Mechanisms of the host immune response and helminth-induced pathology during Trichobilharzia regenti (Schistosomatidae) neuroinvasion in mice.

Helminth neuroinfections represent serious medical conditions, but the diversity of the host-parasite interplay within the nervous tissue often remains poorly understood, partially due to the lack of laboratory models. Here, we investigated the neuroinvasion of the mouse spinal cord by Trichobilharzia regenti (Schistosomatidae). Active migration of T. regenti schistosomula through the mouse spinal cord induced motor deficits in hindlimbs but did not affect the general locomotion or working memory. Histological examination of the infected spinal cord revealed eosinophilic meningomyelitis with eosinophil-rich infiltrates entrapping the schistosomula. Flow cytometry and transcriptomic analysis of the spinal cord confirmed massive activation of the host immune response. Of note, we recorded striking upregulation of the major histocompatibility complex II pathway and M2-associated markers, such as arginase or chitinase-like 3. Arginase also dominated the proteins found in the microdissected tissue from the close vicinity of the migrating schistosomula, which unselectively fed on the host nervous tissue. Next, we evaluated the pathological sequelae of T. regenti neuroinvasion. While no demyelination or blood-brain barrier alterations were noticed, our transcriptomic data revealed a remarkable disruption of neurophysiological functions not yet recorded in helminth neuroinfections. We also detected DNA fragmentation at the host-schistosomulum interface, but schistosomula antigens did not affect the viability of neurons and glial cells in vitro. Collectively, altered locomotion, significant disruption of neurophysiological functions, and strong M2 polarization were the most prominent features of T. regenti neuroinvasion, making it a promising candidate for further neuroinfection research. Indeed, understanding the diversity of pathogen-related neuroinflammatory processes is a prerequisite for developing better protective measures, treatment strategies, and diagnostic tools.

Understanding the host response of farmed fish to blood flukes (Trematoda: Aporocotylidae) for developing new treatment strategies.

Aporocotylids (Trematoda: Digenea), also known as fish blood flukes infect the circulatory system of fish leading to serious health problems and mortality. Aporocotylids are a particular concern for farmed fish as infection intensity can increase within the farming environment and lead to mortalities. In the context of managing these infections, one of the most crucial aspects to consider is the host response of the infected fish against these blood flukes. Understanding the response is essential to improving current treatment strategies that are largely based on the use of anthelmintic praziquantel to manage infections in aquaculture. This review focuses on the current knowledge of farmed fish host responses against the different life stages of aporocotylids. New treatment strategies that are able to provide protection against reinfections should be a long-term goal and is not possible without understanding the fish response to infection and the interactions between host and parasite.

Anti-parasitic activity of garlic (Allium sativum) and onion (Allium cepa) extracts against Dactylogyrus spp. (Monogenean) in Nile tilapia (Oreochromis niloticus): Hematology, immune response, histopathological investigation, and inflammatory cytokine genes of gills.

BACKGROUND: Gills monogenean infestation causes significant mortalities in cultured fishes as a result of respiratory manifestation. Medicinal plants are currently being heavily emphasized in aquaculture due to their great nutritional, therapeutic, antimicrobial activities, and financial value. METHODS: The current study is designed to assess the effect of garlic (Allium sativum) and onion (Allium cepa) extracts as a water treatment on the hematological profile, innate immunity, and immune cytokines expression besides histopathological features of gills of Nile tilapia (Oreochromis niloticus L.) infected with gills monogenetic trematodes (Dactylogyrus sp.). Firstly, the 96-hour lethal concentration 50 (96 h-LC50) of garlic extract (GE) and onion extract (OE) were estimated to be 0.4 g/ L and 3.54 g/ L for GE and OE, respectively. Moreover, the in-vitro anti-parasitic potential for (GE) was found between 0.02 and 0.18 mg/mL and 0.4 to 1.8 mg/mL for OE. For the therapeutic trial, fish (n = 120; body weight: 40-60 g) were randomly distributed into four groups in triplicates (30 fish/group, 10 fish/replicate) for 3 days. Group1 (G1) was not infected or treated and served as control. G2 was infected with Dactylogyrus spp. and not exposed to any treatment. G3, G4 were infected with Dactylogyrus sp. and treated with 1/10 and 1/5 of 96 h LC50 of OE, respectively. G5, G6 were infected with Dactylogyrus sp. and treated with 1/10 and 1/5 of 96 h LC50 of GE, respectively. RESULTS: No apparent signs or behaviors were noted in the control group. Dactylogyrus spp. infected group suffered from clinical signs as Pale color and damaged tissue. Dactylogyrus spp. infection induced lowering of the hematological (HB, MCH, MCHC and WBCs), and immunological variables (lysozyme, nitric oxide, serum Anti- protease activities, and complement 3). the expression of cytokine genes IL-ß and TNF-α were modulated and improved by treatment with A. sativum and A. cepa extracts. The obtained histopathological alterations of the gills of fish infected with (Dactylogyrus spp.) were hyperplasia leading to fusion of the gill filament, lifting of epithelial tissue, aneurism and edema. The results indecated that G4 and G5 is more regenarated epithelium in compare with the control group. CONCLUSION: A. sativum and A. cepa extracts enhance the blood profile and nonspecific immune parameters, and down-regulated the expression level of (IL-1ß and TNF-α).

Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

Proteomic profile and protease activity in the skin mucus of greater amberjack (Seriola dumerili) infected with the ectoparasite Neobenedenia girellae - An immunological approach.

Skin mucus is considered the first barrier against diseases in fish. The skin mucus protein profile of the greater amberjack (Seriola dumerili) and its changes due to experimental infection with Neobenedenia girellae were studied by combining 2-DE-MS/MS and gel-free LC-MS/MS proteomic approaches. The 2-DE results led to the identification of 69 and 55 proteins in noninfected and infected fish, respectively, and revealed that keratins were specifically cleaved in parasitized fish. Therefore, the skin mucus of the infected fish showed a higher protease activity due to, at least in part, an increase of metal-dependent protease and serine-type protease activities. Additionally, through a gel-free LC-MS/MS analysis, 1377 and 1251 different proteins were identified in the skin mucus of healthy and parasitized fish, respectively. The functional analysis of these proteins demonstrated a statistical overrepresentation of ribosomal proteins (a well-known source of antimicrobial peptides) in N. girellae-infected fish. In contrast, the components of membranes and protein transport GO categories were underrepresented after infection. Immune system process-related proteins constituted 2.5% of the total skin mucosal proteins. Among these skin mucosal proteins, 14 and 15 proteins exclusive to non-parasitized and parasitized fish were found, respectively, including specific serine-type proteases and metalloproteases in the parasitized fish. Moreover, the finding of tryptic peptides exclusive to some bacterial genera, obtained by gel-free LC-MS/MS, allowed us to construct a preliminary map of the microbiota living in the mucus of S. dumerili, with Pseudomonas and Paracoccus the most represented genera in both noninfected and infected fish.

The peripheral immune response of mice infected with a neuropathogenic schistosome.

Trichobilharzia regenti (Schistosomatidae) percutaneously infects birds and mammals and invades their central nervous system (CNS). Here, we characterized the peripheral immune response of infected mice and showed how it was influenced by the parasite-induced inflammation in the skin and the CNS. As revealed by flow cytometry, T cells expanded in the spleen and the CNS-draining lymph nodes 7-14 days post-infection. Both T-bet+ and GATA-3+ T cells were markedly elevated suggesting a mixed type 1/2 immune response. However, it dropped after 7 dpi most likely being unaffected by the neuroinflammation. Splenocytes from infected mice produced a high amount of IFN-γ and, to a lesser extent, IL-10, IL-4 and IL-17 after in vitro stimulation by cercarial homogenate. Nevertheless, it had only a limited capacity to alter the maturation status of bone marrow-derived dendritic cells (BMDCs), contrary to the recombinant T. regenti cathepsin B2, which also strongly augmented expression of Ccl5, Cxcl10, Il12a, Il33 and Il10 by BMDCs. Taken together, mice infected with T. regenti developed the mixed type 1/2 immune response, which was driven by the early skin inflammation rather than the late neuroinflammation. Parasite peptidases might play an active role in triggering the host immune response.

Mucosal immune response of Nile tilapia Oreochromis niloticus during Gyrodactylus cichlidarum infection.

Monogenean Gyrodactylus cichlidarum can cause severe mortality of Nile tilapia (Oreochromis niloticus) fry. To date, reports about mucosal immunity of O. niloticus against this parasite have been rare. In order to explore the mucosal immunity of Nile tilapia against G. cichlidarum infection, the expressions of six adaptive immune-related genes and the contents of specific immunoglobulin IgM and IgT in the skin-associated lymphoid tissues (SALT) were dynamically analyzed after primary and secondary infections. The abundances of G. cichlidarum on the hosts after secondary infection were lower than those after primary parasite infection, which implied that hosts could initiate immune protection against G. cichlidarum reinfection to some degree. The transcription levels of TCR-ß and CD4 genes in the skin tissue were significantly up-regulated after primary G. cichlidarum infection, while genes pIgR and IgT were only detected with significant up-regulations during secondary infection. With the exception of pIgR, which had remarkably higher expression in the fish with low parasite loads, all other genes studied tended to have higher mRNA level in the fish with higher parasite loads. The specific IgM content in the skin mucus increased significantly on the 2nd day after the primary exposure, higher than the corresponding value during the secondary exposure, and had significantly positive correlation with the parasite loads during the first parasite infection. These results manifested that acquired immune responses in the SALT of Nile tilapia participated in the resistance against G. cichlidarum infection, underscoring the involvement of mucosal immunity in fish against monogenean infection, and suggesting potential prophylactic treatment of gyrodactylid disease of tilapia.

