Search

American trypanosomiasis is caused by the zoonotic protozoa Trypanosoma cruzi and primarily results in heart disease. Organisms also infect the central nervous system (CNS). The Texas A&M University veterinary teaching hospital archive was searched for dogs with CNS disease with intralesional protozoal amastigotes. This study summarizes 4 cases of dogs with disseminated trypanosomiasis and CNS involvement confirmed by quantitative polymerase chain reaction (qPCR) with T. cruzi primers. Clinical signs included lethargy, respiratory distress, tetraparesis, and seizures. Central nervous system lesions included meningeal congestion (1/4), necrosis with hemorrhage in the spinal cord gray and white matter (2/4), and histiocytic meningoencephalitis (4/4), and meningomyelitis (2/4) with intralesional and intracellular protozoal. Genotyping identified 1 case of T. cruzi discrete typing unit (DTU) TcI and 2 cases as TcIV, both are common variants in the United States. Trypanosomiasis should be considered a differential diagnosis for dogs with CNS signs in T. cruzi -endemic areas.

Naegleria fowleri, a free-living and thermophilic ameba, is the etiological agent of primary amebic meningoencephalitis (PAM). PAM is a rare and highly fatal neurologic disease in humans, and has been rarely documented in animal species. This report describes the pathological and etiological findings of a fatal case of N. fowleri-associated meningoencephalitis in a cow in Southern Brazil. Microscopic findings were consistent with severe, multifocal, hemorrhagic, and necrosuppurative meningoencephalitis associated with a large number of amebic trophozoites compatible with N. fowleri. Brain samples subjected to molecular assays generated a 315 bp fragment, which presented 99% identity with a N. fowleri sequence previously deposited in GenBank. This is the first study reporting the molecular detection of N. fowleri in a case of cattle meningoencephalitis in Latin America, and the obtained sequence represents the first GenBank deposit of N. fowleri identified in Brazil to this day. Additionally, the case reported is the second occurrence of N. fowleri-associated disease in the same city, drawing attention to the local importance of infection by this ameba and potential risk for human infections., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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.

Neurofilaments (NFs) are structural proteins of neurons that are released in significant quantities in the cerebrospinal fluid and blood as a result of neuronal degeneration or axonal damage. Therefore, NFs have potential as biomarkers for neurologic disorders. Neural degeneration increases with age and has the potential to confound the utility of NFs as biomarkers in the diagnosis of neurologic disorders. We investigated this relationship in horses with and without neurological diagnosis. While controlling for horse type (draft, pleasure, and racing), we evaluated the relationship between serum heavy-chain phosphorylated neurofilaments (pNF-H) and age, sex, and serum vitamin E concentrations. Serum pNF-H concentrations increased by 0.002 ng/ml for each year increase in age. There were significant differences in the serum pNF-H concentration among the type of activity performed by the horse. The highest serum pNF-H concentration was found in horses performing heavy work activity (racehorse) and with lower serum pNF-H concentration found among light (pleasure riding) and moderate (draft) activity. There was no significant association between the pNF-H concentration and sex or vitamin E concentration. Serum pNF-H concentration was elevated among horses afflicted with EMND and EPM when compared with control horses without evidence of neurologic disorders. Accordingly, serum pNF-H concentration can serve as a useful biomarker to complement the existing diagnostic work-up of horses suspected of having EPM or EMND., (Copyright © 2019. Published by Elsevier Ltd.)

Naegleria fowleri is a free-living amoeba commonly found in the environment, mainly in fresh water and soil. This protozoon is occasionally involved in cases of fatal central nervous system disease in humans and other animal species. We describe here a case of meningoencephalitis due to Naegleria fowleri in cattle, in southern Brazil. A four-year-old Angus cow presented a clinical history of initial mild neurological signs that progressed to paddling movements, opisthotonus and lateral recumbency after five days. This animal had been kept in an irrigated rice stubble paddock. Grossly, the main lesions consisted of multiple areas of malacia in the right olfactory bulb, piriform lobes, hippocampus, frontal lobe cortex and fornix, along with severe thickening of the mesencephalon and rhombencephalon leptomeninges. Microscopically, severe multifocal necrosuppurative and hemorrhagic meningoencephalitis associated with a large quantity of amoebic trophozoites was present. The latter were confirmed to be Naegleria spp., through immunohistochemistry. Based on the strong congruence with the histopathological data of known cases reported in the literature, a probable association with Naegleria fowleri was established. To our knowledge, this is only the second report of Naegleria fowleri-associated meningoencephalitis in cattle in South America, and it is the first in southern Brazil.

