CD8 T lymphocytes from B-1 cell-deficient mice down-regulates fungicidal activity of macrophages challenged with E. Cuniculi.

BACKGROUND: Encephalitozoon cuniculi is an opportunistic intracellular pathogen that establishes a balanced relationship with immunocompetent individuals depending on the activity of their CD8+ T cells lymphocytes. However, lower resistance to experimental infection with E. cuniculi was found in B-1 deficient mice (Xid), besides increased the number of CD8 T lymphocytes. Here, we evaluated the profile of CD8+ T lymphocytes from Balb/c wild-type (WT) or Balb/c Xid mice (with B-1 cell deficiency) on the microbicidal activity of macrophages challenged with E. cuniculi. METHODS: Naïve CD8 T lymphocytes from WT or Xid mice uninfected and primed CD8 T lymphocytes from WT or Xid mice infected with E cuniculi were co-cultured with macrophages previously challenged with E. cuniculi. We evaluated macrophages viability and microbicidal activity, and CD8 T lymphocytes viability and presence of activating molecules (CD62L, CD69, and CD107a). RESULTS: Macrophages co-cultured with naïve CD8 T lymphocytes from WT demonstrated high microbicidal activity. Naïve CD8 T lymphocytes obtained from WT mice had a higher expression of CD69 and LAMP-1-activating molecules compared to Xid CD8+ T lymphocytes. Primed CD8 T lymphocytes from Xid mice proliferated more than those from WT mice, however, when the expression of the activating molecule CD69 associated with the expression of CD62L was kept low. In conclusion, naïve CD8+ T lymphocytes from Xid mice, deficient in B-1 cells, they had reduced expression of activation molecules and cytotoxic activity.

Distribution of M1 and M2 macrophages in cerebral granulomas caused by Encephalitozoon cuniculi.

Encephalitozoon cuniculi spores cause severe granulomatous inflammation in the brain where mononuclear cells and macrophages infiltrate. Here, we orally infected New Zealand white rabbits with 1 × 106E. cuniculi viable spores to study the recruitment and localization of macrophages in brain granulomas. At day 30 post-infection, the positive phenotype markers iNOS (M1) and Arg-1 (M2) were located in the periphery and center of granulomas, respectively. Live intracytoplasmic spores were found only in positive Arg-1 cells. This is the first work to describe the recruitment and distribution of M1 and M2 macrophages in the brain granulomas of rabbits infected with E. cuniculi.

Opportunistic pneumonia caused by E. cuniculi in mice immunosuppressed with cyclophosphamide.

Opportunistic fungal pneumonia is a cause of concern in immunocompromised patients due to its high morbidity and mortality rates. One such opportunistic agent affecting immunocompromised patients is the microsporidia called Encephalitozoon cuniculi. This study aimed to evaluate pneumonia caused by E. cuniculi in mice treated with the immunosuppressive agent cyclophosphamide (Cy). This study also aimed to describe the immune cells associated with the microsporidial pneumonia. C57BL/6 mice were infected intravenously with E. cuniculi spores and treated with Cy (75 mg/kg/week, intraperitoneally). Thirty days post-infection, the fungal burden (qPCR), histopathological lesions, cytokine production, and the phenotype of the immune cells in the lung parenchyma were evaluated. Histologically, interstitial pneumonia with lymphocytic infiltrate was observed in the infected animals. The infiltrate mainly consisted of CD8+ and CD4+ T lymphocytes, with reduced populations of B lymphocytes and macrophages. The production of tumor necrosis factor-alpha (TNF-α) was significant in the animals of the infected groups. Also, the fungal burden was higher in the Cy-treated animals, which was confirmed by the immunohistochemical observation of spores. These results demonstrated that E. cuniculi infection of C57BL/6 mice caused lymphocytic interstitial pneumonia (characterized by a predominant lymphocytic infiltrate), which was aggravated by Cy-induced immunosuppression. Thus, these results can be used to understand the different pathological, immunological, and therapeutic aspects of lymphocytic interstitial pneumonia.

Mice with genetic and induced B-cell deficiency as a model for disseminated encephalitozoonosis.

