Occurrence of Arcellidae (Amorphea, Arcellinida) in a coastal stream in the State of Rio de Janeiro

The aim of this study was to elaborate a taxonomic survey on the testate amoebae of the Family Arcellidaein coastal streams in the State of Rio de Janeiro. Six samplingswere conducted in Ubatiba coastal stream (Maricá, RJ). In total, 130 liters water were filtered through a conical net of 60-μm mesh and preserved in 4% formalin. Organisms were identified with the aid of an inverted trinocular microscope. The morphological characteristics (lobose testate amoebae with shell composed of granular chitinoid elements) of each species were recorded. The species were described and illustrated. For the verification of new taxa records of Arcellidae in Rio de Janeiro and their distribution in Brazil, a search based on indexarticles by Scopus, Web of Science and Google Scholar using the keywords "tecamebas", "testate amoebae", "Arcella", "Arcellidae", "Brazil" and "Rio de Janeiro", was performed. Eight Arcellidaespecies were recorded. Some ecological and taxonomic information was provided. Due to the small amount of information on testate amoebae, this study is important because it reduces the knowledge gap regarding this community in the State of Rio de Janeiro. Furthermore, we suggest new studies on species identification to be conducted to expand regional knowledge about these organisms.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways.

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways.

Increasing importance of Balamuthia mandrillaris.

Balamuthia mandrillaris is an emerging protozoan parasite, an agent of granulomatous amoebic encephalitis involving the central nervous system, with a case fatality rate of >98%. This review presents our current understanding of Balamuthia infections, their pathogenesis and pathophysiology, and molecular mechanisms associated with the disease, as well as virulence traits of Balamuthia that may be potential targets for therapeutic interventions and/or for the development of preventative measures.

Neoparamoeba perurans is a cosmopolitan aetiological agent of amoebic gill disease.

Previously we described a new member of the Neoparamoeba genus, N. perurans, and showed that it is an agent of amoebic gill disease (AGD) of Atlantic salmon Salmo salar cultured in southeast Tasmania, Australia. Given the broad distribution of cases of AGD, we were interested in extending our studies to epizootics in farmed fish from other sites around the world. Oligonucleotide probes that hybridise with the 18S rRNA of N. perurans, N. branchiphila or N. pemaquidensis were used to examine archival samples of AGD in Tasmania as well as samples obtained from 4 host fish species cultured across 6 countries. In archival samples, N. perurans was the only detectable amoeba, confirming that it has been the predominant aetiological agent of AGD in Tasmania since epizootics were first reported. N. perurans was also the exclusive agent of AGD in 4 host species across 6 countries. Together, these data show that N. perurans is a cosmopolitan agent of AGD and, therefore, of significance to the global mariculture industry.

First cases of amoebic gill disease (AGD) in Norwegian seawater farmed Atlantic salmon, Salmo salar L., and phylogeny of the causative amoeba using 18S cDNA sequences.

Amoebic gill disease (AGD) was observed in seawater farmed Atlantic salmon at four geographically distant locations on the western coast of Norway. To the best of our knowledge, these are the first detected AGD outbreaks in Norway. The outbreaks lasted for 7-12 weeks in late autumn 2006 and were for the most part concurrent. The crude, cumulative mortality was in the range of 12-20% at three farms and 82% at a fourth. The histopathology showed uniform parasomal amoebae in lesions characteristic for AGD. Another gill disease, proliferative gill inflammation (PGI), was also present to a variable degree and the distinction between the two gill problems is discussed. Seawater temperatures were 3.5 degrees C higher than average before disease outbreaks, which subsided in early winter. The geographical and time pattern of these outbreaks strongly indicates simultaneous infection from the marine environment. Two contiguous 18S cDNA sequences, obtained by reverse transcriptase PCR from gill tissue with AGD-related lesions, showed highest similarity (99.2%) to a newly recognized species designated Neoparamoeba perurans and maximum likelihood analysis demonstrates that they represent Norwegian strains of this Neoparamoeba lineage.

Phylogeny of Neoparamoeba strains isolated from marine fish and invertebrates as inferred from SSU rDNA sequences.