Background: Accurate antemortem EPM diagnosis requires evidence of intrathecal antibody production. Some advocate the use of acute phase proteins in addition to serology, which alone results in substantial false positives., Hypothesis/objectives: The purpose of this study was to determine if serum C-reactive protein (CRP) or serum amyloid A (SAA) concentrations were elevated in cases of equine protozoal myeloencephalitis (EPM) compared to other neurological diseases., Animals: 25 clinical cases of equine neurological disease: EPM (10), cervical vertebral stenotic myelopathy (CVSM) (10), neuroborreliosis (2), equine motor neuron disease (1), degenerative myelopathy (1), and leukoencephalomalacia (1)., Methods: Serum and CSF CRP and SAA were measured. Selection criteria included neurologic disease, antemortem diagnosis of EPM or CVSM, or postmortem diagnosis of EPM, CVSM, or other neurologic disease, and availability of serological results and archived samples for testing., Results: Serum SAA and serum CRP levels were generally undetectable or low in horses with EPM (median CRP ≤0.1 mg/L, ≤0.1-14.4 mg/L; median SAA ≤0.1 mg/L, ≤0.1-6.11 mg/L) and CVSM (median CRP ≤0.1, ≤0.1-2.41 mg/L; median SAA ≤0.1mg/L, ≤0.1-13.88 mg/L). CSF CRP and SAA for horses with EPM (median CRP 3.35 mg/l, 0.19-13.43 mg/l; median SAA ≤0.1 mg/L, ≤0.1-2.4 mg/L) and CVSM (median CRP 4.015 mg/L, 0.16-9.62 mg/L; median SAA 0.62 mg/L, ≤0.1-2.91 mg/L) were also undetectable or low. Kruskal-Wallis test showed no statistically significant differences between serum CRP (P = .14), serum SAA (P = .79), spinal fluid CRP (P = .65), or spinal fluid SAA between horses with EPM and CVSM (P = .52)., Conclusion: Neither SAA nor CRP in serum or CSF aid diagnosis of EPM., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

A 1-year-old Siberian Husky dog with acute-onset of seizures, recumbency, paddling, and muscular fasciculations was autopsied. A locally extensive hemorrhagic and malacic focus was noted in the right cerebral frontal cortex, and severe necrotizing and hemorrhagic, neutrophilic meningoencephalitis was diagnosed microscopically. Amoebic trophozoites and cysts were identified within the affected cerebral parenchyma and confirmed by indirect immunofluorescence assay and real-time PCR as Balamuthia mandrillaris. B. mandrillaris is found in soil and water and the infection has been reported in both immunocompromised and immunocompetent humans and rarely in the dog., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study we describe a case of neurological leishmaniasis in a dog, reported in an endemic area, with signs of multifocal intracranial lesions involving the vestibular system and the cerebellum. Serum biochemistry revealed a decrease of albumin-globulin ratio caused by an increase of alfa2-, beta-, and gamma- globulin, while antibody titers were highly positive for Leishmania spp. Magnetic resonance images of the brain were consistent with a granulomatous in ammation/infection. Cerebrospinal fluid revealed a marked mononuclear pleocytosis and was positive to the Pandy Test, as well as to a Leishmania spp. -speci c polymerase chain reaction (PCR) assay. Sequencing of the PCR products revealed the highest similarity with several strains belonging to the Leishmania infantum / Leishmania chagasi group. Clinical response to treatment for systemic leishmaniasis was supportive of diagnosis. This report focuses on an atypical form of canine leishmaniasis and suggests that in endemic geographic areas leishmaniasis has to be considered for di erential diagnosis in neurological manifestations. Also, cerebrospinal liquor should always be tested when neurological symptoms are present in order to di erentiate neurological leishmaniasis from unrelated neurological signs in Leishmania positive patients.

A 3-year-old sheep was examined after an acute onset of hind limb paralysis and ataxia. At necropsy, central nervous system, pulmonary and intestinal hyperaemia and ecchymoses in the aortic arch were observed. Main microscopic lesions were confined to the heart, cerebrum and cerebellum. There were a multifocal mild myocarditis and nonsuppurative meningoencephalitis together with protozoal cysts in the heart and the brain. Protozoal cystic structures were observed within many of the myocardial fibers as well as in the cerebrum and cerebellum. Using light microscopy it could not be morphologically determined whether these organisms were Toxoplasma (T.) gondii or Neospora (N.) caninum. Additional diagnostic methods like immunohistochemistry and polymerase chain reaction provided differentiation of Sarcocystis from T. gondii and N. caninum. Transmission electron microscopy demonstrated characteristic features of Sarcocystis sp. as previously described. This is the first confirmed diagnosis of Sarcocystis sp. in the central nervous system of a sheep from Turkey.

Migration of parasitic worms through the host tissues, which may occasionally result in fatal damage to the internal organs, represents one of the major risks associated with helminthoses. In order to track the parasites, traditionally used 2D imaging techniques such as histology or squash preparation do not always provide sufficient data to describe worm location/behavior in the host. On the other hand, 3D imaging methods are widely used in cell biology, medical radiology, osteology or cancer research, but their use in parasitological research is currently occasional. Thus, we aimed at the evaluation of suitability of selected 3D methods to monitor migration of the neuropathogenic avian schistosome Trichobilharzia regenti in extracted spinal cord of experimental vertebrate hosts. All investigated methods, two of them based on tracking of fluorescently stained larvae with or without previous chemical clearing of tissue and one based on X-ray micro-CT, exhibit certain limits for in vivo observation. Nevertheless, our study shows that the tested methods as ultramicroscopy (used for the first time in parasitology) and micro-CT represent promising tool for precise analyzing of parasite larvae in the CNS. Synthesis of these 3D imaging techniques can provide more comprehensive look at the course of infection, host immune response and pathology caused by migrating parasites within entire tissue samples, which would not be possible with traditional approaches., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

