Increasing the known biodiversity of cnidarian parasites of bryconid fishes from South America: two novel Myxobolus species with ultrastructure and ssrDNA-based phylogeny.

This study increases the known biodiversity of cnidarian parasites in neotropical bryconid fishes. Two novel Myxobolus species are described based on morphology, ultrastructure and small subunit ribosomal DNA (ssrDNA) sequencing: Myxobolus vetuschicanus n. sp. infecting fins of Salminus franciscanus and Myxobolus mineirus n. sp. infecting the mesentery of Brycon orthotaenia from the São Francisco River basin, Minas Gerais State, Brazil. Ultrastructural analysis of the two species revealed an asynchronous sporogenesis process, with germinative cells and young developmental stages of myxospores in the periphery of the plasmodia. In M. vetuschicanus n. sp., the plasmodia were surrounded by a layer of fibroblasts and in M. mineirus n. sp., the plasmodial membrane had direct contact with the host tissue. The phylogenetic analysis based on the ssrDNA of Henneguya/Myxobolus species showed that the two novel Myxobolus species grouped in subclades together with other parasite species of bryconid fishes.

Characterization and gene expression analysis of pacu (Piaractus mesopotamicus) inducible nitric oxide synthase (iNOS) following Aeromonas dhakensis infection.

Nitric oxide (NO) is an important effector molecule which is involved in a myriad of biological processes, including immune responses against pathogens such as parasites, virus and bacteria. During the inflammatory processes in vertebrates, NO is produced by the inducible nitric oxide synthase (iNOS) enzyme in practically all nucleated cells to suppress or kill intracellular pathogens. The aim of the present study was to characterize the full coding region of the iNOS gene of pacu (Piaractus mesopotamicus), an economically and ecologically important South American fish species, and to analyze mRNA expression levels following intraperitoneal infection with the pathogenic bacterium Aeromonas dhakensis by means of quantitative real time PCR (qPCR). The results showed that the pacu iNOS transcript is 3237 bp in length, encoding a putative protein composed of 1078 amino acid residues. The amino acid sequence showed similarities ranging from 69.03% to 94.34% with other teleost fish and 57.70% with the human iNOS, with all characteristic domains and cofactor binding sites of the enzyme detected. Phylogenetic analysis showed that the iNOS from the red-bellied piranha, another South American characiform, was the closest related sequence to the pacu iNOS. iNOS transcripts were constitutively detected in the liver, spleen and head kidney, and there was a significant upregulation in the liver and spleen at 12, 24 and 48 h after infection with A. dhakensis. No significant variations were observed in the head kidney during the periods analyzed. These results show that iNOS expression was induced by A. dhakensis infection and suggest that this enzyme may be involved in the response to this bacterium in pacu.

Expression of immune genes in systemic and mucosal immune tissues of channel catfish vaccinated with live theronts of Ichthyophthirius multifiliis.

Ichthyophthiriasis caused by Ichthyophthirius multifiliis (Ich) has a worldwide distribution and affects most freshwater fishes. Fish surviving natural infection and/or immunized with Ich develop strong innate and adaptive immune responses. However, there is a lack of the knowledge regarding immune gene expression patterns in systemic and mucosal immune tissues, and how immune genes interact and lead to innate and adaptive immune protection against Ich infection in fish. The objective of this study was to investigate the expression of innate and adaptive immune-related genes in systemic (liver, spleen) and mucosal (gill, intestine) tissues of channel catfish over time following vaccination with live Ich theronts. The vaccinated fish showed significantly higher antibody titers and survival (95%) than those of mock immunized fish. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 h (h4) to 2 days (d2) post-vaccination in systemic immune tissues. Immune cell receptor genes (CD4, CD8-α, MHC I, MHC II ß, TcR-α, and TcR-ß) were more highly upregulated and remained upregulated for longer duration in systemic tissues than in mucosal tissues of the vaccinated fish. The cytokine genes IL-1ßa and IFN-γ were rapidly upregulated in both systemic and mucosal tissues of vaccinated fish, with peak expression from h4 to d1 post-vaccination. Toll-like receptor genes TLR-1 and TLR-9 showed relatively stable upregulation in the gill of immunized fish following vaccination. Results of this study revealed the molecular immune responses in mucosal and systemic tissues of vaccinated fish and demonstrated that Ich vaccination resulted in innate and adaptive immune responses against Ich infection.

Detection and quantification of virulent Aeromonas hydrophila in channel catfish tissues following waterborne challenge.

The aim of this study was to understand the pathogenesis of motile aeromonas septicemia caused by an emergent, high virulent Aeromonas hydrophila (vAh) in channel catfish, Ictalurus punctatus Adipose fin clipped catfish were challenged with vAh using a waterborne challenge method, and the distribution of vAh over a time course was detected and quantified using real-time polymerase chain reaction. The results showed that 77.8% of fish died within 48 h post challenge with mean day to death of 1.5 days. At 2 h post challenge, vAh (inferred from genomic DNA copies or genome equivalents) was detected in all external and internal tissues sampled. Gill had the highest vAh cells at 1 h post challenge. Spleen harbored the most vAh cells among internal organs at 4 h post challenge. The tissues/organs with most vAh cells detected at 8 h post challenge were adipose fin, blood, intestine, kidney and skin, while liver showed the highest vAh cells at 24 h post challenge. These results suggest that vAh was able to rapidly proliferate and spread, following wound infection, through the fish blood circulation system and cause mortality within 8-24 h.

