Phagocytotic activity and gene expression of leukocytes isolated from Astyanax lacustris.

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris-an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research-using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1ß, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1ß and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

Characterization of a new strain of Aeromonas dhakensis isolated from diseased pacu fish (Piaractus mesopotamicus) in Brazil.

This study provides a detailed description and characterization of a strain of Aeromonas dhakensis isolated from a diseased juvenile Piaractus mesopotamicus obtained from the fish farm of the National Center for Continental Fish Research and Conservation (CEPTA/ICMBio), in the state of São Paul, Brazil. Biochemical tests using the VITEK 2 automated bacterial identification system identified the isolate to genus level; however, further molecular analysis of the 16S rRNA, gyrB and rpoD genes showed that the strain belonged to the species A. dhakensis. As expected, the isolated A. dhakensis strain was resistant to ampicillin and ampicillin/sulbactam, as resistance to ampicillin is a typical characteristic of the genus Aeromonas. Resistance to cefoxitin and meropenem was also observed, but the strain was susceptible to most of the tested antibiotics. The isolated strain of A. dhakensis caused acute haemorrhagic septicaemia in experimentally infected P. mesopotamicus, with a fifty per cent lethal dose of 1.14 × 105  CFU/fish. This is the first report of the occurrence of an A. dhakensis strain causing an infection in a fish species of South America, providing important epidemiologic data relating to this important pathogenic species.

Phylogenetic and host-parasite relationship analysis of Henneguya multiplasmodialis n. sp. infecting Pseudoplatystoma spp. in Brazilian Pantanal wetland.

A new species of the genus Henneguya (Henneguya multiplasmodialis n. sp.) was found infecting the gills of three of 89 specimens (3.3%) of Pseudoplatystoma corruscans and two of 79 specimens (2.6%) of Pseudoplatystoma reticulatum from rivers in the Pantanal wetland, Brazil. Partial sequencing of the 18S rDNA gene of the spores obtained from one plasmodium from the gills of P. corruscans and other one from the gills of P. reticulatum, respectively, resulted in a total of 1560 and 1147 base pairs. As the spores of H. multiplasmodialis n. sp. resemble those of Henneguya corruscans, which is also a parasite of P. corruscans, sequencing of the 18S rDNA gene of the spores of H. corruscans found on P. corruscans caught in the Brazilian Pantanal wetland was also provided to avoid any taxonomic pendency between these two species, resulting in 1913 base pairs. The sequences of H. multiplasmodialis n. sp. parasite of P. corruscans and P. reticulatum and H. corruscans did not match any of the Myxozoa available in the GenBank. The similarity of H. multiplasmodialis n. sp. obtained from P. corruscans to that from P. reticulatum was of 99.7%. Phylogeny revealed a strong tendency among Henneguya species to form clades based on the order and/or family of the host fish. H. multiplasmodialis n. sp. clustered in a clade with Henneguya eirasi and H. corruscans, which are also parasites of siluriforms of the family Pimelodidae and, together with the clade composed of Henneguya spp. parasites of siluriforms of the family Ictaluridae, formed a monophyletic clade of parasites of siluriform hosts. The histological study revealed that the wall of the plasmodia of H. multiplasmodialis n. sp. were covered with a stratified epithelium rich in club cells and supported by a layer of connective tissue. The interior of the plasmodia had a network of septa that divided the plasmodia into numerous compartments. The septa were composed of connective tissue also covered on both sides with a stratified epithelium rich in club cells. Inflammatory infiltrate was found in the tissue surrounding the plasmodia as well as in the septa.

Host-parasite-environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp. parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil.

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 ± 1.8 µm in total length, 12.9 ± 0.8 µm in body length, 3.4 ± 0.3 µm in width, 3.1 ± 0.1 µm in thickness and 24.6 ± 2.2 µm in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 ± 0.5 µm in length and 0.7 ± 0.1 µm in width. Polar filaments had 12-13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size.

Light, electron microscopy and histopathology of Myxobolus salminus n. sp., a parasite of Salminus brasiliensis from the Brazilian Pantanal.

In this report, we describe the morphology and histopathology of Myxobolus salminus n. sp., a parasite of the gill filaments of wild Salminus brasiliensis (dourado) from the Brazilian Pantanal. The small polysporic plasmodia were approximately 100 microm in diameter and the development was asynchronous. The mature spores were oval to pear shaped and had a smooth wall. The spore measurements were (mean+/-S.D., with range in parentheses): length 10.1+/-0.4 microm (9.6-10.5), width 6.1+/-0.4 microm (5.8-6.6) and thickness 5.0+/-0.6 microm (4.7-5.3). The polar capsules were elongated and of equal size: length 4.6+/-0.2 microm (4.3-4.8) and width 1.7+/-0.1 microm (1.5-1.9). The histological analysis revealed numerous plasmodia in the blood vessels of the gill filaments. The site of parasite development was the wall of the large-caliber blood vessel of the gill filament, with progressive growth towards the lumen, resulting in the obstruction of blood flow, congestion and perivascular edema. The ultrastructural study revealed that the plasmodial wall was composed of two membranes, had numerous pinocytic canals and was in direct contact with the basement membrane of the vessel. The development of the parasite was asynchronous, with mature spores, immature spores and young developmental stages randomly distributed throughout the plasmodium. The prevalence of the parasite was 4.4%, with male and female fish being infected.