RESUMO
Balamuthia mandrillaris is a pathogenic protozoan that causes a rare but almost always fatal infection of the central nervous system and, in some cases, cutaneous lesions. Currently, the genomic data for this free-living amoeba include the description of several complete mitochondrial genomes. In contrast, two complete genomes with draft quality are available in GenBank, but none of these have a functional annotation. In the present study, the complete genome of B. mandrillaris isolated from a freshwater artificial lagoon was sequenced and assembled, obtaining an assembled genome with better assembly quality parameter values than the currently available genomes. Afterward, the genome mentioned earlier, along with strains V039 and 2046, were subjected to functional annotation. Finally, comparative genomics analysis was performed, and it was found that homologous genes in the core genome potentially involved in the virulence of Acanthamoeba spp. and Trypanosoma cruzi. Moreover, eleven of fifteen genes were identified in the three strains described as potential target genes to develop new treatment approaches for B. mandrillaris infections. These results describe proteins in this protozoan's complete genome and help prioritize which target genes could be used to develop new treatments.
Assuntos
Acanthamoeba , Balamuthia mandrillaris , Balamuthia mandrillaris/genética , Virulência/genética , Hibridização Genômica Comparativa , Acanthamoeba/genética , GenômicaRESUMO
Mexico ranks second in shrimp (Litopenaeus vannamei) production of in Latin America with significant annual growth, however, during 2011 shrimp production fell by almost 50 % due to the presence of the white spot syndrome virus (WSSV). In this context, a life cycle analysis (LCA) and data envelopment analysis (DEA) were performed on 76 commercial farms severely affected by the presence of WSSV in northwestern Mexico. The application of this combined methodology allowed a detailed quantification of different environmental impact categories. During the presence of WSSV, there was a negative effect on the feed conversion ratio (FCR) (>40 %), higher consumption of seawater (38 %), and energy (38 %). Consequently, operational outputs related to the discharge of nitrogen and phosphorus increased by 60 and 57 %, respectively. Similarly, CO2 emissions, increased by 38 % relative to a typical year of production. Overall, the main critical points in the impact categories analyzed are related to food (98 %), use of diesel (23 %), and rearing (24 %), dominating pollutants emissions in all categories. Consequently, an improvement scenario was evaluated related to innovation in the formulation of foods supplied with immunostimulants, which confer protection against pathogenic microorganisms. This scenario lead to a reduction environmental impact of about 82 %. The results of this analysis will be a useful resource in the design of mitigation strategies with innovation processes that allow maintaining yields for shrimp producers in this region and at the same time reduce the environmental impacts generated.
Assuntos
Penaeidae , Vírus da Síndrome da Mancha Branca 1 , Animais , México , Agricultura , Água do MarRESUMO
Free-living amoebae (FLA) are protozoa widely distributed in the environment, found in a great diversity of terrestrial biomes. Some genera of FLA are linked to human infections. The genus Acanthamoeba is currently classified into 23 genotypes (T1-T23), and of these some (T1, T2, T4, T5, T10, T12, and T18) are known to be capable of causing granulomatous amoebic encephalitis (GAE) mainly in immunocompromised patients while other genotypes (T2, T3, T4, T5, T6, T10, T11, T12, and T15) cause Acanthamoeba keratitis mainly in otherwise healthy patients. Meanwhile, Naegleria fowleri is the causative agent of an acute infection called primary amoebic meningoencephalitis (PAM), while Balamuthia mandrillaris, like some Acanthamoeba genotypes, causes GAE, differing from the latter in the description of numerous cases in patients immunocompetent. Finally, other FLA related to the pathologies mentioned above have been reported; Sappinia sp. is responsible for one case of amoebic encephalitis; Vermamoeba vermiformis has been found in cases of ocular damage, and its extraordinary capacity as endocytobiont for microorganisms of public health importance such as Legionella pneumophila, Bacillus anthracis, and Pseudomonas aeruginosa, among others. This review addressed issues related to epidemiology, updating their geographic distribution and cases reported in recent years for pathogenic FLA.