Toll-like signaling pathway in the transcriptome of Littorina littorea.

The transcriptome of the caenogastropod mollusk Littorina littorea was scanned for the presence of sequences encoding Toll-like receptors (TLRs) and corresponding proteins involved in downstream TLR signaling pathway. In the transcriptomic snapshots of hemocytes and kidney tissues, 45 complete TLRs encoded by 35 genes were identified. Out of the 59 non-TLR molecules involved in a canonical TLR signaling pathway, 35 genes were classified as homologous and could be placed within the TLR-mediated MyD88-and MAPK-dependent circuitries. No reference vertebrate adapters TIRAP, TRIF and TRAM were identified in the transcriptome. The results of RNA-seq experiments with an immune challenge (rediae of the digenean Himasthla elongata) indicate that four TLRs (LlTLR1, 3, 5 and 8) and a set of upregulated genes involved in signal transduction (LlMyd88, LlTNFα, LlCASP8, LlFADD, LlNFKBIA (IkBα), LlIRAK1, LlSTAT1, LlMAPK14 (P38), LlMAP2K1 (MEK1/2), LlIRF3 and LlIRF5) may participate in the anti-digenean immune response of L. littorea.

Relationship between Southern Bluefin Tuna, Thunnus maccoyii, melanomacrophage centres and Cardicola spp. (Trematoda: Aporocotylidae) infection.

Southern Bluefin Tuna (SBT), Thunnus maccoyii, is ranched off Port Lincoln, South Australia and is Australia's second largest economic finfish aquaculture industry. The biggest threats to SBT health identified by the industry are the blood flukes Cardicola forsteri and C. orientalis (Trematoda: Aporocotylidae). Melanomacrophage centres (MMCs) are aggregations of pigmented macrophage like cells present in spleen, kidney and liver of teleost fish. The aim of this study was to quantify MMCs in SBT anterior kidney, liver and spleen to investigate changes in relation to Cardicola spp. Infection. Samples were collected at the end of ranching from pontoons where SBT were treated with PZQ and pontoons with untreated SBT. SBT MMC percentage of surface area cover was highest in SBT spleen and lowest in the liver. Significant positive correlations were identified between SBT MMC area and SBT size in all three organs (p < 0.05). MMC area and parasite infection showed significant positive correlations in the kidney and spleen for Cardicola spp. gill egg counts, and in the kidney for C. forsteri DNA from SBT hearts and gills (p < 0.05). MMCs area increased with increased intensity of Cardicola spp. Infection and MMCs have the potential to be used as an indicator to assess health effects that Cardicola spp. have on SBT.

Increased parasite resistance of greater amberjack (Seriola dumerili Risso 1810) juveniles fed a cMOS supplemented diet is associated with upregulation of a discrete set of immune genes in mucosal tissues.

The main objective of this study was to determine the effect of two forms of mannan oligosaccharides (MOS: Bio-Mos® and cMOS: Actigen®, Alltech Inc, USA) and their combination on greater amberjack (Seriola dumerili) growth performance and feed efficiency, immune parameters and resistance against ectoparasite (Neobenedenia girellae) infection. Fish were fed for 90 days with 5 g kg-1 MOS, 2 g kg-1 cMOS or a combination of both prebiotics, in a Seriola commercial base diet (Skretting, Norway). At the end of the feeding period, no differences were found in growth performance or feed efficiency. Inclusion of MOS also had no effect on lysozyme activity in skin mucus and serum, but the supplementation of diets with cMOS induced a significant increase of serum bactericidal activity. Dietary cMOS also reduced significantly greater amberjack skin parasite levels, parasite total length and the number of parasites detected per unit of fish surface following a cohabitation challenge with N. girellae, whereas no effect of MOS was detected on these parameters. Of 17 immune genes studied cMOS dietary inclusion up-regulated hepcidin, defensin, Mx protein, interferon-γ (IFNγ), mucin-2 (MUC-2), interleukin-1ß (IL-1B), IL-10 and immunoglobulin-T (IgT) gene expression in gills and/or skin. MOS supplementation had a larger impact on spleen and head kidney gene expression, where piscidin, defensin, iNOS, Mx protein, interferons, IL-1ß, IL-10, IL-17 and IL-22 were all upregulated. In posterior gut dietary MOS and cMOS both induced IL-10, IgM and IgT, but with MOS also increasing piscidin, MUC-2, and IL-1ß whilst cMOS induced hepcidin, defensin and IFNγ. In general, the combination of MOS and cMOS resulted in fewer or lower increases in all tissues, possibly due to an overstimulation effect. The utilization of cMOS at the dose used here has clear benefits on parasite resistance in greater amberjack, linked to upregulation of a discrete set of immune genes in mucosal tissues.