Encephalitozoon cuniculi, an intracellular pathogen, lives in a balanced relationship with immunocompetent individuals based on the activity of T lymphocytes. We previously highlighted the greater susceptibility of B-1 cell-deficient mice (XID mice) to encephalitozoonosis. This study aimed to develop a model of disseminated and severe encephalitozoonosis in mice with combined immunodeficiency to elucidate the role of B cells. To address this objective, cyclophosphamide (Cy)-treated BALB/c and XID mice were inoculated with E. cuniculi, followed by the evaluation of the immune response and histopathological lesions. Immunosuppressed BALB/c mice manifested no clinical signs with an increase in the populations of T lymphocytes and macrophages in the spleen. Immunosuppressed and infected XID mice revealed elevated T cells, macrophages populations, and pro-inflammatory cytokines levels (IFN-γ, TNF-α, and IL-6) with the presence of abdominal effusion and lesions in multiple organs. These clinical characteristics are associated with extensive and severe encephalitozoonosis. The symptoms and lesion size were reduced, whereas B-2 and CD4+ T cells populations were increased in the spleen by transferring B-2 cells adoptive to XID mice. Moreover, B-1 cells adoptive transfer upregulated the peritoneal populations of B-2 cells and macrophages but not T lymphocytes and decreased the symptoms. Herein, we speculated the consistency in the development of severe and disseminated encephalitozoonosis in mice with genetic deficiency of Bruton's tyrosine kinase (Btk) associated with Cy immunosuppression develop with that of the models with T cell deficiency. Taken together, these data emphasized the crucial role of B cells in the protective immune response against encephalitozoonosis.

Encefalitozoonose em coelho (Oryctolagus cuniculus) / Encephalitozoonosis in rabbit (Oryctolagus cuniculus)

Background: Encephalitozoonosis is caused by the protozoan Encephalitozoon cuniculi, in rabbits, and can affect humans. The disease can be fatal and difficult to diagnose. It can be asymptomatic or cause vestibular neurological disease, paralysis, uveitis in addition to chronic kidney disease in rabbits. The transmission of the microorganism's spores occurs by ingestion, inhalation, or by the transplacental route. The aim of this work is to report a case of encephalitozoonosis in a pet rabbit (Oryctolagus cuniculus). Case: An Oryctolagus cuniculus with a history of paraparesis of the thoracic and pelvic limbs was referred for necropsy, the evolution of the clinical picture happened in one day. After death, a necropsy was performed. Organ fragments were collected, fixed, and processed routinely for histology. Macroscopically, there was evidence of hepatic lobes, without injury to the other organs. Microscopically it was observed in the white and gray substance of the telencephalon multiple circumscribed granulomas composed of a necrotic center surrounded by macrophages, giant multinucleated cells in addition to lymphocytes and plasmocytes in the periphery, delimited by fibrous connective tissue. Around the vessels, perivascular cuffs with 2 to 4 layers of lymphocytic infiltrate were observed. Besides, special staining of Schiff's Periodic Acid (PAS) and Ziehl-Neelsen was performed, in which numerous cylindrical, eosinophilic structures of approximately 2.5 x 1.0 µm were observed, compatible with E. cuniculi spores. Besides, histiocytic lymphoblasts pericoronitis was noted in the liver. There were no relevant changes in the kidney. Discussion: The diagnosis of encephalitozoonosis in rabbits was based on clinical and anatomopathological findings. Tetraparesis was the predominant sign in the present case and was justified by telencephalic lesions. This clinical sign is included in the literature but is less common than the syndrome such as head tilt and paralysis. The diagnosis of the disease is usually made by post mortem examination when it is possible to identify the spores in the lesions. Multifocal granulomatous encephalitis was the most significant finding in this case, which is also consistent with other studies. The pathogenesis of granulomatous lesions is still controversial. It is known that spores allow phagocytosis by macrophages, which induce the production of interleukins and other cytokines by TCD4 + lymphocytes, thereby activating the action of TCD8 + (cytotoxic) lymphocytes. Natural killer cells, granulocytes, other macrophages, and B lymphocytes are also recruited. Although there is such an inflammatory response, the antibodies produced are not efficient to eliminate the agent from the host organism, however, they contribute to the process of opsonization and consequent phagocytosis, facilitating the destruction of the microsporidium by macrophages. The neurological form was predominant in this case, with no chronic or ocular renal forms, possibly due to the rapid clinical evolution. Special stains were useful for visualizing intralesional spores. Although PAS staining is considered to be of little use, it was relevant in this case. The visualization of the agent made it possible to distinguish differential diagnoses, among them vestibular syndrome secondary to otitis due to pasteurellosis, toxoplasmosis, neoplasms, traumas, or diseases of the spine. Thus, a diagnosis of encephalitozoonosis was made in a rabbit through clinical and anatomopathological correlation using Ziehl-Neelsen and PAS stains.

