findings in 11 dogs with presumptively or definitively diagnosed NA. MRI of the brain and/or spinal cord was performed using high-field (1.5 T) or low-field (0.25 T) scanners using various combinations of transverse, sagittal, dorsal and three-dimensional (3D) T1-weighted (T1W), transverse, sagittal and dorsal T2-weighted (T2W), T2W fluid-attenuated inversion recovery (FLAIR) and T2*-weighted (T2*W) gradient echo (GRE), dorsal T2W short tau inversion recovery (STIR) and post-gadolinium transverse, sagittal, dorsal and 3D T1W and transverse T2W FLAIR sequences. In 4/6 cases where the brain was imaged, changes consistent with diffuse meningoencephalitis were observed. Evidence of meningeal involvement was evident even when not clinically apparent. The spinal cord was imaged in 9 dogs, with evidence of meningitis and myelitis detected in regions consistent with the observed neuroanatomical localization. Pathognomonic changes of neural larva migrans, as described in some human patients with NA, were not detected. NA should be considered in the differential diagnosis of dogs with MRI evidence of focal or diffuse meningitis, myelitis and/or encephalitis, especially in areas where A. cantonensis is endemic. If not precluded by imaging findings suggestive of brain herniation, cerebrospinal fluid (CSF) collection for cytology, fluid analysis, real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) testing should be considered mandatory in such cases after the MRI studies.

Diagnostic evaluation of fatal Balamuthia mandrillaris meningoencephalitis in a captive Bornean orangutan (Pongo pygmaeus) with identification of potential environmental source and evidence of chronic exposure.

A female Bornean orangutan (Pongo pygmaeus) aged 11 years and 6 months was examined by veterinarians after caretakers observed lethargy and facial grimacing. Within 72 h the primate had left-sided hemiparesis that worsened over the next week. An MRI revealed a focal right-sided cerebral mass suspected to be a neoplasm. Ten days after onset of clinical signs, the orangutan died. On postmortem exam, the medial right parietal lobe was replaced by a 7 × 4 × 3.5 cm focus of neuromalacia and hemorrhage that displaced the lateral ventricle and abutted the corpus callosum. Histopathology of the cerebral lesion revealed pyogranulomatous meningoencephalitis with intralesional amoeba trophozoites and rare cysts. Fresh parietal lobe was submitted to the Centers for Disease Control and Prevention lab for multiplex free-living amoebae real-time PCR and detected Balamuthia mandrillaris DNA at a high burden. Mitochondrial DNA was sequenced, and a 760-bp locus 19443F/20251R was compared to several human infections of B. mandrillaris and shown to be identical to the isolates from four human cases of encephalitis: 1998 in Australia, 1999 in California, 2000 in New York, and 2010 in Arizona. Indirect immunofluorescent antibody testing of stored serum samples indicated exposure to B. mandrillaris for at least 2 years prior to death. Within 1 week of the orangutan's death, water from the exhibit was analyzed and identified the presence of B. mandrillaris DNA, elucidating a possible source of exposure. B. mandrillaris, first reported in a mandrill in 1986, has since occurred in humans and animals and is now considered an important emerging pathogen.

Disseminated Neospora caninum infection in a dog with severe colitis.

A 12-y-old spayed female Schipperke dog with a previous diagnosis of inflammatory bowel disease was presented with a 2-mo history of severe colitis. The patient's condition progressed to hepatopathy, pneumonia, and dermatitis following management with prednisolone and dexamethasone sodium phosphate. Colonic biopsies identified severe necrosuppurative colitis with free and intracellular parasitic zoites. Postmortem examination confirmed extensive chronic-active ulcerative colitis, severe acute necrotizing hepatitis and splenitis, interstitial pneumonia, ulcerative dermatitis, myelitis (bone marrow), and mild meningoencephalitis with variable numbers of intracellular and extracellular protozoal zoites. PCR on samples of fresh colon was positive for Neospora caninum. Immunohistochemistry identified N. caninum tachyzoites in sections of colon, and a single tissue cyst in sections of brain. Administration of immunosuppressive drugs may have allowed systemic dissemination of Neospora from the intestinal tract.

Identification of cross-reactive markers to strengthen the development of immunodiagnostic methods for angiostrongyliasis and other parasitic infections.

Angiostrongylus cantonensis is the main causative agent of eosinophilic meningoencephalitis (EoM) in humans. Molecular diagnostic methods are essential since the identification of larvae in cerebrospinal fluid (CSF) is extremely rare. To date, the detection of a 31 kDa antigen by Western blotting has been the primary immunodiagnostic method for EoM caused by A. cantonensis. However, cross-reactivity with other parasites has been observed. Therefore, we conducted a comparative analysis using sera from individuals with angiostrongyliasis. We also characterized proteins isolated from different cellular sources of A. cantonensis, Toxocara canis, Schistosoma mansoni, and Strongyloides stercoralis with mass spectrometry. A total of 115 cross-reactive proteins were identified. Three of these proteins, heat shock protein, an intermediate filament protein, and galectin 1, represent potential markers for cross-reactivity. In addition, synthetic peptides were generated from previously identified diagnostic targets and tested against sera from individuals infected with several other parasites. As a result, two other markers of cross-reactivity were identified: peptide #4 derived from the 14-3-3 protein and peptide #12 derived from the Lec-5 protein. In contrast, 34 proteins were exclusively present in the Angiostrongylus extracts and represent promising diagnostic molecules for specific identification of A. cantonensis infection. In particular, cytochrome oxidase subunit I is of great interest as a possible immunodiagnostic target for angiostrongyliasis.

