DNA detection of Gyrodactylus spp. in skin mucus of Nile tilapia Oreochromis niloticus.

Monogeneans Gyrodactylus von Nordmann 1832, cause outbreaks of gyrodactylosis in aquaculture settings worldwide. Detection of Gyrodactylus spp. is based on the morphological identification of isolated parasites after fish necropsy. Contributing to the diagnosis of gyrodactylosis, in this study, a non-destructive PCR assay was standardized; the PCR was first performed using genomic DNA of Gyrodactylus spp. isolated from the surface of the Nile tilapia Oreochromis niloticus (Linnaeus 1758), and subsequently tested with mucus samples of infected and uninfected Nile tilapia fish. The primers (Ekgyro1) were designed from the ribosomal Internal Transcriber Spacer (ITS) RNA region (ITS1, 5.8S and ITS2 rRNA gene) of Gyrodactylus cichlidarum Paperna 1968. The positive control group included the DNA of 30 monogeneans Gyrodactylus spp. The heterologous control group included 75 monogeneans Cichlidogyrus Paperna 1960, 75 protozoans Ichthyophthirius multifiliis Fouquet 1876 and 75 Trichodina Ehrenberg 1830. PCR products of each parasite and from the external mucus samples (described as P and M respectively), were sequenced. The average DNA concentration of the ectoparasites was of 13.5 ng/µl. The PCR test had an analytical sensitivity of 0.0039 ng µl-1 of DNA of Gyrodactylus spp. No cross-reactions were observed with the heterologous group. The sensitivity and specificity of the PCR test were of 100% either with genomic DNA or with DNA from mucus samples. Six DNA consensus sequences with sizes ranging from 568 bp to 571 bp were obtained and the BLAST analysis matched with DNA sequences of G. cichlidarum.

Gastropod parasitic nematodes (Phasmarhabditis sp.) are attracted to hyaluronic acid in snail mucus by cGMP signalling.

Phasmarhabditis hermaphrodita is a parasitic nematode of terrestrial gastropods that has been formulated into a biological control agent for farmers and gardeners to kill slugs and snails. In order to locate slugs it is attracted to mucus, faeces and volatile cues; however, there is no information about whether these nematodes are attracted to snail cues. It is also unknown how wild isolates of P. hermaphrodita or different Phasmarhabditis species behave when exposed to gastropod cues. Therefore, we investigated whether P. hermaphrodita (commercial and wild isolated strains), P. neopapillosa and P. californica were attracted to mucus from several common snail species (Cepaea nemoralis, Cepaea hortensis, Arianta arbustorum and Cornu aspersum). We also examined whether snails (C. aspersum) collected from different locations around the UK differed in their attractiveness to wild isolates of P. hermaphrodita. Furthermore, we also investigated what properties of snail mucus the nematodes were attracted to, including hyaluronic acid and metal salts (FeSO4, ZnSO4, CuSO4 and MgSO4). We found that the commercial strain of P. hermaphrodita responded poorly to snail mucus compared to wild isolated strains, and C. aspersum collected from different parts of the UK differed in their attractiveness to the nematodes. We found that Phasmarhabditis nematodes were weakly attracted to all metals tested but were strongly attracted to hyaluronic acid. In a final experiment we also showed that pharmacological manipulation of cyclic guanosine monophosphate (cGMP) increased chemoattraction to snail mucus, suggesting that the protein kinase EGL-4 may be responsible for Phasmarhabditis sp. chemoattraction.

Direct detection of Tritrichomonas foetus in cattle genital fluid trough loop mediated isothermal amplification of elongation factor 1 alpha 1.