RESUMO
The Chapala Lake is one of the most polluted lakes in Mexico, due to the in flow of effluents from several industrial plants, the lake accumulates pollutants such as chromium(VI) which is considered important for aquatic ecosystem. This study aimed was to evaluate the ability to decrease the concentration of chromium (VI) by Lysinibacillus macroides 2(1B)104A, isolated from sediments of the Chapala Lake. The strain was identified through 16S rRNA sequencing and phylogenetic analysis. Results showed that this strain grows in concentrations of 50, 100, 200 and 300 mgL-1 Cr(VI), in pH ranging 6 to 7, showing 79.508% reduction in concentration 50 mgL-1, determining that the reduction occurs extracellularly. Likewise, it was observed that Lysinibacillus macroides reduced the concentration of Cr(IV) in the broth, it was not observed that the bacteria could sequester Cr(VI) in the membrane or intracellularly. However, it reduced the concentration of Cr(VI) in the broth. Lysinibacillus macroides 2(1B)104A isolate showed having the ability that decrease the concentration of Cr(VI), which makes it a viable options for bioremediation of water polluted with this metal.
Assuntos
Ecossistema , Lagos , Bacillaceae , Cromo/análise , Concentração de Íons de Hidrogênio , México , Oxirredução , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Host genetics and diet can exert an influence on microbiota and, therefore, on feeding efficiency. This study evaluated the effect of genetic line (fast-growth and high-resistance) in Pacific white shrimp (Litopenaeus vannamei) on the hepatopancreatic microbiota and its association with the feeding efficiency in shrimp fed with diets containing different protein sources. Shrimp (2.08 ± 0.06 g) from each genetic line were fed for 36 days with two dietary treatments (animal and vegetable protein). Each of the four groups was sampled, and the hepatopancreatic metagenome was amplified using specific primers for the variable V4 region of the 16S rRNA gene. The PCR product was sequenced on the MiSeq platform. Nineteen bacterial phyla were detected, of which Proteobacteria was the most abundant (51.0 72.5 %), Bacteroidetes (3.6 23.3 %), Firmicutes (4.2 13.7 %), Actinobacteria (1.9 12.1 %), and Planctomycetes (1.3 9.5 %). Diet was the most influential factor in the taxonomic composition of the microbiota, while genetic line was not a strong influential factor. The results suggest that the taxonomic profile of the bacteria colonizing shrimp hepatopancreas was determined by the diet consumed, similar to what occurs in the intestine. Shrimp in the fast-growth line had greater feeding efficiency regardless of the diet supplied. Finally, the results suggest that Proteobacteria influenced (p < 0.05) the feeding efficiency of shrimp fed with a vegetable diet. Nevertheless, further studies are required to explore how shrimp genetic linediet interaction influences microbiota for probiotic development and functional food formulation for farmed shrimp according to the genetic line.(AU)
Assuntos
Animais , Proteínas Alimentares/administração & dosagem , Dieta Rica em Proteínas/efeitos adversos , Dieta Rica em Proteínas/veterinária , Ração Animal/análiseRESUMO
Host genetics and diet can exert an influence on microbiota and, therefore, on feeding efficiency. This study evaluated the effect of genetic line (fast-growth and high-resistance) in Pacific white shrimp (Litopenaeus vannamei) on the hepatopancreatic microbiota and its association with the feeding efficiency in shrimp fed with diets containing different protein sources. Shrimp (2.08 ± 0.06 g) from each genetic line were fed for 36 days with two dietary treatments (animal and vegetable protein). Each of the four groups was sampled, and the hepatopancreatic metagenome was amplified using specific primers for the variable V4 region of the 16S rRNA gene. The PCR product was sequenced on the MiSeq platform. Nineteen bacterial phyla were detected, of which Proteobacteria was the most abundant (51.0 72.5 %), Bacteroidetes (3.6 23.3 %), Firmicutes (4.2 13.7 %), Actinobacteria (1.9 12.1 %), and Planctomycetes (1.3 9.5 %). Diet was the most influential factor in the taxonomic composition of the microbiota, while genetic line was not a strong influential factor. The results suggest that the taxonomic profile of the bacteria colonizing shrimp hepatopancreas was determined by the diet consumed, similar to what occurs in the intestine. Shrimp in the fast-growth line had greater feeding efficiency regardless of the diet supplied. Finally, the results suggest that Proteobacteria influenced (p < 0.05) the feeding efficiency of shrimp fed with a vegetable diet. Nevertheless, further studies are required to explore how shrimp genetic linediet interaction influences microbiota for probiotic development and functional food formulation for farmed shrimp according to the genetic line.