Albendazole-Schisandrin B Co-Therapy on Angiostrongylus cantonensis-Induced Meningoencephalitis in Mice.

Abstract: Currently, Angiostrongylus cantonensis infections are predominantly treated with albendazole. However, the use of albendazole can provoke certain neurological symptoms as a result of the immune response triggered by the dead worms. Therefore, treatment usually involves co-administration of corticosteroids to limit the inflammatory reaction. Corticosteroids play a useful role in suppressing inflammation in the brain; however, long-term usage or high dosage may make it problematic.Schisandrin B, an active ingredient from Schisandra chinensis, has been shown to have anti-inflammatory effects on the brain. This study aimed to investigate the effects and potential of schisandrin B in combination with albendazole to treat Angiostrongylus-induced meningoencephalitis. Here, we show that albendazole-schisandrin B co-treatment suppressed neuroinflammation in Angiostrongylus-infected mice and increased the survival of the mice. Accordingly, albendazole-schisandrin B co-treatment significantly inhibited inflammasome activation, pyroptosis, and apoptosis. The sensorimotor functions of the mice were also repaired after albendazole-schisandrin B treatment. Immune response was shown to shift from Th2 to Th1, which reduces inflammation and enhances immunity against A. cantonensis. Collectively, our study showed that albendazole-schisandrin B co-therapy may be used as an encouraging treatment for Angiostrongylus-induced meningoencephalitis.

First case of fatal equine meningoencephalitis caused by Halicephalobus gingivalis in Mexico.

Aberrant nematode larval migration in the CNS of horses is rare but frequently fatal; one of the main etiological agents involved in this illness is Halicephalobus gingivalis. This soil nematode has been associated with several fatal equine meningoencephalitis reports worldwide; however, it had never been diagnosed in horses of Mexico. A 10 year-old Andalusian horse presented dysphagia, fever, weakness, prostration and ataxia; the patient expired during the medical attention. Post mortem examination was performed and no gross alterations were found. Histopathology revealed meningoencephalitis, vasculitis and intralesional adult nematodes, larvae and eggs compatible with Halicephalobus spp. A polymerase chain reaction (PCR) for the nuclear large subunit ribosomal RNA gene (LSU rDNA) of nematodes was performed from formalin-fixed and paraffin wax-embedded sections of brain. Posterior nucleotide sequence analysis of the amplified fragment identified the agent as H. gingivalis. To our knowledge, this is the first confirmed report of Halicephalobiasis in Mexico.

Angiostrongylus cantonensis activates inflammasomes in meningoencephalitic BALB/c mice.

Angiostrongylus cantonensis is a metastrongyloid nematode that causes eosinophilic meningoencephalitis in humans. A high infestation of A. cantonensis can cause permanent brain damage or even death. The inflammasome is an oligomeric molecular platform that can detect microbial pathogens and activate inflammatory cytokines. The recognition of larval surface antigens by pattern recognition receptors (PRRs) can cause oligomerization of the NOD-like receptor (NLR) or absent in melanoma 2 (AIM2) with the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) to form a caspase-1-activating scaffold. Activated caspase-1 converts pro-inflammatory cytokines into their mature, active forms. Helminths infection has been shown to activate NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasomes. In this study, we aimed to investigate the mechanism of inflammasome activation upon A. cantonensis infection in a mouse model. This study provides evidence that A. cantonensis infection can activate NLRP1B and NLRC4 inflammasomes and promote pyroptosis to cause meningoencephalitis.

Identification of differential protein recognition pattern between Naegleria fowleri and Naegleria lovaniensis.

Many pathogenicity factors are involved in the development of primary amoebic meningoencephalitis (PAM) caused by N fowleri. However, most of them are not exclusive for N fowleri and they have not even been described in other nonpathogenic Naegleria species. Therefore, the objective of this work was to identify differential proteins and protein pattern recognition between Naegleria fowleri and Naegleria lovaniensis using antibodies anti-N fowleri as strategy to find vaccine candidates against meningoencephalitis. Electrophoresis and Western blots conventional and 2-DE were performed for the identification of antigenic proteins, and these were analysed by the mass spectrometry technique. The results obtained in 2-DE gels and Western blot showed very notable differences in spot intensity between these two species, specifically those with relative molecular weight of 100, 75, 50 and 19 kDa. Some spots corresponding to these molecular weights were identified as actin fragment, myosin II, heat shock protein, membrane protein Mp2CL5 among others, with differences in theoretical post-translational modifications. In this work, we found differences in antigenic proteins between both species, proteins that could be used for a further development of vaccines against N fowleri infection.