Molecular immune response of channel catfish immunized with live theronts of Ichthyophthirius multifiliis.

The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. The fish surviving natural infections or immunized with live theronts develop strong specific and non-specific immune responses. Little is known about how these immune genes are induced or how they interact and lead to specific immunity against Ichthyophthirius multifiliis in channel catfish Ictalurus punctatus. This study evaluated the differential expression of immune-related genes, including immunoglobulin, immune cell receptor, cytokine, complement factor and toll-like receptors in head kidney from channel catfish at different time points after immunization with live theronts of I. multifiliis. The immunized fish showed significantly higher anti-Ich antibody expressed as immobilization titer and ELISA titer than those of control fish. The vast majority of immunized fish (95%) survived theront challenge. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 hour (h4) to 2 days (d2) post immunization. Expression of immune cell receptor genes (CD4, CD8-α, MHC I, MHC II ß, TcR-α, and TcR-ß) showed up-regulation from h4 to d6 post immunization, indicating that different immune cells were actively involved in cellular immune response. Cytokine gene expression (IL-1ßa, IL-1ßb, IFN-γ and TNF-α) increased rapidly at h4 post immunization and were at an up-regulated level until d2 compared to the bovine serum albumin control. Expression of complement factor and toll-like receptor genes exhibited a rapid increase from h4 to d2 post immunization. Results of this study demonstrated differential expression of genes involved in the specific or non-specific immune response post immunization and that the vaccination against Ich resulted in protection against infection by I. multifiliis.

The morphological and molecular characterization of Henneguya rotunda n. sp., a parasite of the gill arch and fins of Salminus brasiliensis from the Mogi Guaçu River, Brazil.

A new species of myxosporea (Henneguya rotunda n. sp.) was found in the membrane of the gill arch and the fins of Salminus brasiliensis in the Mogi Guaçu River, municipality of Pirassununga, São Paulo state, Brazil. Morphological and morphometric analyses using light microscopy revealed parasites with similar characteristics at both infection sites. The mature spores found infecting the fins had oval spore body with 7.1 ± 0.2 µm in length, 5.6 ± 0.2 µm in width, 3.7 ± 0.1 µm in thickness, 16.4 ± 1.2 µm in length of the caudal process, and 23.6 ± 1.1 µm in total length of the spore. In a frontal view, the polar capsule was observed to be symmetrical with 3.4 ± 0.2 µm in length and 1.8 ± 0.1 µm in width. Mature spores contain six to seven turns of the polar filaments. The morphometric data concerning the spores obtained from plasmodia from the membrane of the gill arch were similar to those from the fins. Ultrastructure analysis revealed that the plasmodial wall was formed by a single membrane and had numerous pinocytotic canals connecting the outside of the plasmodia to the ectoplasm zone. Beyond that, various electron-translucent vesicles also were observed at the periphery of the plasmodium. The molecular analyses of the 18S rDNA gene from the spores obtained from the gill arch membrane and fin membrane showed that these sequences shared 100% similarity. Phylogenetic studies using maximum parsimony and maximum likelihood methods demonstrated the polyphyletic clustering of the myxosporean parasites of characiform fishes. H. rotunda n. sp. clustered as a sister species of Myxobolus pantanalis, also a parasite of S. brasiliensis.

Morphology and 18S rDNA sequencing identifies Henneguya visibilis n. sp., a parasite of Leporinus obtusidens from Mogi Guaçu River, Brazil.

During a survey of myxozoan parasites of freshwater fish from the Mogi Guaçu River in São Paulo State, Brazil, plasmodia of Henneguya visibilis n. sp. were found on the fins of Leporinus obtusidens (Characiformes: Anostomidae). The plasmodia, which were observed on five out of eight (62.5%) L. obtusidens examined, were 400-1,000 µm long. Mature spores were elongated with a spore body 10.8 ± 0.6 µm long and 3.9 ± 0.2 µm wide, a caudal process 18 ± 1.2 µm long, and a total spore length of 26.8 ± 1.1 µm. Polar capsules were elongated 4.9 ± 0.3 µm long and 1.4 ± 0.1 µm wide. Histological examination indicated that the plasmodia developed in the connective tissue, and no inflammatory infiltrate was observed at the infection site. Ultrastructural analysis showed a plasmodium wall with a single membrane and several pinocytotic canals. Sporogenesis occurred from the periphery to the center of the plasmodia. Phylogenetic analysis of the 18S rDNA sequence using maximum likelihood and maximum parsimony methods showed H. visibilis n. sp. positioned in a sub-clade composed of Henneguya/Myxobolus parasites of several freshwater fish families.