We characterised 9 strains selected from primary isolates referable to Paramoeba/Neoparamoeba spp. Based on ultrastructural study, 5 strains isolated from fish (amoebic gill disease [AGD]-affected Atlantic salmon and dead southern bluefin tuna), 1 strain from netting of a floating sea cage and 3 strains isolated from invertebrates (sea urchins and crab) were assigned to the genus Neoparamoeba Page, 1987. Phylogenetic analyses based on SSU rDNA sequences revealed affiliations of newly introduced and previously analysed Neoparamoeba strains. Three strains from the invertebrates and 2 out of 3 strains from gills of southern bluefin tunas were members of the N. branchiphila clade, while the remaining, fish-isolated strains, as well as the fish cage strain, clustered within the clade of N. pemaquidensis. These findings and previous reports point to the possibility that N. pemaquidensis and N. branchiphila can affect both fish and invertebrates. A new potential fish host, southern bluefin tuna, was included in the list of farmed fish endangered by N. branchiphila. The sequence of P. eilhardi (Culture Collection of Algae and Protozoa [CCAP] strain 1560/2) appeared in all analyses among sequences of strain representatives of Neoparamoeba species, in a position well supported by bootstrap value, Bremer index and Bayesian posterior probability. Our research shows that isolation of additional strains from invertebrates and further analyses of relations between molecular data and morphological characters of the genera Paramoeba and Neoparamoeba are required. This complexity needs to be considered when attempting to define molecular markers for identification of Paramoeba/Neoparamoeba species in tissues of fish and invertebrates.

Cytopathogenicity of Balamuthia mandrillaris, an opportunistic causative agent of granulomatous amebic encephalitis.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis in humans and other mammals. Balamuthia mandrillaris is highly cytopathic but, in contrast to the related Acanthamoeba, does not feed on bacteria and seems to feed only on eukaryotic cells instead. Most likely, the cytopathogenicity of B. mandrillaris is inseparable from its infectivity and pathogenicity. To better understand the mechanisms of B. mandrillaris cytopathogenicity, an assay for measuring amebic cytolytic activity was adapted that is based on the release of a reporter enzyme by damaged target cells. The ameba is shown to lyse murine mastocytoma cells very efficiently in a time- and dose-related manner. Furthermore, experiments involving semipermeable membranes and phagocytosis inhibitors indicate that the cytolytic activity of B. mandrillaris is essentially cell contact-dependent. Standard and fluorescence light microscopy, as well as scanning and transmission electron microscopy support and extend these findings at the ultrastructural level.

Amoebic gill disease (AGD)-affected Atlantic salmon, Salmo salar L., are resistant to subsequent AGD challenge.

There is inconsistent evidence of resistance of Atlantic salmon, Salmo salar L., to amoebic gill disease (AGD). Here, evidence is presented that demonstrates that Atlantic salmon exposed and subsequently challenged with AGD are more resistant than naïve control fish. Seventy-three per cent of Atlantic salmon previously exposed to AGD survived to day 35 post-challenge compared with 26% exposed to Neoparamoeba sp. for the first time, yet the gill pathology of surviving naïve control or previously exposed fish was not significantly different. Development of resistance to AGD is associated with anti-Neoparamoeba sp. antibodies that were detectable in serum of 50% of surviving Atlantic salmon previously exposed to AGD. However, anti-Neoparamoeba sp. antibodies were not detectable in cutaneous mucus of resistant fish. Increased resistance of Atlantic salmon after secondary Neoparamoeba sp. infection and detection of specific serum antibodies provides support for the development of a vaccine for AGD.

Concentration effects of Winogradskyella sp. on the incidence and severity of amoebic gill disease.

To study the concentration effects of the bacterium Winogradskyella sp. on amoebic gill disease (AGD), Atlantic salmon Salmo salar were pre-exposed to 2 different doses (10(8) or 10(10) cells 1(-1)) of Winogradskyella sp. before being challenged with Neoparamoeba spp. Exposure of fish to Winogradskyella sp. caused a significant increase in the percentage of AGD-affected filaments compared with controls challenged with Neoparamoeba only; however, these percentages did not increase significantly with an increase in bacterial concentration. The results show that the presence of Winogradskyella sp. on salmonid gills can increase the severity of AGD.

Cultured gill-derived Neoparamoeba pemaquidensis fails to elicit amoebic gill disease (AGD) in Atlantic salmon Salmo salar.

Amoebic gill disease (AGD) affects the culture of Atlantic salmon Salmo salar in the southeast of Tasmania. The disease is characterised by the presence of epizoic Neoparamoeba spp. in association with hyperplastic gill tissue. Gill-associated amoebae trophozoites were positively selected by plastic adherence for culture in seawater, where they proliferated using heat-killed E. coli as a nutrient source. One isolate of gill-harvested amoebae designated NP251002 was morphologically consistent to N. pemaquidensis under light, fluorescence and transmission electron microscopy. Rabbit anti-N. pemaquidensis antiserum bound to NP251002, and N. pemaquidensis small subunit (SSU) ribosomal DNA (18S rDNA) was detected in NP251002 genomic DNA preparations using PCR. A high degree of similarity in the alignment of the NP251002 18S rDNA PCR amplicon sequence with reference isolates of N. pemaquidensis suggested conspecificity. While short-term culture (72 h) of gill-harvested amoebae does not affect the capacity of amoebae to induce AGD, Atlantic salmon challenged with NP251002 after the trophozoites had been 34 and 98 d in culture exhibited neither gross nor histological evidence of AGD. It is not known if NP251002 were avirulent at the time of isolation, had down-regulated putative virulence factors or virulence was inhibited by the culture conditions. Therefore, the time in culture could be a limiting factor in maintaining virulence using the culture technique described here.

