Multiresistance and virulence factors of Staphylococcus aureus isolated from pigs / [Multirresistência e fatores de virulência de Staphylococcus aureus isolados de suínos]

The emergence of livestock-associated methicillin-resistant Staphylococcus aureus strains (LA-MRSA) and the potential role of pigs in the evolution of these strains has led to increased interest in research of these microorganisms. However, this has contributed to a lack of research in the isolation and characterization of methicillin-susceptible S. aureus strains (MSSA). In this study, the prevalence of S. aureus in pigs in the nursery and finishing stages were analyzed. The susceptibility profiles to antibiotics, tolerance to heavy metals, and biofilm production of the isolates were evaluated using phenotypic and genotypic techniques. A total of 1,250 colonies suggestive of Staphylococcus spp. were isolated from 128 pigs, of which 63.6% (n = 795) belonged to this microbial genus. Sixty-seven colonies isolated from 34 animals (26.5%) were confirmed as S. aureus (8.4%). No strains resistant to copper, zinc, or methicillin were detected; however, all strains presented a resistance profile to at least three different classes of antimicrobials and 21 produced biofilms. These data are of concern, as they indicate the need for increased surveillance in the use of antimicrobials as well as reinforce the importance of studies on MSSA strains.(AU)

A emergência de cepas de Staphylococcus aureus resistentes à meticilina associadas à pecuária (LA-MRSA) e o papel potencial dos suínos na evolução dessas cepas têm levado ao aumento do interesse na pesquisa desses microrganismos. No entanto, isso tem contribuído para a falta de estudos sobre o isolamento e a caracterização de cepas de S. aureus sensíveis à meticilina (MSSA). Neste estudo, foi analisada a prevalência de S. aureus em suínos nas fases de creche e terminação. Os perfis de suscetibilidade aos antibióticos, a tolerância a metais pesados e a produção de biofilme dos isolados foram avaliados por meio de técnicas fenotípicas e genotípicas. Um total de 1.250 colônias sugestivas de Staphylococcus spp. foi isolado de 128 suínos, das quais 63,6% (n = 795) pertenciam a esse gênero microbiano. Sessenta e sete colônias isoladas de 34 animais (26,5%) foram confirmadas como S. aureus (8,4%). Nenhuma cepa resistente ao cobre, ao zinco ou à meticilina foi detectada; entretanto, todas as cepas apresentaram perfil de resistência a pelo menos três classes diferentes de antimicrobianos e 21 produziam biofilme. Esses dados são preocupantes, pois indicam a necessidade de maior vigilância no uso de antimicrobianos, bem como reforçam a importância de estudos com cepas de MSSA.(AU)

Renal involvement in canine leishmaniasis: a morphological and immunohistochemical study / Envolvimento renal na leishmaniose canina: estudo morfológico e imunoistoquímico

IgG and IgM deposits in kidneys of dogs with visceral leishmaniasis (VL) were studied in 25 symptomatic dogs (case) and 15 asymptomatic dogs (control) by an immunohistochemical method. All tested dogs were positive for VL by polymerase chain reaction, enzyme-linked immunosorbent assay, and indirect immunofluorescence test. Kidney fragments were submitted to immunohistochemical reaction. Many morphological patterns of distribution of subendothelial granules were identified for IgG and IgM in glomerular capillaries: global, segmental, diffuse, or focal. Intensity of immunohistochemical reaction to IgG was not significantly different when comparing the symptomatic and the asymptomatic animal groups by Fisher's exact test. IgM reactions were significantly different between groups (P<0.01). Deposits of IgM on mesangial cells and in inflammatory interstitial infiltrate were rarely seen, although IgG reactions were frequent at these sites. This study concluded that immunohistochemical reactions for IgM were more intense than those observed for IgG in canine VL, and these reactions were characterized by distribution of subendothelial granules in glomerular capillaries.