Assuntos
Animais , Dieta Rica em Proteínas/efeitos adversos , Dieta Rica em Proteínas/veterinária , Proteínas Alimentares/administração & dosagem , Ração Animal/análiseRESUMO
This short-term study evaluated the effect of non-lethal high CO2 concentration on the transcriptional response of immune-related genes of Pacific white shrimp (Litopenaeus vannamei) cultured in recirculating aquaculture systems (RAS). Two experimental groups were created: high CO2 (47.67±2.04 mg L−1) and low CO2 (2.0±1.93 mg L−1). Shrimp of 8.85±1.20 g were placed randomly at a density equivalent to 100 individuals m−3 and were monitored at 6, 12, 18, and 24 h. The transcriptional response of immune-related genes was analyzed by qPCR. Gene expression of hemocyanin, prophenoloxidase, and heat shock protein 60 was downregulated at 24 h, suggesting affectations on oxygen transportation, melanization, and protein functioning of L. vannamei under high CO2 concentrations. Also, gene up-regulation of lipopolysaccharide- and ß-glucan-binding protein and cytosolic manganese superoxide dismutase can impair the bacterial recognition and antioxidant defense of shrimp exposed to high CO2 concentrations. These results suggest that concentration at about 47 mg L−1 of CO2 can significantly influence the transcriptional response modulation of immune-related genes.
Assuntos
Animais , Transcrição Gênica , Expressão Gênica , Penaeidae/imunologia , Dióxido de Carbono/administração & dosagem , Aquicultura/métodosRESUMO
Two amoeboid organisms were obtained from water samples taken from a thermal spring, "Agua Caliente", in Northwestern Mexico. The isolates were obtained when samples were cultivated at 37 °C on non-nutrient agar coated with Escherichia coli. The initial identification of the isolates was performed morphologically using light microscopy. The samples were found to have trophozoite morphology consistent with members of the genus Stenamoeba, a genus derived in 2007 from within the abolished polyphyletic genus Platyamoeba. Further analysis was performed by sequencing PCR products obtained using universal eukaryotic primers for the small subunit ribosomal ribonucleic acid (SSU rRNA) gene. Sequencing primers were designed to allow the comparison of the 18S rRNA gene sequences of the new isolates with previous sequences reported for Stenamoeba. Phylogenetic relationships among sequences from Stenamoeba were determined using Maximum Likelihood analysis. The results showed the two "Agua Caliente" sequences to be closely related, while clearly separating them from those of other Stenamoeba taxa. The degrees of sequence differentiation from other taxa were considered sufficient to allow us to propose that the Mexican isolates represent a new species.
RESUMO
Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.
Assuntos
Acanthamoeba castellanii/ultraestrutura , Balamuthia mandrillaris/ultraestrutura , Peroxinas/genética , Peroxissomos/ultraestrutura , Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Balamuthia mandrillaris/enzimologia , Balamuthia mandrillaris/genética , Catalase/metabolismo , Microscopia Eletrônica de Transmissão , Peroxinas/metabolismo , Peroxissomos/enzimologia , Peroxissomos/genética , FilogeniaRESUMO
Little is known about the prevalence of Balamuthia mandrillaris within the environment due to its difficult isolation, but once an axenic culture is established, it is relatively easy to maintain. As most of the time researchers are interested mainly in isolating B. mandrillaris from environmental samples, the flora that accompanies it becomes second in importance. Therefore, this study aimed to determine which potentially pathogenic free-living amoebae, in addition to B. mandrillaris, could be found co-inhabiting a source of natural thermal water called "Agua Caliente" (Mexico), where this amoeba has previously been detected twice by molecular methods. A third sampling from this same source was carried out to try to isolate B. mandrillaris and other free-living amoebae using 37 and 45⯰C as isolation temperatures. For PCR techniques, specific primers were used for B. mandrillaris, Naegleria fowleri, and Acanthamoeba species, plus a universal primer set for the eukaryotic 18S SSU rRNA gene for other isolated amoebae. PCR products were sequenced for final identification. 42 strains of the primary isolate were obtained, but only 34 could be kept in culture. Of them, 23 strains were identified as Naegleria lovaniensis, eight strains as Acanthamoeba jacobsi, two strains as Stenamoeba sp. and only one was identified as Vermamoeba vermiformis. The isolation of B. mandrillaris was once again not successful, but the presence of potentially pathogenic and nonpathogenic free-living amoebae is reported for the first time in this type of water in Mexico thanks to molecular methodology.