Balamuthia mandrillaris stimulates interleukin-6 release in primary human brain microvascular endothelial cells via a phosphatidylinositol 3-kinase-dependent pathway.

Balamuthia mandrillaris is an emerging protozoan parasite that can cause fatal granulomatous encephalitis. Haematogenous spread is a likely route prior to entry into the central nervous system (CNS), but it is not clear how circulating amoebae cross the blood-brain barrier. Using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, we determined HBMEC inflammatory response to B. mandrillaris and the underlying mechanisms associated with this response. We demonstrated that HBMEC incubated with B. mandrillaris released significantly higher levels of interleukin-6 (IL-6) (>400 pg/ml) as compared with less than 50 pg/ml in HBMEC incubated alone. Western blotting assays determined that B. mandrillaris specifically activates phosphatidylinositol 3-kinase (PI3K). By using LY294002, a PI3K inhibitor, as well as by using HBMEC expressing dominant-negative PI3K, we have identified PI3K as an important mediator of B. mandrillaris-mediated IL-6 release. We conclude that B. mandrillaris induces HBMEC signalling pathways, which lead to IL-6 release. This is the first time PI3K has been shown to play a crucial role in B. mandrillaris-mediated IL-6 release in HBMEC.

Balamuthia mandrillaris, an opportunistic agent of granulomatous amebic encephalitis, infects the brain via the olfactory nerve pathway.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis (GAE) in humans and other mammals. Its supposed routes of infection have been largely assumed from what is known about Acanthamoeba spp. and Naegleria fowleri, other free-living amebae and opportunistic encephalitis agents. However, formal proof for any migratory pathway, from GAE patients or from animal models, has been lacking. Here, immunodeficient mice were infected with B. mandrillaris amebae by intranasal instillation, the most likely natural portal of entry. By means of classical and immunohistology, the amebae are shown to adhere to the nasal epithelium, progress along the olfactory nerves, traverse the cribriform plate of the ethmoid bone, and finally infect the brain. A similar invasion pathway has been described for N. fowleri. The data suggest that the olfactory nerve pathway is a likely route for natural infection of the brain by B. mandrillaris amebae.

In vitro interactions between Neoparamoeba sp. and Atlantic salmon epithelial cells.

Neoparamoeba sp., including the putative aetiological agent of amoebic gill disease in cultured fish (N. pemaquidensis), were incubated in vitro with an Atlantic salmon gill epithelium (RGE-2) cell line. Proliferation by the amoeba population was dependent upon culture osmolarity; no growth occurred at 330 mm x kg(-1) but a sixfold increase was observed at 1000 mm x kg(-1). At 780 mm x kg(-1) there was a fourfold increase in the amoeba population but a concurrent decrease in RGE-2 cell density that was significantly greater than that caused by the high culture osmolarity alone. This apparent cytopathic effect (CPE) developed rapidly and resulted in complete cytolysis of the monolayer in 5 days. CPE occurred in multiple foci and presented as cell vacuolation, rounding and clumping, and the rapid clearance of large areas of the cell monolayer. The possibility that CPE is because of the presence of Neoparamoeba sp. derived cytolytic products is discussed in the context of the pathology of the disease in vivo and the occurrence of secreted cytopathogenic compounds in other amoeba species.

Experimental amoebic gill disease of Atlantic salmon, Salmo salar L.: further evidence for the primary pathogenic role of Neoparamoeba sp. (Page, 1987).

Amoebic gill disease (AGD) has been attributed to infection by Neoparamoeba sp. The causal mechanisms for AGD lesion development and the primary pathogenic role of Neoparamoeba sp. require elucidation. Three groups of Atlantic salmon were exposed to viable gill isolated amoebae, to sonicated amoebae, or to sea water containing viable amoebae without direct contact with gill epithelia. Fish were removed 8 days post-exposure and the gills assessed histologically for AGD. AGD occurred only when fish were exposed to viable trophozoites. Consequently, in an accompanying experiment, infection was evaluated histologically at 12, 24 and 48 h post-exposure in three groups of salmon, one group being mechanically injured 12 h prior to exposure. A progressive host response and significant increase (P < 0.001) in the numbers of attached amoebae was apparent over the 48-h duration in undamaged hemibranchs in both treatment groups. There were no significant differences to mucous cell populations. Attachment of Neoparamoeba sp. to damaged gill filaments was significantly reduced (P < 0.05) by 48 h post-exposure. These data further confirm and describe the primary pathogenic role of Neoparamoeba sp. and the early host response in AGD. Preliminary evidence suggests that lesions resulting from physical gill damage are not preferentially colonized by Neoparamoeba sp.