CASE DESCRIPTION A 22-year-old male gorilla (Gorilla gorilla gorilla) housed in a zoo was evaluated for signs of lethargy, head-holding, and cervical stiffness followed by development of neurologic abnormalities including signs of depression, lip droop, and tremors. CLINICAL FINDINGS Physical examination under general anesthesia revealed a tooth root abscess and suboptimal body condition. A CBC and serum biochemical analysis revealed mild anemia, neutrophilia and eosinopenia consistent with a stress leukogram, and signs consistent with dehydration. Subsequent CSF analysis revealed lymphocytic pleocytosis and markedly increased total protein concentration. TREATMENT AND OUTCOME Despite treatment with antimicrobials, steroids, and additional supportive care measures, the gorilla's condition progressed to an obtunded mentation with grand mal seizures over the course of 10 days. Therefore, the animal was euthanized and necropsy was performed. Multifocal areas of malacia and hemorrhage were scattered throughout the brain; on histologic examination, these areas consisted of necrosis and hemorrhage associated with mixed inflammation, vascular necrosis, and intralesional amoebic trophozoites. Tan foci were also present in the kidneys and pancreas. Immunohistochemical testing positively labeled free-living amoebae within the brain, kidneys, eyes, pancreas, heart, and pulmonary capillaries. Subsequent PCR assay of CSF and frozen kidney samples identified the organism as Balamuthia mandrillaris, confirming a diagnosis of amoebic meningoencephalitis. CLINICAL RELEVANCE Infection with B mandrillaris has been reported to account for 2.8% of captive gorilla deaths in North America over the past 19 years. Clinicians working with gorillas should have a high index of suspicion for this diagnosis when evaluating and treating animals with signs of centrally localized neurologic disease.

Primary amoebic meningoencephalitis (PAM) is a fatal infection in most cases, caused by the amoeba flagellate Naegleria fowleri. This report describes the first cases of PAM in Algeria, in a cow and a ewe from Batna, north-eastern Algeria. The death of both ruminants occurred a week after the first clinical manifestations. The cerebrospinal fluid, after staining with May-Grünwald-Giemsa, showed the presence of amoebae cells. Histological sections revealed numerous amoebae in all parts of the brain. The presence of N. fowleri was confirmed using a species-specific real-time PCR in histological tissue sections. The two PAM cases were reported during the hot season, and the source of infection is very likely the water where the cattle came to drink. Particular attention should be focused on this type of infection in aquatic environments when the temperature is high and preventive measures must be taken to avoid the proliferation of N. fowleri., (© M. Benterki et al., published by EDP Sciences, 2016.)

Scuticociliatosis is an economically important, frequently fatal disease of marine fish in aquaculture, caused by histophagous ciliated protozoa in the subclass Scuticociliatida of the phylum Ciliophora. A rapidly lethal systemic scuticociliate infection is described that affected aquarium-captive zebra sharks (Stegostoma fasciatum), Port Jackson sharks (Heterodontus portusjacksoni), and a Japanese horn shark (Heterodontus japonicus). Animals died unexpectedly or after a brief period of lethargy or behavioral abnormality. Gross findings included necrohemorrhagic hepatitis and increased volumes of celomic fluid. Histologically, 1 or more of a triad of necrotizing hepatitis, necrotizing meningoencephalitis, and thrombosing branchitis were seen in all cases, with necrotizing vasculitis or intravascular fibrinocellular thrombi. Lesions contained variably abundant invading ciliated protozoa. Molecular identification by polymerase chain reaction from formalin-fixed tissues identified these as the scuticociliate Philasterides dicentrarchi (syn. Miamiensis avidus), a novel and potentially emergent pathogen in sharks.

A 14-year-old Morgan gelding was presented for progressive weakness and muscle atrophy. The horse was initially diagnosed with equine protozoal myelitis based on history, physical examination, and laboratory diagnostics. Despite therapy, the horse declined clinically and was euthanized. Necropsy revealed a rare form of neurotropic lymphoma, described in this report.