Variation of the CD4, CD8, and MHC II cell population in granulomas of immunocompetent and immunosuppressed rabbits in Encephalitozoon cuniculi infection.

Encephalitozoon cuniculi (E. cuniculi) is a fungi-related, obligate, zoonotic, spore-forming intracellular eukaryotic microorganism. This emerging pathogen causes granulomas in brain and kidneys of infected individuals. The objective of this study was to detect the distribution of CD4, CD8 and MHCII-positive cells within granulomas in these organs in infected immunocompetent (group A) and infected immunosuppressed (group B) New Zealand white rabbits using immunohistochemistry. In brain, labeled CD4 immune cells were mainly located in the periphery of granulomas in group B. Kidneys of groups A and B, displayed CD4-positive in granulomas and were significant different when compared to brain. CD8 immune cells in brain and kidneys were disseminated in the granulomas in groups A and B; however, no significant difference was observed. MHCII-positive cells were more numerous in brain sections of group B and were significantly different when compared to kidney sections. Granulomas were not observed in control animals of group C and D. In conclusion, we identified CD4-positive cells in both the brain and kidneys of immunocompetent and immunosuppressed animals; CD8-positive cells were more numerous in brain of immunosuppressed rabbits and MHCII cells were more predominant in brain of immunocompetent rabbits. Apparently, the immunosuppression stimulated a change in the cellular phenotype of Th1- to Th2-like granulomas in brain and kidneys by an unknown mechanism. These results increase our understanding of CD4, CD8 and MHCII positive cells within the E. cuniculi granuloma microenvironment and will help in future microsporidian granulomas studies of both immunocompetent and immunosuppressed individuals.

B-1 cell-mediated modulation of M1 macrophage profile ameliorates microbicidal functions and disrupt the evasion mechanisms of Encephalitozoon cuniculi.

Here, we have investigated the possible effect of B-1 cells on the activity of peritoneal macrophages in E. cuniculi infection. In the presence of B-1 cells, peritoneal macrophages had an M1 profile with showed increased phagocytic capacity and index, associated with the intense microbicidal activity and a higher percentage of apoptotic death. The absence of B-1 cells was associated with a predominance of the M2 macrophages, reduced phagocytic capacity and index and microbicidal activity, increased pro-inflammatory and anti-inflammatory cytokines production, and higher percentual of necrosis death. In addition, in the M2 macrophages, spore of phagocytic E. cuniculi with polar tubular extrusion was observed, which is an important mechanism of evasion of the immune response. The results showed the importance of B-1 cells in the modulation of macrophage function against E. cuniculi infection, increasing microbicidal activity, and reducing the fungal mechanisms involved in the evasion of the immune response.

Effects of Homeopathic Phosphorus on Encephalitozoon cuniculi-Infected Macrophages In-Vitro.

INTRODUCTION: Encephalitozoon cuniculi (E. cuniculi), a fungus that acts as an intracellular pathogen, causes a marked neurological syndrome in many host species and is a zoonotic concern. Although no well-established treatment for this syndrome is known, previous successful clinical experience using homeopathic phosphorus has been described in which symptom remission with no mortality occurred in 40/42 animals by means of unknown immunological mechanisms. The latter observation was the main motivation for this study. OBJECTIVE: To verify, in an in-vitro model, if macrophages infected with E. cuniculi can change in function after treatment with different potencies of phosphorus. MATERIALS AND METHODS: RAW 264.7 macrophages were infected with E. cuniculi in-vitro and treated with various homeopathic potencies of phosphorus. The vehicle was used as a control solution (0.06% succussed ethanol). After 1 and 24 hours, the following parameters were analyzed: parasite internalization (by the Calcofluor staining method), lysosome activity (by the acridine orange method), cytokine/chemokine production (by the MAGPIX system), and cell ultrastructure. Automatic image analysis was used when applicable, and the experiments were performed in triplicate. RESULTS: Treatment with vehicle alone increased interleukin (IL)-6, tumor necrosis factor alpha and monocyte chemotactic protein -1 production (p ≤ 0.05) and reduced the number of internalized parasites (p ≤ 0.001). A progressive and time-dependent increase in RANTES (regulated on activation, normal T-cell expressed and secreted) and lysosome activity (p ≤ 0.002) was observed only after treatment with the highest potency of phosphorus (Phos 200cH), together with decreased apoptosis rate, intense parasite digestion, and the presence of non-internalized spores. CONCLUSIONS: Phos 200 cH has a modulatory action on the activity of infected macrophages, especially a specific increase in RANTES, a key element in the prognosis of E. cuniculi-infected and of immunosuppressed patients bearing infections.