Testing for Tritrichomonas foetus and exclusion of infected animals is an effective way of improving the reproductive efficiency in a herd. Conventional PCR is inherently more specific than the culture method and quantitative PCR can significantly increase the detection limit. Loop Mediated Isothermal DNA Amplification (LAMP) is gaining interest because the method does not require expensive equipment, specificity and sensitivity can be as high as quantitative PCR. The object of this study was to develop a sensitive and friendly test for point-of-care detection of T. foetus. The LAMP test that targeted T. foetus elongation factor 1 alpha 1 sequences showed high specificity. Sensitivity was 100-1000 times higher than that reached through culture, polymerase chain reaction or with a previously developed LAMP for 5.8 ribosomal sequences. Moreover, T. foetus detection could be performed without DNA purification from infected cervical vaginal mucus (CVM) or smegma samples. The tf-ef1a1 LAMP method was tested for field detection with paper strips soaked in CVM from infected cows and the results were observed 90 min later. Direct detection of T. foetus in CVM with the tf-ef1a1 LAMP showed high sensitivity and specificity, and an overall diagnostic odds ratio of 56 (CI: 13.3-235.0). The tf-ef1a1 LAMP showed great potential for diagnosis and control of T. foetus in resource-challenged regions.

The use of a mucus trap by Dinophysis acuta for the capture of Mesodinium rubrum prey under culture conditions.

A capture mechanism observed in a culture of the dinoflagellate Dinophysis acuta when preying on the ciliate Mesodinium rubrum (also sometimes referred to as Myrionecta rubra) is described. The dinoflagellate released cohesive clumps of mucilage into the culture media. When M. rubrum cells came into contact with this mucilage, they were immediately immobilized, but remained alive for a short period of time. Observations of D. acuta cells 'visiting and probing' trapped M. rubrum cells were made and at a critical point D. acuta cells removed individual M. rubrum cells from the mucus to swim away with them. The removal of M. rubrum from the mucus coincided with the cells losing all their cilia and becoming swollen, presumably signifying the death of the cell. These changes may enable the D. acuta peduncle to penetrate the ciliate cell cortex. It is hypothesized that toxins produced by D. acuta play a role in the immobilization process within the mucilage trap.

Occurrence of immune cells in the intestinal wall of Squalius cephalus infected with Pomphorhynchus laevis.

A sub-population of 34 specimens of chub, Squalius cephalus, was sampled from the River Brenta (Northern Italy) and examined for ecto- and endo-parasites. Pomphorhynchus laevis (Acanthocephala) was the only enteric helminth encountered. Immunofluorescence and ultrastructural studies were conducted on the intestines of chub. Near the site of parasite's attachment, mucous cells, mast cells (MCs), neutrophils and rodlet cells (RCs) were found to co-occur within the intestinal epithelium. The numbers of mucous cells, MCs and neutrophils were significantly higher in infected fish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the lectin Dolichos Biflorus Agglutinin (DBA) and the macrophage-specific MAC387 monoclonal antibody, with parallel transmission electron microscopy, revealed that epithelial MCs often made intimate contact with the mucous cells. Degranulation of a large number of MCs around the site of the acanthocephalan's attachment and in proximity to mucous cells was also documented. MCs and neutrophils were abundant in the submucosa. Immune cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions are discussed.

Release of lungworm larvae from snails in the environment: potential for alternative transmission pathways.