Primary amoebic meningoencephalitis is an acute disease of the central nervous system of humans and animals caused by Naegleria fowleri. This report describes a case of meningoencephalitis caused by N. fowleri in a crossbred, one-year-old bovine with progressive neurological signs. At necropsy there was thickening of the meninges and multifocal areas of malacia in the thalamus, caudal and rostral colliculi, parietal and occipital cortex, and cerebellum. Histologically there was multifocal necrosupurative meningoencephalitis associated with areas of malacia containing amoebic trophozoites. Immunohistochemistry of the brain was positive for N. fowleri. The disease should be included among the differential diagnosis of diseases of the central nervous system in cattle in areas where animals are exposed to hot, stagnant water., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinases (MMPs) are a group of calcium- and zinc-dependent endopeptidases that are involved in maintaining the extracellular matrix. MMP-2 and MMP-9 are thought to be related to the disruption of the blood-brain-barrier (BBB) by their ability to cleave type IV collagen, the main component of the basal membrane. To establish the presence of MMP-2 and MMP-9 in the pathogenesis of canine cerebral leishmaniasis, we examined the levels of these metalloproteinases in the cerebrospinal fluid (CSF) and serum of dogs with visceral leishmaniasis and neurological symptoms (n=16) and in the CSF and serum of uninfected healthy dogs (n=10) using zymography. In the CSF of dogs with cerebral leishmaniasis there was a massive presence of active MMP-2, whereas only the levels of both proMMP-2 and proMMP-9 were elevated in the serum. Although the detected MMP activity in the CSF might merely be related to CNS inflammation, these enzymes may also play a collaborative role in the disease progression. Both MMP-2 and MMP-9 are known to target critical constituents of the BBB, and once activated, they may promote cerebral barrier breakdown, allowing the entrance of inflammatory cells and proteins within the nervous system milieu., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: A 12-year-old female western lowland gorilla died in a zoological garden in Germany after exhibiting general neurological signs., Methods: Balamuthia mandrillaris was identified as causative agent by indirect immunofluorescent staining of brain sections and confirmed by PCR and respective sequencing., Results: The animal suffered from a chronic progressive necrotizing amebic meningoencephalitis., Conclusion: This is the first case of Balamuthia amebic encephalitis in Germany., (© 2011 John Wiley & Sons A/S.)

A 5-month-old mongrel puppy with a history of respiratory disease presented with progressive neurologic dysfunction. Hematologic results included leukocytosis (neutrophilia with a left shift) and lymphopenia. A mass in the right forebrain, identified by magnetic resonance imaging, was biopsied during decompressive craniectomy. The histologic diagnosis was granulomatous meningoencephalitis with intralesional amoebae. The dog died within 24 hours of surgery. At necropsy, a well-demarcated granuloma was confined to the cerebrum, but granulomatous pneumonia was disseminated through all lobes of the lung. Concurrent infections included canine distemper, canine adenoviral bronchiolitis, and oral candidiasis. Canine distemper virus probably caused immunosuppression and increased susceptibility to secondary infections.

A 9-year-old female spayed Shetland Sheepdog was presented to the Kansas State University Veterinary Medical Teaching Hospital for evaluation following a 3-week history of left rear limb lameness that had progressed to generalized ataxia. Multifocal or diffuse brain lesions were suspected based on physical examination findings. Cerebrospinal fluid (CSF) contained 52 nucleated cells/μL composed of mixed inflammatory cells. Treatment with prednisone and cyclosporine was initiated based on a presumptive diagnosis of granulomatous meningoencephalitis. Thirteen days later the dog was nonambulatory and mentally obtunded. Repeat CSF analysis revealed 298 nucleated cells/μL with 61% eosinophils. Rare protozoal tachyzoites consistent with Neospora caninum, Toxoplasma gondii, or Sarcocystis spp. were found extracellularly and within macrophages and an eosinophil. Despite cessation of prednisone and cyclosporine therapy and provision of supportive care, the dog died 6 days later. Examination of brain tissue sections revealed multifocally extensive, necrotizing, histiocytic, and lymphoplasmacytic meningoencephalitis with numerous protozoal zoites and cysts. Immunohistochemical analysis of brain tissue using a monoclonal antibody specific for N. caninum confirmed the diagnosis of neosporosis. Similar but less severe lesions were noted in the spinal cord, although organisms were not found. This case emphasizes the value of repeated CSF analysis when therapy is ineffective and the importance of excluding infectious causes of meningoencephalitis before commencement of immunosuppressive therapy., (©2010 American Society for Veterinary Clinical Pathology.)

This article reports the results of necropsy, parasitologic, microbiologic, histopathologic, immunohistochemical, indirect immunofluorescence, biomolecular, and serologic investigations on 8 striped dolphins (Stenella coeruleoalba) found stranded from August to December 2007 on the Ligurian Sea coast of Italy. Severe, nonsuppurative meningoencephalitis was found in 4 animals, as characterized by prominent perivascular mononuclear cell cuffing and macrophage accumulations in neuropil. These lesions were associated with mild lymphocytic-plasmacytic infiltration of choroid plexuses in 1 dolphin. Toxoplasma gondii cysts and zoites, confirmed by immunohistochemical labeling, were scattered throughout the brain parenchyma of 2 of the 4 dolphins. No viral inclusions were seen in the brain of any animal. Other findings included severe bronchointerstitial pneumonia and pulmonary atelectasis, consolidation, and emphysema. Parasites were identified in a variety of organs, including lung (Halocerchus lagenorhynchi). Microbiologic and serologic examinations for Brucella spp were negative on all 8 dolphins. The 4 animals with meningoencephalitis had serum antibodies against T gondii (titers ranging from 1:80 to 1:320) but not against morbillivirus. In contrast, the other 4 dolphins were seropositive for morbillivirus (with titers ranging from 1:10 to 1:40) but seronegative for T gondii. No morbillivirus antigen or nucleic acid was detected in the tissues of any dolphin. It is concluded that the severe lung and brain lesions were the cause of death and that T gondii was the likely etiologic agent of the cerebral lesions. Morbillivirus infection was not considered to have contributed to death of these animals.