Quantitative analysis of TNF-α, IL-4, and IL-10 expression, nitric oxide response, and apoptosis in Encephalitozoon cuniculi-infected rabbits.

The expression of tumor necrosis factor (TNF) -α, interleukin (IL) -4 and IL-10, as well as apoptosis and nitric oxide (NO) levels were measured in the brain and kidneys of immunocompetent and immunosuppressed New Zealand White rabbits infected with Encephalitozoon cuniculi. All of the animals had clinical signs histopathological lesions compatible with encephalitozoonosis and were E. cuniculi-positive by using a carbon immunoassay test. Encephalitozoon cuniculi infection promoted the expression of TNF-α and NO production in the kidneys of infected rabbits, and a synergic effect was observed in animal treated with dexamethasone. The IL-4 expression was similar in the brain and kidneys of infected rabbits, regardless of their immunologic status. The IL-10 mRNA expression in the brain of infected immunosuppressed rabbits was elevated when compared with positive controls. Apoptosis of granuloma mononuclear-like cells was detected in immunocompetent E. cuniculi-infected rabbits, but it was more evident in infected-immunosuppressed animals. Nitric oxide levels were elevated both in immunocompetent and immunosuppressed infected animals, but it was more apparent in the kidneys. These data suggest that modulation of the immune response by E. cuniculi could contribute to the survival of the parasite within phagocytic cells in granulomas via an as yet undetermined mechanism.

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis.

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8+ T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID + B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-γ, TNF-α and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.

Diabetes mellitus increases the susceptibility to encephalitozoonosis in mice.

Microsporidiosis are diseases caused by opportunistic intracellular fungi in immunosuppressed individuals, as well as in transplanted patients, the elderly and children, among others. Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia and decreased T cell response, neutrophil function, humoral immunity failure, increasing the susceptibility to infections. Here, we investigated the susceptibility of streptozotocin (STZ)-induced type I diabetic and/or immunosuppressed mice to encephalitozoonosis by Encephalitozoon cuniculi. Microscopically, granulomatous hepatitis, interstitial pneumonia and pielonephritis were observed in all infected groups. STZ treatment induced an immunossupressor effect in the populations of B (B-1 and B2) and CD4+ T lymphocytes. Moreover, infection decreased CD4+ and CD8+ T lymphocytes and macrophages of DM mice. Furthermore, infection induced a significant increase of IL-6 and TNF-α cytokine serum levels in DM mice. IFN-γ, the most important cytokine for the resolution of encephalitozoonosis, increased only in infected mice. In addition to the decreased immune response, DM mice were more susceptible to encephalitozoonosis, associated with increased fungal burden, and symptoms. Additionally, cyclophosphamide immunosuppression in DM mice further increased the susceptibility to encephalitozoonosis. Thus, microsporidiosis should be considered in the differential diagnosis of comorbidities in diabetics.

Histochemical study of Encephalitozoon cuniculi spores in the kidneys of naturally infected New Zealand rabbits.