Caracterizou-se a deposição de IgG e IgM em rins de cães com leishmaniose visceral (LV) pelo uso da técnica imunoistoquímica. Foram estudados rins de 25 cães sintomáticos (caso) e de 15 cães assintomáticos (controle). Todos os animais foram positivos para leishmaniose pela reação em cadeia da polimerase, pelo ELISA e pela imunofluorescência indireta. Fragmentos renais foram submetidos à reação imunoistoquímica. Diversos padrões morfológicos de distribuição de grânulos subendoteliais de IgG e IgM foram identificados nos capilares glomerulares: global, segmentar, difuso ou focal. A intensidade da reação imunoistoquímica da IgG, medida pelo teste exato de Fisher não diferiu entre os grupos sintomáticos e assintomáticos e a intensidade de reação da IgM foi diferente entre os grupos (P<0,01). Depósitos de IgM nas células mesangiais e no infiltrado inflamatório raramente foram visualizados, no entanto as reações IgG foram freqüentemente visualizadas nesses locais. Concluiu-se que as reações de IgM foram mais intensas que as reações de IgG na LV canina e caracterizam-se pela distribuição de grânulos subendoteliais nos capilares glomerulares.

Renal involvement in canine leishmaniasis: a morphological and immunohistochemical study / Envolvimento renal na leishmaniose canina: estudo morfológico e imunoistoquímico

IgG and IgM deposits in kidneys of dogs with visceral leishmaniasis (VL) were studied in 25 symptomatic dogs (case) and 15 asymptomatic dogs (control) by an immunohistochemical method. All tested dogs were positive for VL by polymerase chain reaction, enzyme-linked immunosorbent assay, and indirect immunofluorescence test. Kidney fragments were submitted to immunohistochemical reaction. Many morphological patterns of distribution of subendothelial granules were identified for IgG and IgM in glomerular capillaries: global, segmental, diffuse, or focal. Intensity of immunohistochemical reaction to IgG was not significantly different when comparing the symptomatic and the asymptomatic animal groups by Fisher's exact test. IgM reactions were significantly different between groups (P<0.01). Deposits of IgM on mesangial cells and in inflammatory interstitial infiltrate were rarely seen, although IgG reactions were frequent at these sites. This study concluded that immunohistochemical reactions for IgM were more intense than those observed for IgG in canine VL, and these reactions were characterized by distribution of subendothelial granules in glomerular capillaries.(AU)

Caracterizou-se a deposição de IgG e IgM em rins de cães com leishmaniose visceral (LV) pelo uso da técnica imunoistoquímica. Foram estudados rins de 25 cães sintomáticos (caso) e de 15 cães assintomáticos (controle). Todos os animais foram positivos para leishmaniose pela reação em cadeia da polimerase, pelo ELISA e pela imunofluorescência indireta. Fragmentos renais foram submetidos à reação imunoistoquímica. Diversos padrões morfológicos de distribuição de grânulos subendoteliais de IgG e IgM foram identificados nos capilares glomerulares: global, segmentar, difuso ou focal. A intensidade da reação imunoistoquímica da IgG, medida pelo teste exato de Fisher não diferiu entre os grupos sintomáticos e assintomáticos e a intensidade de reação da IgM foi diferente entre os grupos (P<0,01). Depósitos de IgM nas células mesangiais e no infiltrado inflamatório raramente foram visualizados, no entanto as reações IgG foram freqüentemente visualizadas nesses locais. Concluiu-se que as reações de IgM foram mais intensas que as reações de IgG na LV canina e caracterizam-se pela distribuição de grânulos subendoteliais nos capilares glomerulares.(AU)

Naphthoimidazoles promote different death phenotypes in Trypanosoma cruzi.

SUMMARY: In a screening of 65 derivatives of natural quinones using bloodstream trypomastigotes of Trypanosoma cruzi, the 3 naphthoimidazoles derived from beta-lapachone - N1, N2 and N3--were selected as the most active. Investigation of their mode of action led to the characterization of mitochondrion, reservosomes and DNA as their main targets, and stimulated further studies on death pathways. Ultrastructural analysis revealed both autophagic (autophagosomes) and apoptotic-like (membrane blebbing) phenotypes. Flow cytometry analysis showed, in N2-treated trypomastigotes, a small increase of phosphatidylserine exposure, and a large increase in the percentage of necrosis, caused by N1 or N2. These death phenotypes were not detected in treated epimastigotes. The strong increase in labelling of monodansyl cadaverine, the inhibition of the death process by wortmannin or 3-methyladenine, the overexpression of ATG genes in treated epimastigotes, together with ultrastructural evidence point to autophagy as the predominant phenotype induced by the naphthoimidazoles. However, there are other pathways occurring concomitantly with variable intensities, justifying the need to detail the molecular features involved.

Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection.

Mast cells (MC) secrete diverse pre-stored chemical mediators that are pivotal in inflammatory and fibrotic etiologies, such as Trypanosoma cruzi-induced myocardiopathy. However, due to reduced number of cardiac MC, in situ and in vitro identification, and difficult tissue isolation, these cells are rarely addressed. In this work we optimized the identification of cardiac and peritoneal MC and developed an enzymatic method for MC isolation using control and T. cruzi-infected mice. MC were identified by: toluidine blue (TB); alcian blue (AB)/safranin (S); AB or a mixed solution composed by AB/S/TB. Previous evaluations of cardiac MC in T. cruzi infection were based on TB staining and our results using AB/S/TB solution showed an increase in, at least, five times the detection of MC. This mixed solution may improve the identification of MC populations also from skin, mucosa and tissues that are infected by other pathogens or under the influence of chronic inflammation, leading to more precise results. Furthermore, the appropriate combination of samples (frozen/unfixed/thick slices) and staining protocols can assure the best evaluation of MC. We have also isolated cardiac MC using collagenase and developed a highly efficient 60%/70% Percoll-graded protocol that enriched in, at least, 95% the population of cardiac MC.

Anti-proliferative effect of the essential oil of Cymbopogon citratus (DC) Stapf (lemongrass) on intracellular amastigotes, bloodstream trypomastigotes and culture epimastigotes of Trypanosoma cruzi (Protozoa: Kinetoplastida).

This study analyses the anti-proliferative effect of lemongrass essential oil and its main constituent (citral) on all 3 evolutive forms of Trypanosoma cruzi. Steam distillation was used to obtain lemongrass essential oil, with chemical composition determined by gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS). The IC50/24 h (concentration that reduced the parasite population by 50%) of the oil and of citral upon T. cruzi was determined by cell counting in a Neubauer chamber, while morphological alterations were visualized by scanning and transmission electron microscopy. Treatment with the essential oil resulted in epimastigote growth inhibition with IC50=126.5 microg/ml, while the IC50 for trypomastigote lysis was 15.5 microg/ml. The IC50/48 h for the Association Index (% macrophage infection x number of amastigotes per cell) was 5.1 microg/ml, with a strong inhibition of intracellular amastigote proliferation. Ultrastructural analysis demonstrated cytoplasmic and nuclear extraction, while the plasma membrane remained morphologically preserved. Our data show that lemongrass essential oil is effective against T. cruzi trypomastigotes and amastigotes, and that its main component, citral, is responsible for the trypanocidal activity. These results indicate that essential oils can be promising anti-parasitic agents, opening perspectives to the discovery of more effective drugs of vegetal origin for treatment of parasitic diseases. However, additional cytotoxicity experiments on different cell lines and tests in a T. cruzi-mouse model are needed to support these data.

Mitochondrial disruption and DNA fragmentation in Trypanosoma cruzi induced by naphthoimidazoles synthesized from beta-lapachone.

Three naphthoimidazoles presenting aromatic groups attached to the imidazole ring were the most active against trypomastigotes of Trypanosoma cruzi between 45 derivatives from beta-lapachone. N1 is active against the three forms of the parasite. In this work, we investigated N2 and N3 and analyzed the effect of the three derivatives on metacyclogenesis, endocytosis, and cell cycle. In epimastigotes, N2 and N3 blocked the cell cycle, inhibited succinate cytochrome c reductase, metacyclogenesis, and induced damage to mitochondrion, Golgi, and reservosomes. In treated trypomastigotes, there were alterations in the mitochondrion, nucleus and kinetoplast, and DNA fragmentation. Preincubation with cysteine protease inhibitors reversed the effect of N1, N2, and N3. Such reversion and ultrastructural alterations suggest the involvement of autophagy in parasite death. Ultrastructural, flow cytometry, and biochemical studies suggest that naphthoimidazoles interferes with the energetic metabolism and induces DNA fragmentation.