This study aimed to characterize astrocytic and microglial response in the central nervous system (CNS) of equines experimentally infected with T. evansi. The experimental group comprised males and females with various degrees of crossbreeding, ages between four and seven years. The animals were inoculated intravenously with 10(6) trypomastigotes of T. evansi originally isolated from a naturally infected dog. All equines inoculated with T. evansi were observed until they presented symptoms of CNS disturbance, characterized by motor incoordination of the pelvic limbs, which occurred 67 days after inoculation (DAI) and 124 DAI. The animals in the control group did not present any clinical symptom and were observed up to the 125th DAI. For this purpose the HE histochemical stain and the avidin biotin peroxidase method was used. Lesions in the CNS of experimentally infected horses were those of a wide spread non suppurative meningoencephalomyelitis.The severity of lesions varied in different parts of the nervous system, reflecting an irregular distribution of inflammatory vascular changes. The infiltration of mononuclear cells was associated with anisomorphic gliosis and reactive microglia was identified. The intensity of the astrocytic response in the CNS of the equines infected by T. evansi characterizes the importance of the performance of these cells in this trypanosomiasis. The characteristic gliosis observed in the animals in this experiment suggests the ability of these cells as mediators of immune response. The parasite, T. evansi, was not identified in the nervous tissues.

Background: Equine protozoal myeloencephalitis (EPM) is a serious and often fatal neurologic disease of horses, but few studies have investigated risk factors., Objectives: To evaluate operation- and individual-level factors associated with likelihood of the occurrence of EPM., Animals: Data were collected as part of a study of the US equine industry from 1,178 operations representing 83.9% of horses and 51.6% of operations with > or =3 horses in 28 states., Methods: Probability-based sampling was used to enroll representative operations in a cross-sectional study. Interviews were conducted to collect information regarding health and management of horses. A nested case-control study was used to investigate risk factors among individual horses. Interview data were combined with climate data, human population density, and opossum regional ecology categories. Data were analyzed using logistic regression to identify risk factors for the occurrence of EPM., Results: Owners reported that 95% of EPM cases included in this study were diagnosed by veterinarians. Variables associated with EPM occurrence on premises included opossum regional ecology, reported exposure to small wildlife, climate, terrain, housing, choice of bedding material, method of storing feeds, equine stocking density, and primary use of horses. Among individual horses, age was most strongly associated with disease risk. Associations also were identified with sex, breed, primary use, and participation in competitions., Conclusions and Clinical Importance: Because the risk of EPM occurrence on operations is closely tied to factors that impact exposure to opossums, their feces, and their environment, controlling these exposures may be important in preventing the occurrence of EPM.

Sarcocystis neurona is an important cause of neurological disease in horses (equine protozoal myeloencephalitis, EPM) and sea otters in the United States. In addition, EPM-like disease has been diagnosed in several other land and marine mammals. Opossums are its only definitive hosts. Little genetic diversity among isolates of S. neurona from different hosts has been reported. Here, we used 11 microsatellites to characterize S. neurona DNA isolated from natural infections in 22 sea otters (Enhydra lutris) from California and Washington and in 11 raccoons (Procyon lotor) and 1 striped skunk (Mephitis mephitis) from Wisconsin. By jointly analyzing these 34 isolates with 26 isolates previously reported, we determined that geographic barriers may limit S. neurona dispersal and that only a limited subset of possible parasite genotypes may have been introduced to recently established opossum populations. Moreover, our study confirms that diverse intermediate hosts share a common infection source, the opossum (Didelphis virginiana).

A new species of Myxosporea, Myxobolus neurotropus n. sp., is described from the brain and spinal cord of rainbow trout (Oncorhynchus mykiss) from Duncan Creek, Owyhee County, Idaho. Spores are oval, have 2 pyriform polar capsules, and possess a thick spore wall (sutural rim) with a short intracapsular offshoot. The mean spore dimensions are length 11.8 microm, width 10.8 microm, and thickness 8.8 microm. This myxozoan is compared to other described Myxobolus species found in cranial tissues of salmonids in terms of spore morphology and phylogenetic analysis. Because it is found in brain and spinal cord, it is encountered while performing screening tests for Myxobolus cerebralis, the causative agent of salmonid whirling disease. Where chronic inflammation and granulomatous lesions are associated with M. cerebralis, histological examination shows no host response to M. neurotropus n. sp. A diagnostic polymerase chain reaction (PCR) test is included as an aid in properly identifying the species.

Objective: To identify risk factors for equine protozoal myeloencephalitis (EPM) among horses examined at 11 equine referral hospitals., Design: Case-control study., Animals: 183 horses with EPM, 297 horses with neurologic disease other than EPM (neurologic controls), and 168 horses with non-neurologic diseases (non-neurologic controls) examined at 11 equine referral hospitals in the United States., Procedures: A study data form was completed for all horses. Data were compared between the case group and each of the control groups by means of bivariate and multivariate polytomous logistic regression., Results: Relative to neurologic control horses, case horses were more likely to be > or = 2 years old and to have a history of cats residing on the premises. Relative to non-neurologic control horses, case horses were more likely to be used for racing or Western performance., Conclusions and Clinical Relevance: Results indicated that cats may play a role in the natural epidemiology of EPM, that the disease is less common among horses < 2 years of age relative to other neurologic diseases, and that horses used for particular types of competition may have an increased risk of developing EPM.