Encephalitozoon cuniculi is an important microsporidian pathogen that is considered an emergent, zoonotic, and opportunistic. It infects both domestic and laboratory rabbits, generating severe chronic interstitial and granulomatous nephritis with fibrosis and granulomatous encephalitis. Encephalitozoonosis is diagnosed in paraffin-embedded sections by examining the spores in the host tissues. The spores are difficult to observe when the samples are stained with hematoxylin and eosin (H&E), particularly when there is an inflammatory reaction and tissue damage. The spores are easily mistaken for other microorganisms, such as fungi (yeasts), protozoa, and bacteria. In our study, we used kidney samples from E. cuniculi-positive rabbits and employed 14 recommended histologic stains for detecting microsporidia spores: alcian blue, calcofluor white, Giemsa, Gram, Grocott, H&E, Luna, Luxol fast blue, Masson trichrome, modified trichrome stain (MTS), periodic acid-Schiff reaction (PAS), Van Gieson, Warthin-Starry (WS), and Ziehl-Neelsen (ZN).We concluded that MTS and Gram stain, detected by light microscopy, and calcofluor white stain, detected by ultraviolet light microscopy, are the best stains for detecting spores of E. cuniculi in paraffin-embedded tissues from infected rabbits. These stains were superior to WS, ZN, Giemsa, and PAS for identifying spores without background "noise" or monochromatic interference. Also, they allow individual spores to be discerned in paraffin-embedded tissues. MTS allows observation of the polar tube, polaroplast, and posterior vacuole, the most distinctive parts of the spore.

B-1 cell decreases susceptibility to encephalitozoonosis in mice.

Encephalitozoon cuniculi is an opportunist intracellular pathogen of mammals. The adaptive immune response is essential to eliminate E. cuniculi, but evidence is mounting that the response initiated by the innate immune response may ultimately define whether or not the parasite can survive. B-1 cells may act as antigen-presenting cells or differentiate into phagocytes, playing different roles in many infection models. However, the role of these cells in the dynamics of Encephalitozoon sp. infections is still unknown. To investigate the role of B-1 cells in E. cuniculi infection, BALB/c and BALB/c XID (B-1 cells deficient) mice were infected with E. cuniculi spores. Cytometric analyses of peritoneal cells showed that B-1 cells and macrophages increased significantly in infected BALB/c mice compared to uninfected controls. Despite the increase in the number of CD4+ and CD8+ lymphocytes in XID mice, these animals were more susceptible to infection as evidenced histologically with more prominent inflammatory lesions and parasite burden. Pro-inflammatory cytokines increased in both infected BALB/c and BALB/c XID mice. To confirm B-1 cells role in encephalitozoonosis, we adoptively transferred B-1 cells to BALB/c XID mice and this group showed few symptoms and microscopic lesions, associated with an increased in cytokines. Together, these results suggest that B-1 cells may increase the resistance of BALB/c mice to encephalitozoonosis, evidencing for the first time the important role of B-1 lymphocytes in the control of microsporidia infection.

Interferon γ and interleukin 10 responses in immunocompetent and immunosuppressed New Zealand White rabbits naturally infected with Encephalitozoon cuniculi.

Levels of interferon (IFN)-γ and interleukin (IL)-10 were measured in the serum of immunocompetent and immunosuppressed New Zealand White rabbits naturally infected with Encephalitozoon cuniculi. IFN-γ levels were elevated in infected rabbits, and a synergic effect was observed in animals treated with the immunosuppressive agent dexamethasone (Dex). The role of IL-10 in infected rabbits remains unclear, as IL-10 levels were similar to those of negative controls. Dex appeared to exhibit a proinflammatory effect, as IFN-γ levels were elevated in infected immunosuppressed rabbits. Similarly, Dex exhibited a synergic effect in infected immunosuppressed rabbits, as evidenced by the elevation in IFN-γ production. These data indicate that the immune response to this glucocorticoid should be considered in the design of future animal model studies of immunosuppression.

Encephalitozoon intestinalis Inhibits Dendritic Cell Differentiation through an IL-6-Dependent Mechanism.

Microsporidia are a group of intracellular pathogens causing self-limited and severe diseases in immunocompetent and immunocompromised individuals, respectively. A cellular type 1 adaptive response, mediated by IL-12, IFNγ, CD4+, and CD8+ T cells has been shown to be essential for host resistance, and dendritic cells (DC) play a key role at eliciting anti-microsporidial immunity. We investigated the in vitro response of DC and DC precursors/progenitors to infection with Encephalitozoon intestinalis (Ei), a common agent of human microsporidosis. Ei-exposed DC cultures up-regulated the surface expression of MHC class II and the costimulatory molecules CD86 and CD40, only when high loads of spores were used. A vigorous secretion of IL-6 but not of IL-1ß or IL-12p70 was also observed in these cultures. Ei-exposed DC cultures consisted of immature infected and mature bystander DC, as assessed by MHC class II and costimulatory molecules expression, suggesting that intracellular Ei spores deliver inhibitory signals in DC. Moreover, Ei selectively inhibited the secretion of IL-12p70 in LPS-stimulated DC. Whereas Ei-exposed DC promoted allogeneic naïve T cell proliferation and IL-2 and IFNγ secretion in DC-CD4+ T cell co-cultures, separated co-cultures with bystander or infected DCs showed stimulation or inhibition of IFNγ secretion, respectively. When DC precursors/progenitors were exposed to Ei spores, a significant inhibition of DC differentiation was observed without shifting the development toward cells phenotypically or functionally compatible with myeloid-derived suppressor cells. Neutralization experiments demonstrated that this inhibitory effect is IL-6-dependent. Altogether this investigation reveals a novel potential mechanism of immune escape of microsporidian parasites through the modulation of DC differentiation and maturation.

