Functional annotation and comparative genomics analysis of Balamuthia mandrillaris reveals potential virulence-related genes.

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.

Distribution and Current State of Molecular Genetic Characterization in Pathogenic Free-Living Amoebae.

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.

Dietary Use of Methionine Sources and Bacillus amyloliquefaciens CECT 5940 Influences Growth Performance, Hepatopancreatic Histology, Digestion, Immunity, and Digestive Microbiota of Litopenaeus vannamei Fed Reduced Fishmeal Diets.

An 8-week feeding trial investigated the effect of Fishmeal (FM) replacement by soybean meal (SBM) and poultry by-product meal (PBM) in diets supplemented with DL-Met, MET-MET (AQUAVI®), Bacillus amyloliquefaciens CECT 5940 (ECOBIOL®) and their combinations on growth performance and health of juvenile Litopenaeus vannamei. A total of six experimental diets were formulated according to L. vannamei nutritional requirements. A total of 480 shrimp (0.30 ± 0.04 g) were randomly distributed into 24 tanks (4 repetitions/each diet, 20 shrimp/tank). Shrimp were fed with control diet (CD; 200 g/Kg fishmeal) and five diets with 50% FM replacement supplemented with different methionine sources, probiotic (B. amyloliquefaciens CECT 5940) and their combinations: D1 (0.13% DL-MET), D2 (0.06% MET-MET), D3 (0.19% MET-MET), D4 (0.13% DL-MET plus 0.10% B. amyloliquefaciens CECT 5940 and D5 (0.06% MET-MET plus 0.10% B. amyloliquefaciens CECT 5940). Shrimp fed D3 and D5 had significantly higher final, weekly weight gain, and final biomass compared to shrimp fed CD (p < 0.05). Shrimp fed D2 to D5 increased the hepatopancreas epithelial cell height (p < 0.05). Digestive enzymatic activities were significantly increased in shrimp hepatopancreas' fed D3 (p < 0.05). Meanwhile, shrimp fed D1 had significant downregulation of immune-related genes (p < 0.05). Moreover, shrimp fed D3 and D5 increased the abundance of beneficial prokaryotic microorganisms such as Pseudoalteromonas and Demequina related to carbohydrate metabolism and immune stimulation. Also, shrimp fed D3 and D5 increased the abundance of beneficial eukaryotic microorganism as Aurantiochytrium and Aplanochytrium were related to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production which plays a role in growth promoting or boosting the immunity of aquatic organisms. Therefore, fishmeal could be partially substituted up to 50% by SBM and PBM in diets supplemented with 0.19% MET-MET (AQUAVI®) or 0.06% MET-MET (AQUAVI®) plus 0.10% B. amyloliquefaciens CECT 5940 (ECOBIOL®) and improve the productive performance, health, and immunity of white shrimp. Further research is necessary to investigate synergistic effects of amino acids and probiotics in farmed shrimp diets, as well as to evaluate how SBM and PBM influence the fatty acid composition of reduced fishmeal diets and shrimp muscle quality. Nevertheless, this information could be interesting to develop low fishmeal feeds for aquaculture without affecting the growth and welfare of aquatic organisms.

Effect of dietary protein and genetic line of Litopenaeus vannamei on its hepatopancreatic microbiota

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.

Transcriptional response of immune-related genes in Litopenaeus vannamei cultured in recirculating aquaculture systems with elevated CO2

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.

Effect of dietary protein and genetic line of Litopenaeus vannamei on its hepatopancreatic microbiota

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)

Stenamoeba dejonckheerei sp. nov., a Free-Living Amoeba Isolated from a Thermal Spring.

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.

Acanthamoeba mauritaniensis genotype T4D: An environmental isolate displays pathogenic behavior.

Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5 T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.

Draft Genome Sequence of White Spot Syndrome Virus Isolated from Cultured Litopenaeus vannamei in Mexico.

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.