Assuntos
Amoeba/patogenicidade , Fontes Termais/parasitologia , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Acanthamoeba/patogenicidade , Amoeba/classificação , Amoeba/genética , Amoeba/isolamento & purificação , Balamuthia mandrillaris/classificação , Balamuthia mandrillaris/genética , Balamuthia mandrillaris/isolamento & purificação , Balamuthia mandrillaris/patogenicidade , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Genótipo , Fontes Termais/química , Concentração de Íons de Hidrogênio , México , Naegleria fowleri/classificação , Naegleria fowleri/genética , Naegleria fowleri/isolamento & purificação , Naegleria fowleri/patogenicidade , Filogenia , Reação em Cadeia da Polimerase , Espectrofotometria , TemperaturaRESUMO
The presence of free-living amoebae of the genera Naegleria, Acanthamoeba and Balamuthia, which contain pathogenic species for humans and animals, has been demonstrated several times and in different natural aquatic environments in the northwest of Mexico. With the aim of continuing the addition of knowledge about immunology of pathogenic free-living amoebae, 118 sera from children and adolescents, living in three villages, were studied. Humoral IgG response against B. mandrillaris, N. fowleri and Acanthamoeba sp. genotype T4, was analyzed in duplicate to titers 1: 100 and 1: 500 by enzyme-linked immunosorbent assay (ELISA). Children and adolescents ages ranged between 5 and 16 years old, with a mean of 9 years old, 55% males. All tested sera were positive for the 1: 100 dilution, and in the results obtained with the 1: 500 dilution, 116 of 118 (98.3%) were seropositive for N. fowleri, 101 of 118 (85.6%) were seropositive for Acanthamoeba sp. genotype T4, and 43 of 118 (36.4%) were seropositive for B. mandrillaris. The statistical analysis showed different distributions among the three communities and for the three species of pathogenic free-living amoebae, including age. Lysed and complete cells used as Balamuthia antigens gave differences in seropositivity.
Assuntos
Acanthamoeba/imunologia , Anticorpos Antiprotozoários/sangue , Balamuthia mandrillaris/imunologia , Infecções Protozoárias do Sistema Nervoso Central/epidemiologia , Naegleria fowleri/imunologia , Adolescente , Amebíase/epidemiologia , Amebíase/imunologia , Infecções Protozoárias do Sistema Nervoso Central/imunologia , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Criança , Pré-Escolar , Ecossistema , Encefalite/epidemiologia , Encefalite/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , México/epidemiologia , População RuralRESUMO
T-cell hybridoma assays have been widely used for the in vitro study of antigen processing and presentation because they represent an unlimited source of cells and they bypass the difficulty of maintaining T-cell clones in culture. One of the most widely used methods to assess hybridoma activation is measurement of CTLL-2 cell proliferation, which is dependent on IL-2. However, continuous culture of this cell line results in a loss of sensitivity, and significant interassay variability can occur. Therefore, our goal was to develop a method to assess T-cell hybridoma activation that was fast and sensitive with low variability based on the IL-2 secretion assay. The assay used flow cytometry detection and employed the hen egg lysozyme (HEL)-specific 3A9 hybridoma as a model. The original murine IL-2 secretion assay protocol from Miltenyi Biotec® was tested and modified; the conjugated capture antibody (anti-CD45-anti-IL-2) was added together with the stimulus at the beginning of the antigen presentation assay instead of after antigenic stimulation. With this modification, the percentage of detectable CD4+IL-2+ cells following HEL stimulation rose from 4.5% with the original protocol (0.8% without stimulus) to 94.1% (0.8% without stimulus) with the newly proposed method under the conditions evaluated in this study. This modification allowed us to evaluate the activation of hybridomas directly and more rapidly (~18h) than the reference method that assayed CTLL-2 cell proliferation using the MTT reduction assay (~48h). In conclusion, the proposed method offered a rapid alternative for screening T-cell hybridomas and evaluating their antigen-specific activation.