Microsporidiosis in rabbits of a laboratory animal facility: histopathological findings / Microsporidiose em coelhos de biotério de criação: achados histopatológicos

Encephalitozoonosis is a zoonotic microsporidiosis present in creations of rabbits and one of the most important diseases reported in rabbits. Thirty rabbits from commercial breeding were received for quarantine in a laboratory animal facility for scientific purposes and during the period showed symptoms as: loss of appetite, anorexia, head tilt, mucopurulent conjunctivitis, nasal serous secretion and subcutaneous abscess in the neck, some came to death and the rest were euthanized after not responding to treatment. The histopathological findings showed lesions in kidneys, liver, spleen and brain with the presence of cysts similar to Encephalitozoon in part of the organsidentifying an outbreak of microsporidiosis in rabbits.

A encefalitozoonose é uma microsporidiose zoonótica presente em criações de coelhos e é uma das mais importantes doenças relatadas em coelhos. Trinta coelhos provenientes de criação comercial foram recebidos para quarentena em um biotério de criação para fins científicos e durante o período começaram a apresentar sintomas diversos, como: inapetência, anorexia, torcicolo, conjuntivite mucopurulenta, secreção nasal serosa e abscesso subcutâneo na região do pescoço, vindo alguns a óbito e sendo os restantes eutanasiados após não responderem ao tratamento instituído. Os achados histopatológicos demonstraram lesões nos rins, fígado, baço e cérebro com a presença de cistos semelhantes a Encephalitozoon em parte dos órgãos identificando um surto de microsporidiose em coelhos.

Microsporidiosis in rabbits of a laboratory animal facility: histopathological findings / Microsporidiose em coelhos de biotério de criação: achados histopatológicos

Encephalitozoonosis is a zoonotic microsporidiosis present in creations of rabbits and one of the most important diseases reported in rabbits. Thirty rabbits from commercial breeding were received for quarantine in a laboratory animal facility for scientific purposes and during the period showed symptoms as: loss of appetite, anorexia, head tilt, mucopurulent conjunctivitis, nasal serous secretion and subcutaneous abscess in the neck, some came to death and the rest were euthanized after not responding to treatment. The histopathological findings showed lesions in kidneys, liver, spleen and brain with the presence of cysts similar to Encephalitozoon in part of the organsidentifying an outbreak of microsporidiosis in rabbits. (AU)

A encefalitozoonose é uma microsporidiose zoonótica presente em criações de coelhos e é uma das mais importantes doenças relatadas em coelhos. Trinta coelhos provenientes de criação comercial foram recebidos para quarentena em um biotério de criação para fins científicos e durante o período começaram a apresentar sintomas diversos, como: inapetência, anorexia, torcicolo, conjuntivite mucopurulenta, secreção nasal serosa e abscesso subcutâneo na região do pescoço, vindo alguns a óbito e sendo os restantes eutanasiados após não responderem ao tratamento instituído. Os achados histopatológicos demonstraram lesões nos rins, fígado, baço e cérebro com a presença de cistos semelhantes a Encephalitozoon em parte dos órgãos identificando um surto de microsporidiose em coelhos. (AU)

[Prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi in HIV positive patients to Maracaibo, Venezuela]. / Prevalencia de Encephalitozoon intestinalis y Enterocytozoon bieneusi en pacientes VIH positivos de Maracaibo, Venezuela.