Morphology and preliminary enzyme characterization of the salivary glands from the predatory bug Podisus nigrispinus (Heteroptera: Pentatomidae).

Podisus nigrispinus (Dallas) is a common predator in agricultural and natural systems in Neotropical America. Its feeding strategy involves extra-oral digestion and to better understand this process its salivary glands were extracted and subjected to morphological and preliminary enzyme characterization. The salivary glands of P. nigrispinus are formed by a pair of main and accessory gland complexes. The main salivary glands are further divided into an anterior and a posterior lobe. The compartmentalization of the salivary gland complex is likely to be important for the production, activation and release of the digestive enzymes used in the extra-oral digestion of prey items. Proteases and lipase, important digestive enzymes involved in zoophagy, were detected in the salivary glands of P. nigrispinus. The prevailing trypsin-like protease activity was characterized by using the serine-protease substrate N-alpha-benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and the trypsin inhibitors tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. The KM value obtained for trypsin-like activity was 1.57 mm and the different peaks of optimum pH and temperature activity suggest the presence of multiple forms of this enzyme in P. nigrispinus. Detection of amylase activity in the salivary glands of this predator suggests its ability to digest starch and obtain nutrients from plants, which may have adaptative value under prey scarcity.

Polymerase chain reaction in detecting Leishmania sp in symptomatic and asymptomatic seropositive dogs

In human and canine renal histological studies of visceral leishmaniasis (VL), the etiological agent is rarely found in situ. The objective of this study was to evaluate PCR in identifying the etiological agent in spleen, liver, lymph node, and kidneys of VL-seropositive dogs. Twenty-five symptomatic (case group) and 15 asymptomatic (control group) VL-seropositive dogs of different breeds, sexes, and ages from Teresina, Piauí State, Brazil, were used. Serologic diagnosis was made by enzyme-linked immunosorbent assay and indirect immunofluorescence test. Animals were subjected to euthanasia and necropsy. Renal fragments were immersed in buffered formaldehyde solution. Spleen, liver, lymph node, and kidney samples were collected and frozen at -70ºC until DNA extraction. After dehydration and diaphanization, renal fragments were infiltrated and embedded in paraffin, cut at 3 mm, and stained with hematoxylin-eosin (HE). DNA amplification used an automatic thermocycler with specific Leishmania primers. All case-group dogs and 2 controls showed positive results in spleen, liver, or lymph node PCRs. There was a significant difference by Fisher exact test. In symptomatic seropositive dogs, renal histopathological evaluation showed one animal (4%) with amastigote forms of Leishmania in inflammatory infiltrate, and kidney PCRs detected Leishmania DNA in eight animals (32%). The conclusion was that PCR is more precise than the conventional histopathology in detecting the Leishmania parasite in kidney

Renal involvement in visceral leishmaniasis dogs

Visceral leishmaniasis (VL) is a zoonosis that affects both animals and man. Dogs are the etiological agent’s main reservoir. The aim of this study was to evaluate the clinical laboratory aspects and renal histopathology of VL dogs. Thirty-four symptomatic (case) and 17 asymptomatic (control) VL seropositive dogs of different breeds, sexes, and ages from Teresina, Piauí State, Brazil, were used. Diagnosis was confirmed by enzyme-linked immunosorbent assay and indirect immunofluorescence test. Clinical and laboratory tests included blood cell count and renal function analysis (urea and creatinine). Animals were subjected to euthanasia and necropsy. Renal fragments were prepared by the usual histological techniques and stained with hematoxylin-eosin and periodic acid-Schiff. Physical examination showed that lymph node hypertrophy (85.29%) and skin lesions (35.29%) were frequent in the case group. Anemia was found in 55.88% of the case and in 11.76% of the control group. There was a significant difference between groups by Fisher’s exact test. Two case-group dogs showed azotemia. Renal histopathological evaluation showed that 61.76% case and 17.65% control-group dogs had membranoproliferative glomerulonephritis. Mesangial proliferative glomerulonephritis was seen in 32.35% case and 64.70% control-group animals. There was a significant difference for both types of glomerulonephritis between groups. Amastigote forms of Leishmania were found in the renal parenchyma, in the inflammatory infiltrate of one case-group dog. We concluded that, in canine VL, regardless of the clinical signs at physical examination, the kidneys are frequently compromised

Reservosome: an endocytic compartment in epimastigote forms of the protozoan Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). Correlation between endocytosis of nutrients and cell differentiation.