Assuntos
Biomarcadores/análise , Imunoensaio/métodos , Interleucina-2/metabolismo , Ativação Linfocitária , Linfócitos T/citologia , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/citologia , Linhagem Celular , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Hibridomas , Camundongos , Camundongos Endogâmicos C3H , Muramidase/imunologiaRESUMO
The first genome sequence of a Mexican white spot syndrome virus is presented here. White spot syndrome is a shrimp pandemic virus that has devastated production in Mexico for more than 10 years. The availability of this genome will greatly aid epidemiological studies worldwide, contributing to the molecular diagnostic and disease prevention in shrimp farming.
RESUMO
Since the first report of Balamuthia mandrillaris as a causative agent of granulomatous amoebic encephalitis in humans, the environmental niche of this amoeba was assumed to be restricted to soil and dust. A single isolation from water was recently made independently by us from Northern Mexico. Now we report the isolation of 8 new strains of B. mandrillaris from Mexico. This continues the pattern of an excess of isolates from North America, compared to other parts of the world. All of the new isolates are environmental isolates, 7 from water samples and one from soil. The identity of each isolate was confirmed by PCR and by examining the sequences of the mitochondrial 16S-like rRNA gene. Success in amplification was determined using comparisons of amplifications of DNA from the strain CDC: V039 and the water strain (ITSON-BM1) as positive controls. The DNA sequences of the new isolates were compared to older strains from clinical cases using phylogenetic analysis, showing very high sequence similarity. The similarity among the new isolates and with previous clinical and environmental isolates of B. mandrillaris was also examined using biochemical and immunological studies. High homogeneity of total protein products, and similarity in antigenic moiety among the eight new isolates and two controls was found. Taken together, the molecular and biochemical studies indicate very low levels of genetic variation within B. mandrillaris.
Assuntos
Balamuthia mandrillaris/genética , Água Doce/parasitologia , Proteínas de Protozoários/química , Solo/parasitologia , Balamuthia mandrillaris/classificação , Balamuthia mandrillaris/isolamento & purificação , Western Blotting , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , México , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
A survey was designed to know the concentration of Naegleria fowleri in recreational areas in Hornos, Sonora, during a year. Samples were taken monthly at La Isleta and Las Palmas and the total amoeba counts were obtained by the most probable number method (MPN). The identification of N. fowleri was made by PCR. The maximum concentration of total thermophilic amoebae was 9175 MPN/L for La Isleta and 3477 MPN/L for Las Palmas. Thermophilic Naegleria were present mainly during summer and fall. October's concentrations were up to 201 MPN/L, at both places. The maximum concentrations of N. fowleri were 201 MPN/L and 18 MPN/L for La Isleta and Las Palmas respectively, and were isolated from August to October. The presence of N. fowleri in these particular natural bodies of water reinforces the need for adaptation of preventive measures to avoid cases of primary amoebic meningoencephalitis.
Assuntos
Água Doce/parasitologia , Naegleria fowleri/crescimento & desenvolvimento , México , Naegleria fowleri/isolamento & purificação , Recreação , Estações do Ano , TemperaturaRESUMO
BACKGROUND: Primary amebic meningoencephalitis (PAM) is an emerging disease with a rapidly fatal outcome. Only eight reports of cured cases have appeared in the medical literature to date. METHODS: A 10-year-old boy developed PAM caused by Naegleria fowleri 1 week after swimming in an irrigation canal. He was admitted to our hospital after 9 h of severe headache and vomiting, fever, ataxic gait, mild confusion, and seizures were evident. Trophozoites were identified in the cerebrospinal fluid (CSF). Treatment with intravenous (i.v.) dexamethasone, amphotericin B, fluconaloze, and oral rifampicin was started. After several hours of conflicting clinical signs, recovery began, and on the third day he was conscious again. Hospital discharge occurred on day 23, after a normal brain CT scan. There was no sequel to the disease during the following 12 months. RESULTS: The amebas present in the CSF were identified and confirmed as N. fowleri after observation of wet mounts and of cultures seeded on 1.5% non-nutrient agar plates covered with Escherichia coli, vegetative and cystic forms, enflagellation experiments in distilled water at 98 degrees F, temperature tolerance testing and by indirect immunofluorescence using N. fowleri LEE antibody. The genotype was determined by PCR amplification and sequencing of the internal transcribed spacers (ITS) including the 5.8S rDNA. CONCLUSIONS: Early treatment of PAM by i.v. administration of amphotericin B and fluconazole, and oral administration of rifampicin can offer some hope of cure for this devastating disease.