Protozoal meningoencephalitis is considered to be an important cause of mortality in the California sea otter (Enhydra lutris). Thirty nine of 344 (11.3%) California (CA) and Washington state (WA) sea otters examined from 1985 to 2004 had histopathological evidence of significant protozoal meningoencephalitis. The aetiological agents and histopathological changes associated with these protozoal infections are described. The morphology of the actively multiplicative life stages of the organisms (tachyzoites for Toxoplasma gondii and merozoites for Sarcocystis neurona) and immunohistochemical labelling were used to identify infection with S. neurona (n=22, 56.4%), T. gondii (n=5, 12.8%) or dual infection with both organisms (n=12, 30.8%). Active S. neurona was present in all dual infections, while most had only the latent form of T. gondii. In S. neurona meningoencephalitis, multifocal to diffuse gliosis was widespread in grey matter and consistently present in the molecular layer of the cerebellum. In T. gondii meningoencephalitis, discrete foci of gliosis and malacia were more widely separated, sometimes incorporated pigment-laden macrophages and mineral, and were found predominantly in the cerebral cortex. Quiescent tissue cysts of T. gondii were considered to be incidental and not a cause of clinical disease and mortality. Protozoal meningoencephalitis was diagnosed more frequently in the expanding population of WA sea otters (10 of 31, 32.3%) than in the declining CA population (29 of 313, 9.3%). Among sea otters with protozoal meningoencephalitis, those that had displayed neurological signs prior to death had active S. neurona encephalitis, supporting the conclusion that S. neurona is the most significant protozoal pathogen in the central nervous system of sea otters.

Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish(-1)). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin-trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P<0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.

Objective: To determine gene transcription for cytokines in nucleated cells in CSF of horses without neurologic signs or with cervical stenotic myelopathy (CSM), West Nile virus (WNV) encephalitis, equine protozoal myeloencephalitis (EPM), or spinal cord trauma., Animals: 41 horses (no neurologic signs [n = 12], CSM [8], WNV encephalitis [9], EPM [6], and spinal cord trauma [6])., Procedures: Total RNA was extracted from nucleated cells and converted into cDNA. Gene expression was measured by use of real-time PCR assay and final quantitation via the comparative threshold cycle method., Results: Cytokine genes expressed by nucleated cells of horses without neurologic signs comprised a balance between proinflammatory tumor necrosis factor-alpha (TNF-alpha), anti-inflammatory cytokines (interleukin [IL]-10 and transforming growth factor [TGF]-beta), and Th1 mediators (interferon [IFN]-gamma). Cells of horses with CSM mainly expressed genes for TNF-alpha, TGF-beta, and IL-10. Cells of horses with WNV encephalitis mainly expressed genes for IL-6 and TGF-beta. Cells of horses with EPM mainly had expression of genes for IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, and TGF-beta. Cells from horses with spinal cord trauma had expression mainly for IL-6; IFN-gamma; TGF-beta; and less frequently, IL-2, IL-10, and TNF-alpha. Interleukin-8 gene expression was only detected in CSF of horses with infectious diseases., Conclusions and Clinical Relevance: Despite the small number of CSF samples for each group, results suggest distinct gene signatures expressed by nucleated cells in the CSF of horses without neurologic signs versus horses with inflammatory or traumatic neurologic disorders.

Trypanotolerance is the capacity of certain West-African, taurine breeds of cattle to remain productive and gain weight after trypanosome infection. Laboratory studies, comparing Trypanosoma congolense infections in trypanotolerant N'Dama cattle (Bos taurus) and in more susceptible Boran cattle (Bos indicus), confirmed the field observations. Experiments using haemopoietic chimeric twins, composed of a tolerant and a susceptible co-twin, and T cell depletion studies suggested that trypanotolerance is composed of two independent traits. The first is a better capacity to control parasitaemia and is not mediated by haemopoietic cells, T lymphocytes or antibodies. The second is a better capacity to limit anaemia development and is mediated by haemopoietic cells, but not by T lymphocytes or antibodies. Weight gain was linked to the latter mechanism, implying that anaemia control is more important for survival and productivity than parasite control. Anemia is a marker for a more complex pathology which resembles human haemophagocytic syndrome: hepatosplenomegaly, pancytopenia and a large number of hyperactivated phagocytosing macrophages in bone marrow, liver and other tissues. Thus, mortality and morbidity in trypanosome-infected cattle are primarily due to self-inflicted damage by disproportionate immune and/or innate responses. These features of bovine trypanotolerance differ greatly from those in murine models. In mice, resistance is a matter of trypanosome control dependent on acquired immunity. However, a model of anaemia development can be established using C57BL/6J mice. As in cattle, the induction of anaemia was independent of T cells but its development differed with different trypanosome strains. Identification of the molecular pathways that lead to anaemia and haemophagocytosis should allow us to design new strategies to control disease.