BACKGROUND: Gastropod-borne parasites may cause debilitating clinical conditions in animals and humans following the consumption of infected intermediate or paratenic hosts. However, the ingestion of fresh vegetables contaminated by snail mucus and/or water has also been proposed as a source of the infection for some zoonotic metastrongyloids (e.g., Angiostrongylus cantonensis). In the meantime, the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior are increasingly spreading among cat populations, along with their gastropod intermediate hosts. The aim of this study was to assess the potential of alternative transmission pathways for A. abstrusus and T. brevior L3 via the mucus of infected Helix aspersa snails and the water where gastropods died. In addition, the histological examination of snail specimens provided information on the larval localization and inflammatory reactions in the intermediate host. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-four specimens of H. aspersa received ~500 L1 of A. abstrusus and T. brevior, and were assigned to six study groups. Snails were subjected to different mechanical and chemical stimuli throughout 20 days in order to elicit the production of mucus. At the end of the study, gastropods were submerged in tap water and the sediment was observed for lungworm larvae for three consecutive days. Finally, snails were artificially digested and recovered larvae were counted and morphologically and molecularly identified. The anatomical localization of A. abstrusus and T. brevior larvae within snail tissues was investigated by histology. L3 were detected in the snail mucus (i.e., 37 A. abstrusus and 19 T. brevior) and in the sediment of submerged specimens (172 A. abstrusus and 39 T. brevior). Following the artificial digestion of H. aspersa snails, a mean number of 127.8 A. abstrusus and 60.3 T. brevior larvae were recovered. The number of snail sections positive for A. abstrusus was higher than those for T. brevior. CONCLUSIONS: Results of this study indicate that A. abstrusus and T. brevior infective L3 are shed in the mucus of H. aspersa or in water where infected gastropods had died submerged. Both elimination pathways may represent alternative route(s) of environmental contamination and source of the infection for these nematodes under field conditions and may significantly affect the epidemiology of feline lungworms. Considering that snails may act as intermediate hosts for other metastrongyloid species, the environmental contamination by mucus-released larvae is discussed in a broader context.

Effects of single and repeated infections with Neoparamoeba perurans on antibody levels and immune gene expression in Atlantic salmon (Salmo salar).

Amoebic gill disease (AGD) is the main health problem for the salmon industry in Tasmania, Australia and is now reported in most salmon producing countries. Antibody and gene expression responses to the pathogen, Neoparamoeba perurans, have been studied independently following primary exposure; however, the effects of sequential reinfection, which can often occur during net-pen culture of salmon, remain unclear. The association between the transcription of immunoglobulin (Ig) and their systemic and mucosal antibody levels in regards to AGD is unknown. Herein, we assessed the antibody responses as well as Ig transcription in the gills of Atlantic salmon infected only once and also sequentially with N. perurans. After four successive AGD challenges, no significant differences in plasma or skin mucus levels of IgM were observed between AGD-naïve and challenged fish. However, IgM gene expression in gill lesions of AGD-affected fish increased up to 31 d after infection, while no changes in IgT, TCR and CD8 transcription were observed. Changes at IgM transcription level did not match the lack of antibody response in mucus, which is possibly explained by weak correlations existing between protein and mRNA abundances in cells and tissues. In the second experiment, which investigated Ig responses to AGD at the transcriptional as well as antibody production level in salmon after a single infection, the levels of serum or skin mucus IgM antibody were not affected and no changes in the IgM or IgT transcription were induced.

Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

The external surfaces of fish, such as gill and skin, are covered by mucus, which forms a thin interface between the organism and water. Amoebic gill disease (AGD) is a parasitic condition caused by Neoparamoeba perurans that affects salmonids worldwide. This disease induces excessive mucus production in the gills. The host immune response to AGD is not fully understood, and research tools such as genomics and proteomics could be useful in providing further insight. Gill and skin mucus samples were obtained from Atlantic salmon (Salmo salar) which were infected with N. perurans on four successive occasions. NanoLC tandem mass spectrometry (MS/MS) was used to identify proteins in gill and skin mucus of Atlantic salmon affected by AGD. A total of 186 and 322 non-redundant proteins were identified in gill and skin mucus respectively, based on stringent filtration criteria, and statistics demonstrated that 52 gill and 42 skin mucus proteins were differentially expressed in mucus samples from AGD-affected fish. By generating protein-protein interaction networks, some of these proteins formed part of cell to cell signalling and inflammation pathways, such as C-reactive protein, apolipoprotein 1, granulin, cathepsin, angiogenin-1. In addition to proteins that were entirely novel in the context in the host response to N. perurans, our results have confirmed the presence of protein markers in mucus that have been previously predicted on the basis of modified mRNA expression, such as anterior gradient-2 protein, annexin A-1 and complement C3 factor. This first proteomic analysis of AGD-affected salmon provides new information on the effect of AGD on protein composition of gill and skin mucus. Future research should focus on better understanding of the role these components play in the response against infection with N. perurans.