Reservosomes are large membrane-bound organelles found at the posterior end of epimastigote forms of Trypanosoma cruzi, but absent in amastigotes and trypomastigotes. We have transferred bloodstream trypomastigotes to LIT medium supplemented with gold-labelled transferrin in order to analyse, at the ultrastructural level, the occurrence of reservosomes and endocytosis during the trypomastigote to epimastigote differentiation. After 24 h, the trypomastigotes differentiated into amastigotes, which adhered to each other forming large clusters. Electron-dense vesicles were detected close to the Golgi complex in cells with intermediary characteristics between amastigotes and epimastigotes, but typical reservosomes at the posterior cell tip were still absent. Transferrin-gold complexes were observed only bound to the surface of clustered cells. After 72 h, epimastigotes were observed being released from the clusters and free-swimming epimastigotes appeared, containing electron-dense vesicles at their posterior region. Typical reservosomes, labelled with transferrin-gold, were observed only in free-swimming epimastigotes. When fully differentiated epimastigotes were incubated with transferrin-gold complexes and then processed for the immunocytochemical detection of cysteine proteinase, all reservosomes were positive for the enzyme, but co-localization of both markers did not occur in all organelles. Our data demonstrate that in T. cruzi epimastigotes endocytosis is strongly related to reservosome biogenesis during the trypomastigote to epimastigote differentiation process.

Ultrastructure of the salivary glands of Rhodnius domesticus Neiva & Pinto, 1923 (Hemiptera: Reduviidae).

The fine structure of the salivary glands of the triatomine bug Rhodnius domesticus was investigated. Stereomicroscopy and scanning electron microscopy showed that each salivary gland pair contains two close and independent units: the larger is reddish and elongated (principal gland), while the smaller is round and translucent (accessory gland). The accessory gland opens at the base of the main excretion duct, which arises at the medial portion of the principal gland. An accessory duct emerges at the base of the main excretion duct, above the accessory gland opening, and runs towards the digestive tract. Transmission electron microscopy showed that both gland units are formed by a single layer of epithelial gland cells, surrounded by a thick basal lamina containing tracheolae and muscle cell fibers. Adjacent gland cells are interconnected by interdigitations of their lateral plasma membranes and by septate junctions. Microvilli are present at the apical domain of the gland cell plasma membrane, which allow faster diffusion of the saliva towards the gland lumen. Several mitochondria, abundant endoplasmic reticulum profiles and usually one elongated nucleus are observed in the gland cells. According to standard nomenclatures, the salivary gland cells can be classified as type I cells, secreting the saliva into a large gland lumen.

Haemoglobin biosynthesis site in rabbit embryo erythroid cells

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.

Morphological, biochemical and molecular characterization of Herpetomonas samuelpessoai camargoi n. subsp., a trypanosomatid isolated from the flower of the squash Cucurbita moschata.

We report the morphological, biochemical and molecular characteristics of a trypanosomatid isolated from the flower of Cucurbita moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of Phytomonas-specific molecular markers based on spliced-leader and ribosomal genes as well as by monoclonal antibodies specific for Phytomonas argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetomonas. Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. samuelpessoai than to any other species. However, the presence of spined flagellates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate-specific RAPD fingerprints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Herpetomonas samuelpessoai camargoi n. subsp. is proposed.

Spread of methicillin-resistant Staphylococcus aureus belonging to the Brazilian epidemic clone in a general hospital and emergence of heterogenous resistance to glycopeptide antibiotics among these isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) infections have been increasing at an alarming rate world-wide. MRSA epidemics due to the clonal spread of multi-resistant isolates have been described. In this paper we show the absolute predominance of MRSA strains from the Brazilian epidemic clone in a hospital in the Northeast region of Brazil and the emergence of a vancomycin and teicoplanin heterogeneous resistant subpopulation among these isolates.

Low temperature blocks fluid-phase pinocytosis and receptor-mediated endocytosis in Trypanosoma cruzi epimastigotes.