Microsporidioses are considered emerging and opportunistic infections in immunocompromised individuals worldwide. The purpose of this study was to identify the species of intestinal microsporidia in patients with HIV-AIDS from the Servicio Autónomo Hospital Universitario de Maracaibo, Venezuela (SAHUM). Fecal samples were collected from 50 patients with confirmed diagnosis of HIV, during the years 2007 and 2008; the CD4 values were obtained from 42 patients. The samples were analyzed by separate PCRs to identify Encephalitozoon intestinalis and Enterocytozoon bieneusi. Microsporidia species showed a 36% prevalence: ten patients had Encephalitozoon intestinalis, four Enterocytozoon bieneusi and four both species. An inverse and statistically significant relationship between the CD4 count and the presence of microsporidia in the fecal sample was also found. It is remarkable the high prevalence of microsporidia species observed in the HIV patients studied, with a predominance of E. intestinalis.

Effect of three drugs against Encephalitozoon cuniculi infection in immunosuppressed mice.

Microsporidia comprise a large group of obligate intracellular parasites. The microsporidian Encephalitozoon cuniculi causes disseminated infection in immunosuppressed patients with HIV, cancer, or transplants and in the elderly. In vivo and in vitro studies on the effectiveness of drugs are controversial. Currently, there is no effective treatment. We tested albendazole, albendazole sulfoxide, metronidazole, and cyclosporine in mice immunosuppressed with cyclophosphamide and inoculated by the intraperitoneal route with 10(7) E. cuniculi spores. One week after experimental inoculation, the mice were treated with albendazole, albendazole sulfoxide, metronidazole, and cyclosporine. Histological and morphometric analyses were performed to compare the treated groups. The state of immunosuppression was evaluated by phenotyping CD4(+) and CD8(+) T cells by flow cytometry. Nontreated mice showed acute disseminated and fatal encephalitozoonosis. The treatment with benzimidazoles significantly reduced infection until 30 days posttreatment (p.t.), but at 60 days p.t., the infection had recurred. Metronidazole decreased infection by a short time, and cyclosporine was not effective. All animals were immunosuppressed by all the experiments, as demonstrated by the low number of CD4(+) and CD8(+) T cells. We conclude that no drug was effective against E. cuniculi, but the benzimidazoles controlled the infection transiently.

Prevalencia de Encephalitozoon intestinalis y Enterocytozoon bieneusi en pacientes VIH positivos de Maracaibo, Venezuela / Prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi in HIV positive patients to Maracaibo, Venezuela

Los microsporidios pueden provocar infecciones emergentes y oportunistas en individuos inmunocomprometidos de todo el mundo. Se realizó éste estudio para identificar las especies de microsporidios intestinales presentes en pacientes con VIH-SIDA del Servicio Autónomo Hospital Universitario de Maracaibo (SAHUM). Se recolectaron 50 muestras fecales de individuos con diagnóstico confirmado de VIH durante los años 2007-2008; se obtuvieron las cifras de CD4 de solo 42 pacientes. Las muestras se analizaron mediante PCR separadas para la identificación de Encephalitozoon intestinalis y Enterocytozoon bieneusi. Las especies de microsporidios presentaron un 36% de prevalencia, 10 pacientes presentaron Encephalitozoon intestinalis, 4 Enterocytozoon bieneusi y 4 ambas especies. Se determinó una relación inversamente proporcional y estadísticamente significativa entre el contaje de CD4 y la presencia de microsporidios en la muestra fecal. Es destacable la elevada prevalencia de especies de microsporidios observada en los pacientes VIH estudiados, donde predominó E. intestinalis.

Microsporidioses are considered emerging and opportunistic infections in immunocompromised individuals worldwide. The purpose of this study was to identify the species of intestinal microsporidia in patients with HIV-AIDS from the Servicio Autónomo Hospital Universitario de Maracaibo, Venezuela (SAHUM). Fecal samples were collected from 50 patients with confirmed diagnosis of HIV, during the years 2007 and 2008; the CD4 values were obtained from 42 patients. The samples were analyzed by separate PCRs to identify Encephalitozoon intestinalis and Enterocytozoon bieneusi. Microsporidia species showed a 36% prevalence: ten patients had Encephalitozoon intestinalis, four Enterocytozoon bieneusi and four both species. An inverse and statistically significant relationship between the CD4 count and the presence of microsporidia in the fecal sample was also found. It is remarkable the high prevalence of microsporidia species observed in the HIV patients studied, with a predominance of E. intestinalis.