Pathways for transmission of angiostrongyliasis and the risk of disease associated with them.

Angiostrongylus cantonensis, the rat lungworm, is a major cause of eosinophilic meningitis in humans. This short paper reviews what is known about the pathways of infection and assesses the probable importance of each in causing disease. Rats are the definitive hosts. People can become infected by eating, both deliberately and inadvertently, raw or under-cooked intermediate hosts (snails or slugs) or paratenic hosts such as freshwater shrimp, crabs and frogs. Food preparation prior to cooking can leave debris from which infection can also occur. It may be possible to become infected by consuming snail/slug slime (mucus) on produce or by transferring mucus from hands to mouth after handling snails/slugs. Infection from consuming drinking water contaminated by snails/slugs and infection via open wounds may be theoretically possible but no cases have been reported. The severity of the disease is probably related to the number of infective larvae ingested as well as to the precise location of the worms in the host and the host's inflammatory response. Strategies for reducing human infection should include snail and slug control to reduce chances of accidental ingestion, cooking of intermediate and paratenic hosts, and public education on food preparation.

Early host-pathogen interactions in marine bivalves: evidence that the alveolate parasite Perkinsus marinus infects through the oyster mantle during rejection of pseudofeces.

Parasites have developed myriad strategies to reach and infect their specific hosts. One of the most common mechanisms for non-vector transmitted parasites to reach the internal host environment is by ingestion during feeding. In this study, we investigated the mechanisms of oyster host colonization by the alveolate Perkinsus marinus and focused on how oysters process infective waterborne P. marinus cells during feeding in order to determine the portal(s) of entry of this parasite to its host. We also compared the infectivity of freely-suspended cells of P. marinus with that of cells incorporated into marine aggregates to link changes in particle processing by the feeding organs with infection success and route. Finally, we evaluated the effect of oyster secretions (mucus) covering the feeding organs on P. marinus physiology because these host factors are involved in the processing of waterborne particles. The ensemble of results shows a unique mechanism for infection by which the parasite is mostly acquired during the feeding process, but not via ingestion. Rather, infection commonly occurs during the rejection of material as pseudofeces before reaching the mouth. The pseudofeces discharge area, a specialized area of the mantle where unwanted particles are accumulated for rejection as pseudofeces, showed significantly higher parasite loads than other host tissues including other parts of the mantle. Aggregated P. marinus cells caused significantly higher disease prevalence and infection intensities when compared to freely-suspended parasite cells. Mucus covering the mantle caused a quick and significant increase in parasite replication rates suggesting rapid impact on P. marinus physiology. A new model for P. marinus acquisition in oysters is proposed.

Parâmetros hematológicos e alterações histopatológicas em bijupirá (Rachycentron canadum Linnaeus, 1766) com amyloodiniose / Parameters hematological and histopathologic alterations in cobia (Rachycentron canadum Linnaeus, 1766) com amyloodiniose

O objetivo do trabalho foi descrever os parâmetros hematológicos e as alterações histopatológicas em bijupirás infectados por Amyloodinium ocellatum. Um grupo de 27 peixes foi anestesiado para coleta de amostras de sangue e eutanasiados para coleta de muco e fragmentos de tecido cutâneo e branquial. Foram avaliadas a prevalência e a intensidade parasitária da infecção, assim como os valores de parâmetros hematológicos e alterações histopatológicas. A prevalência parasitária nas brânquias foi de 100% e no muco foi de 80,8% e as intensidades parasitárias médias foram de 683,5 nas brânquias, e 67,1 no muco cutâneo. Os valores médios dos parâmetros hematológicos foram: eritrócitos 4,3x10(6)µL; VG 26%; VGM 64,2fL; proteína plasmática 5,8mg/dL; trombócitos 5,2 x10³/µL e leucócitos 3,6 x10³/µL. Além disso, foram verificadas hiperplasia do epitélio respiratório acompanhada de fusão lamelar, descolamento do epitélio, dilatação do seio venoso, formação de aneurisma, ruptura do epitélio lamelar, hemorragia, necrose, reação inflamatória linfocítica. O parasito foi observado nas lamelas branquiais, o VMA variou do grau discreto ao severo e o IAH foi de 76,8. A pesquisa assume importância por se tratar dos primeiros estudos em Rachycentron canadum, um peixe que se destaca com potencial ao cultivo.