Objective: To assess the use of CSF testing with an indirect fluorescent antibody test (IFAT) for diagnosis of equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona., Sample Population: Test results of 428 serum and 355 CSF samples from 182 naturally exposed, experimentally infected, or vaccinated horses., Procedure: EPM was diagnosed on the basis of histologic examination of the CNS. Probability distributions were fitted to serum IFAT results in the EPM+ and EPM-horses, and correlation between serum and CSF results was modeled. Pairs of serum-CSF titers were generated by simulation, and titer-specific likelihood ratios and post-test probabilities of EPM at various pretest probability values were estimated. Post-test probabilities were compared for use of a serum-CSF test combination, a serum test only, and a CSF test only., Results: Post-test probabilities of EPM increased as IFAT serum and CSF titers increased. Post-test probability differences for use of a serum-CSF combination and a serum test only were < or = 19% in 95% of simulations. The largest increases occurred when serum titers were from 40 to 160 and pre-test probabilities were from 5% to 60%. In all simulations, the difference between pre- and post-test probabilities was greater for a CSF test only, compared with a serum test only., Conclusions and Clinical Relevance: CSF testing after a serum test has limited usefulness in the diagnosis of EPM. A CSF test alone might be used when CSF is required for other procedures. Ruling out other causes of neurologic disease reduces the necessity of additional EPM testing.

Diclazuril is a triazine-based antiprotozoal agent which may have clinical application in the treatment of equine protozoal myeloencephalomyelitis (EPM). In this study, the use of the sodium salt diclazuril to increase the apparent bioavailability of diclazuril for the treatment and prophylaxis of EPM and various other Apicomplexan mediated diseases is described. In this study, diclazuril sodium salt was synthesized and administered to horses as diclazuril sodium salt formulations. The absorption, distribution, and clearance of diclazuril sodium salt in the horse are described. Diclazuril was rapidly absorbed, with peak plasma concentrations occurring at 8-24 hours following an oral mucosal administration of diclazuril sodium salt. The mean oral bioavailability of diclazuril as Clinacox was 9.5% relative to oral mucosal administration of diclazuril sodium salt. Additionally, diclazuril in DMSO administered orally was 50% less bioavailable than diclazuril sodium salt following an oral mucosal administration. It was also shown that diclazuril sodium salt has the potential to be used as a feed additive for the treatment and prophylaxis of EPM and various other Apicomplexan mediated diseases.

Naegleria fowleri causes an acute and rapidly fatal central nervous system infection called primary amebic meningoencephalitis (PAM) in healthy children and young adults. We describe here the identification of N. fowleri isolated from the brain of one of several cows that died of PAM based on sequencing of the internal transcribed spacers, including the 5.8S rRNA genes.

A single-round polymerase chain reaction (PCR) diagnostic assay was developed from a small subunit ribosomal DNA (SSU rDNA) gene sequence to detect the myxozoan parasite Kudoa neurophila, the causative agent of myxozoan disease in the hatchery reared marine finfish, striped trumpeter Latris lineata (Forster). The assay was developed for use as a disease control management tool in a hatchery system specifically designed to research and produce marine finfish such as striped trumpeter juveniles for aquaculture. The assay is sufficiently species specific and sensitive enough to detect a small fragment of the parasite's SSU rDNA. At the lower limits of detection, the test is consistently positive to an estimated 0.1 spore or 60 fg of parasite DNA per 25 microl PCR reaction in serial dilution and positive to an estimated 0.1 spore in 25 mg of infected fish CNS tissue (4 spores g(-1). Specifically, the test is capable of detecting early stages of the life cycle within the fish host and consequently diagnosing an infection not normally detected using traditional histological techniques. The test is also effective for screening water supplies and prey species cultures throughout the hatchery system to determine bio-security efficacy, to assist in identification of an alternate or other primary fish host, to indicate the location of potential disease reservoirs, and to enable a targetted approach to disease prevention.

A free-ranging juvenile fisher (Martes pennanti) with ataxia, lethargy, stupor, and intermittent, whole-body tremors was examined postmortem. Microscopically, the fisher had protozoal meningoencephalitis caused by Sarcocystis neurona, which was confirmed by immunohistochemistry, polymerase chain reaction (PCR) and restriction fragment length polymorphism testing, and genetic sequencing. Sarcocysts found in the skeletal muscle of the fisher were negative for S. neurona by PCR, but were morphologically similar to previous light and electron microscopy descriptions of S. neurona. This is the first report of clinical neural S. neurona infection in a fisher.

Equine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neurona in horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 x 10(5) sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 x 10(8) merozoites of the WSU-1 isolate of S. neurona. Despite prolonged parasitemia and persistent infection of visceral tissues (skeletal muscle, cardiac muscle, lung, liver, and spleen) as demonstrated by PCR and culture, SCID horses did not develop neurologic signs after oral or i.v. infection. S. neurona was undetectable in the neuronal tissues of SCID horses by either PCR, immunohistochemistry, or culture. In contrast, although parasitemia was undetectable in orally infected IC horses and of only short duration in i.v. infected IC horses, four of six IC horses developed neurologic signs. S. neurona was detectable by PCR and/or culture of neural tissue but not visceral tissue of IC horses with neurologic disease. Infected SCID horses are unable to clear S. neurona from visceral tissues, but the infection does not result in neurologic signs; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but frequently experience neuroinvasion and exhibit clinical signs of neurologic disease.