An undescribed species of Sarcocystis associated with necrotizing meningoencephalitis in naturally infected backyard chickens in the Midwest of Brazil.

Sarcocistys -associated menigoencephalitis is virtually an unrecognized cause of neurological disease in chickens. An undescribed species of Sarcocystis cause fatal infection in two backyard chickens in the Midwest of Brazil. Infected chickens presented anorexia, weight loss, incoordination, ataxia and opisthotonos. Yellow necrotic foci in the gray and white matter of the telencephalon were the main gross lesion. Microscopically, necrotizing granulomatous and heterophilic meningoencephalitis with intralesional Sarcocystis -like schizonts and mezoites were observed in the central nervous system. Molecular analysis of frozen brain samples of the two chickens was identical and the protozoan was named Sarcocystis sp. Chicken-2016-DF-BR. Complete nested PCR- sequence of Sarcocystis sp. Chicken-2016-DF-BR was equally similar to Sarcocystis anasi (EU553477) and Sarcocystis albifronsi (EU502868). This is the first report of Sarcocistys -associated meningoencephalitis with molecular characterization in backyard chickens.

First molecular characterization of Sarcocystis neurona causing meningoencephalitis in a domestic cat in Brazil.

Sarcocystis neurona is the main agent associated with equine protozoal myeloencephalitis (EPM). Apart from horses, S. neurona has been occasionally described causing neurologic disease in several other terrestrial animals as well as mortality in marine mammals. Herein, we describe the clinical, pathological, and molecular findings of a fatal case of S. neurona-associated meningoencephalitis in a domestic cat. The causing agent was analyzed by multilocus genotyping, confirming the presence of S. neurona DNA in the tissue samples of the affected animal. Significant molecular differences were found in relation to S. neurona isolates detected in other regions of the Americas. In addition, the parasite was identical to Sarcocystis sp. identified in opossum sporocysts in Brazil at molecular level, which suggests that transmission of. S. neurona in Brazil might involve variants of the parasite different from those found elsewhere in the Americas. Studies including more samples of S. neurona would be required to test this hypothesis, as well as to assess the impact of this diversity.

Next-generation sequencing specifies Angiostrongylus eosinophilic meningoencephalitis in infants: Two case reports.

RATIONALE: Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis (AEM) in infants is a very rare but fatal disease. Utilization of genetic assay to detect the cerebral parasite plays an important role for the treatment of the infection. PATIENT CONCERNS: Two infants (<2 years) presented with cough, intermittent fever, mental fatigue, and poor diet. DIAGNOSIS: The patients were under clinical examination and laboratory test including cardiac ultrasound, chest X-ray, blood or cerebrospinal fluid (CSF) cell counting, serum enzyme-linked immunosorbent assay (ELISA), head magnetic resonance imaging (MRI) and next-generation sequencing (NGS) on DNA from CSF. Due to hypereosinophils in patients' peripheral blood and CSF, and abundant DNA sequences from A cantonensis in CSF, the patients were diagnosed with Angiostrongylus eosinophilic meningoencephalitis. INTERVENTIONS: The patients were treated with albendazole to deworm, and methylprednisolone to reduce inflammation. OUTCOME: The patients were completely recovered from AEM without relapse after 10-day treatment. LESSONS: ELISA and MRI are not sufficiently accurate for the diagnosis of AEM in infants. NGS can specify the infection by the cerebral parasite and offers a new effective approach for the early and precise diagnosis of AEM in infants.

Proteasome serves as pivotal regulator in Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis.

Proteasome primarily degrades the unneeded or damaged proteins by proteolysis. Disruption of the brain barrier and its resulting meningoencephalitis caused by Angiostrongylus cantonensis are important pathological events in non-permissive hosts. In this study, the results showed upregulated proteasome during A. cantonensis infection. Occludin degradation and matrix metalloproteinase-9 (MMP-9) activity were significantly increased in infected mice than in uninfected mice. Moreover, confocal immunoflourescence microscopy showed that occludin was co-localized with MMP-9. The infected-mice were treated with proteasomal activity inhibitor MG132 by 1.5 and 3.0 mg/kg/day, which resulted in significantly reduced protein levels of phosphorylated IκBα (P<0.05) compared with the untreated control. The phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) showed similar result. In addition, MMP-9 activity and occludin degradation were reduced because of MG132 treatment. These results suggested that the proteasome in A. cantonensis infection degraded phosphorylated IκBα, modulated phosphorylated NF-κB, and then regulated the activation of MMP-9 and occludin degradation. Proteasome alterations were presented in eosinophilic meningitis of BALB/c mice and may contribute to the pathophysiology of eosinophilic meningitis by increasing occludin degradation. This molecule would serve as pivotal regulator in A. cantonensis-induced eosinophilic meningoencephalitis.