The aim of this study was to describe the hematological parameters and histopathologic alterations in cobia infected by Amyloodinium ocellatum. A group of 27 fish were anesthetized to collect blood samples and euthanatized to collect mucus and tissue fragments of skin and gills. The prevalence and parasitic intensity of the infection, besides hematologic parameters and histopathologic alterations, was measured. Parasite prevalence in the gills was 100% and in the mucus 80.8%, and the average intensity of infection in gills and skin was 683.5 e 67.1 respectively. The mean values of hematological parameters were: erythrocytes 4.3x10(6)μL; PCV 26%, MCV 64.2 fL, plasma protein 5.8mg/dL, thrombocytes 5.2x10³/μL and leukocytes 3.6x10³/μL . Furthermore was found hyperplasia of the respiratory epithelium accompanied by lamellar fusion, detachment of the epithelium, venous sinus dilatation, aneurysm formation and rupture of the lamellar epithelium, hemorrhage, necrosis and lymphocytic inflammatory reaction. The parasite was observed between the gills lamellae, the AMV ranged from mild to severe and AHI values were 76.8. The study assumes importance because it is the first study in Rachycentron canadum, a fish that stands out with potential for growing.

Biostatic activity of piscine serum and mucus on myxozoan fish infective stages.

Since the basis of host specificity in Myxozoa, i.e. the differential disposition and extinction of erroneously penetrated myxozoan infective stages in non-susceptible fish hosts, remains puzzling, we aimed to explore the role of the innate immune system in this issue. In a comparative incubation challenge of actinospore sporoplasms of the freshwater parasite species Myxobolus cerebralis, Henneguya nuesslini and Myxobolus pseudodispar to isolates of host and non-host muci and blood sera, we measured cellular disintegration proportions and times by means of a double staining viability assay utilizing fluorescent dyes. After their activation, emerging primary and secondary sporoplasm cells were evaluated microscopically for physical integrity and onset of cell death due to exposure. Impairment by any mucus used was not detected up to 100 min of exposure. All parasites showed significantly increased cellular breakdown in non-susceptible host serum compared to the respective substrates from susceptible host fish. Except for M. cerebralis, the serum of the susceptible host was considerably less effective over time. In this species, both the primary and the secondary cells were affected in much shorter times than in the other two representatives. Inhibition of protease activity did not affect carp serum effect on M. cerebralis stages. We suggest the active components to be complement or complement induced factors since heat inactivation and withdrawal of bivalent metal ions lowered serum activity significantly. The study marks the first in vitro viability challenge of activated myxozoan transmission stages with teleost derived immune factors.

Non-lethal detection of DNA from Cichlidogyrus spp. (Monogenea, Ancyrocephalinae) in gill mucus of the Nile tilapia Oreochromis niloticus.

Infection of Nile tilapia Oreochromis niloticus by monogeneans of the genus Cichlidogyrus is harmful. Currently, diagnosis of this infection is based on invasive techniques and the identification of isolated parasites by their morphology. To facilitate diagnosis, we have developed a non-lethal polymerase chain reaction (PCR) test for detection of Cichlidogyrus spp. DNA in the gill mucus of O. niloticus, using 5 pairs of specific primers based on Cichlidogyrus sclerosus 28S rRNA (Cicly 1 to Cicly 5) which generate fragments of approximately 188, 180, 150, 159 and 189 bp, respectively. PCR specificity was tested using genomic DNA extracted individually from 175 isolated Cichlidogyrus spp., 75 Gyrodactylus cichlidarum and 75 endopararasitic Enterogyrus spp., as well as from 75 protozoans Trichodina spp. The Cicly primers were used to detect Cichlidogyrus spp. DNA in mucus from the gills of 23 Nile tilapia confirmed to be infected with the parasite. Negative controls consisted of 45 uninfected Nile tilapia. The limit of sensitivity of the assay was 1.2 ng of purified parasite DNA. The Cicly primers did not amplify DNA from the mucus of non-infected Nile tilapia, G. cichlidarum, Trichodina spp. or Enterogyrus spp. In all cases, the sensitivity and specificity of the test were 100%. The sequences of all the amplified fragments showed a high similarity to that of the 28S rRNA region of C. sclerosus (93 to 100% identical to GenBank Accession No. DQ157660.1). We provide evidence for a safe and non-invasive DNA-based diagnostic method for the presence of Cichlidogyrus in the gill mucus of O. niloticus.