Gold-labeled albumin and transferrin were used to follow at the ultrastructural level the early events and the effect of low temperature on protein uptake by Trypanosoma cruzi epimastigotes. In parasites incubated for 5 min at 28 degrees C with protein-gold complexes, extracellular markers were found only at the cytostome and/or the flagellar pocket regions, whereas intracellular gold particles were detected inside small uncoated vesicles located nearby. Within 10 min, labeling was also observed in uncoated vesicles close to the nucleus. Only after 30 min could the tracers be detected in the reservosomes. Weak labeling in the cytostome and flagellar pocket of parasites incubated at 4 degrees C with the albumin-gold solution indicated that albumin uptake occurred by fluid-phase pinocytosis. On the other hand, intense labeling at the cytostome was observed in parasites incubated at 4 degrees C with gold-labeled transferrin, showing that receptor-mediated endocytosis occurs mainly at this site. Both proteins were absent from the cells at 4 degrees C and 12 degrees C. Raising the temperature from 12 degrees C to 28 degrees C led to transferrin labeling in intracellular vesicles dispersed throughout the cytoplasm, but not in reservosomes. Our results suggest that low temperatures affect the transport and pinching of endocytic vesicles as well as the rate of delivery of transferrin to reservosomes.

Differentiation of Trypanosoma cruzi epimastigotes: metacyclogenesis and adhesion to substrate are triggered by nutritional stress.

Differentiation of Trypanosoma cruzi epimastigotes to metacyclic trypomastigotes occurs in the insect rectum, after adhesion of the epimastigotes to the intestinal wall. We investigated the effect of the nutritional stress on the metacyclogenesis process in vitro by incubating epimastigotes in the chemically defined TAU3AAG medium supplemented with different nutrients. Addition of fetal bovine serum induced epimastigote growth but inhibited metacyclogenesis. In this medium, few parasites attached to the substrate. Ultrastructural analysis demonstrated reservosomes at the posterior end of the epimastigotes. Incubation of the cells in TAU3AAG medium containing gold-labeled transferrin resulted in high endocytosis of the marker by both adhered and free-swimming epimastigotes. No intracellular gold particles could be detected in trypomastigotes. Addition of transferrin gold complexes to adhered epimastigotes cultivated for 4 days in TAU3AAG medium resulted in decrease of both metacyclogenesis and adhesion to the substrate, as compared with parasites maintained in transferrin-free medium. Adhesion to the substrate is triggered by nutritional stress, and proteins accumulated in reservosomes are used as energy source during the differentiation. A close relationship exists among nutritional stress, endocytosis of nutrients, adhesion to the substrate, and cell differentiation in T. cruzi epimastigotes.

Immunocytochemical mapping of the hemoglobin biosynthesis site in amphibian erythroid cells.

During the past 25 years, several studies have attempted to determine the site of integration of the heme and the four globin chains in vertebrate erythroid cells that is important in the formation of the hemoglobin molecule. Mitochondrion-like organelles or hemosomes were pointed out as responsible for this task. We performed several experiments to investigate this hypothesis. The intracellular distribution of hemoglobin in amphibian erythroid cells was detected by post-embedding immuno-electron microscopy, using a polyclonal anti-human hemoglobin-proteinA-gold complex. Hemoglobin mapping showed an intense labeling in the cell cytoplasm, but none in cytoplasmic structures such as endoplasmic reticulum, mitochondria, mitochondrion-like organelles, Golgi complex, ribosomes or ferruginous inclusions. The mitochondrial fraction obtained according to the protocol described for some authors, showed by ultrastructural examination that this fraction has a heterogeneous content, also composed by microvesicles rich in cytoplasmic hemoglobin, an artifact generated by mechanical action during cell fractionation. Thus, when this fraction is lysed and its content submitted to electrophoresis, hemoglobin bands would be found inevitably, causing false-positive results, erroneously attributed to hemoglobin content of mitochondrion-like organelles. Our data do not confirm the hypothesis that the final hemoglobin biosynthesis occurs inside mitochondrion-like organelles. They suggest that the hemoglobin molecule be assembled in the erythrocyte cytoplasm outside of mitochondria or hemosomes.