Bilateral chronic granulomatous nephritis and meningoencephalitis were diagnosed on necropsy of a 2-year-old male Great Dane dog. The causative agent was identified as Balamuthia mandrillaris, based on morphologic features, immunohistochemical staining, and deoxyribonucleic acid detection using the polymerase chain reaction with newly designed primer pairs. Trophozoite and cystic forms of the amoeba were evident within the kidneys and brain parenchyma. This is the first report on a B. mandrillaris infection in a dog.

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.

A Pacific harbor seal (Phoca vitulina richardsii) was found on the central California coast with neurologic signs and labored breathing, which were unresponsive to treatment. Necropsy revealed a nonsuppurative necrotizing meningoencephalitis, a multilocular thymic cyst, and nonsuppurative cystitis and renal pyelitis. Microscopic examination revealed protozoans in the brain, thymic cyst, and bladder mucosa. Ultrastructurally, the protozoal tachyzoites were different from those of Neospora caninum, Toxoplasma gondii, and Sarcocystis neurona; the rhoptries were small and had electron-dense contents, and the organism divided by endodyogeny. Specific antibodies were not detected in serum using agglutination (N. caninum, T. gondii) and immunoblot assays (S. neurona). Immunohistochemistry for these organisms was negative. Polymerase chain reaction on brain tissue using specific primers did not amplify T. gondii deoxyribonucleic acid. The meningoencephalitis in this seal thus appears to have been caused by a novel protozoan.

Striped trumpeter, Latris lineata, are being experimentally cultured by the Tasmanian Aquaculture and Fisheries Institute at Taroona, Tasmania. Fish surviving over 30 days post-hatching have frequently developed nervous aberrations associated with a severe granulomatous meningoencephalomyelitis. The myxozoan parasite Pentacapsula neurophila sp.n. was revealed as the parasite causing the disease in the striped trumpeter juveniles. Measurements made of isolated spores indicated that the organism was distinct from all previously described Pentacapsula species. This is the first report of a marine myxozoan parasite of the genus Pentacapsula in Australian waters.

Equine protozoal myeloencephalitis developed in a three-year-old male Thoroughbred racehorse imported from the United States. The animal showed astasia five days after the onset of ataxia. Histopathologically, focal nonpurulent myelitis accompanied by hemorrhage and perivascular infiltration was observed in the fourth and fifth cervical spinal cord. Immunohistochemically, shizonts were occasionally observed and were positive for anti-Sarcocystis neurona (S. neurona) antiserum. S. neurona-specific antibodies were detected in the serum and cerebrospinal fluid by Western blot. This is the first equine protozoal myeloencephalitis case in Japan.

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by infection of the central nervous system with the protozoan parasite Sarcocystis neurona. A herd-level analysis of a cross-sectional study of serum antibodies to S. neurona in Michigan equids was conducted, using data collected in 1997 for study that included 1121 equids from 98 Michigan horse farms. Our objective was to identify specific herd-level risk factors associated with seropositivity. We tested associations between herd seroprevalence and various farm-management practices (including feed-storage methods and wildlife control). Multivariable models were developed for three strata based on relative opossum abundance (opossum districts). Herd seroprevalence ranged from 0 to 100% (median=57%). No risk factor was significantly associated with herd seroprevalence at P< or = 0.05 in all opossum districts. Our results suggest that equids living in areas with large opossum populations might be infected with S. neurona from multiple sources.

Sarcocystis sp. schizonts were found in the cerebellum of a northern gannet (Morus bassanus), exhibiting neurologic signs, found on the Florida (USA) east coast. Based upon molecular characterization of DNA isolated from the brain of the gannet, this Sarcocystis sp. appeared to be closely related, if not identical, to an unnamed Sarcocystis sp. typified by isolates 1085 and 1086 collected from feces of a Virginia opossum (Didelphis virginiana) on the east coast of Florida. Because the life cycle of this parasite appears to be land based, urban waste discharge to marine/estuarine environments may be a source of infection for marine species.

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM) in horse in the Americas. The only known definitive host for this parasite in the United States is the opossum (Didelphis virginiana); however, despite the importance of the disease, the epidemiology of the parasite in the definitive host is poorly understood. To begin addressing these data gaps, potential risk factors were evaluated for their association with the presence of sporocysts of S. neurona in opossums live-trapped in March 1999 and November 1999 to May 2000. Sporocysts of S. neurona were found in 19 of the 72 animals examined. Potential risk factors evaluated were locality, trap date, age, gender, the presence of young in the pouch of females, and body condition score. Variables that were associated with the presence of S. neurona sporocysts were used in logistic regression analysis. Of the factors examined, season and body condition score were associated with increased odds of an animal harboring sporocysts.