Fish mucous cocoons: the 'mosquito nets' of the sea.

Mucus performs numerous protective functions in vertebrates, and in fishes may defend them against harmful organisms, although often the evidence is contradictory. The function of the mucous cocoons that many parrotfishes and wrasses sleep in, while long used as a classical example of antipredator behaviour, remains unresolved. Ectoparasitic gnathiid isopods (Gnathiidae), which feed on the blood of fish, are removed by cleaner fish during the day; however, it is unclear how parrotfish and wrasse avoid gnathiid attacks at night. To test the novel hypothesis that mucous cocoons protect against gnathiids, we exposed the coral reef parrotfish Chlorurus sordidus (Scaridae) with and without cocoons to gnathiids overnight and measured the energetic content of cocoons. Fish without mucous cocoons were attacked more by gnathiids than fish with cocoons. The energetic content of mucous cocoons was estimated as 2.5 per cent of the fish's daily energy budget fish. Therefore, mucous cocoons protected against attacks by gnathiids, acting like mosquito nets in humans, a function of cocoons and an efficient physiological adaptation for preventing parasite infestation that is not used by any other animal.

Release of infectious cells from epidermal ulcers in Ichthyophonus sp.-infected Pacific herring (Clupea pallasii): evidence for multiple mechanisms of transmission.

A common clinical sign of ichthyophoniasis in herring and trout is "sandpaper" skin, a roughening of the epidermis characterized by the appearance of small papules, followed by ulceration and sloughing of the epithelium; early investigators hypothesized that these ulcers might be a means of transmitting the parasite, Ichthyophonus sp., without the necessity of ingesting an infected host. We examined the cells associated with the epidermal lesions and confirmed that they were viable Ichthyophonus sp. cells that were readily released from the skin into the mucous layer and ultimately into the aquatic environment. The released cells were infectious when injected into the body cavity of specific-pathogen-free herring. Our hypothesis is that different mechanisms of transmission occur in carnivorous and planktivorous hosts: Planktonic feeders become infected by ingestion of ulcer-derived cells, while carnivores become infected by ingestion of whole infected fish.

Some characteristics of host-parasite relationship for Cryptocaryon irritans isolated from South China.

A survey on the host range for the parasitic ciliate Cryptocaryon irritans was carried out among the major maricultured fish species in the Huizhou region of Guangdong Province in South China, and some characteristics of its host-parasite relationship were described. The survey showed that all ten investigated species of fish (representing six different families) were infected with C. irritans with similar susceptibility. In chemoattraction assays, sera and mucus collected from investigated fish strongly attracts C. irritans theronts. Sera collected from infected orange-spotted groupers and yellow spotted grunts (Plectorhynchus cinctus) could immobilize C. irritans theronts, and their immobilization titers were 1:40 and 1:6.7, respectively. The surface antigens of C. irritans were demonstrated by indirect immunofluorescence and immunostaining assays using immune orange-spotted grouper serum and a monoclonal antibody against grouper IgM.

Myxozoan transmission via actinospores: new insights into mechanisms and adaptations for host invasion.

Various mechanisms that enable and improve transmission success of myxozoan actinospore stages towards fish hosts are described, based upon a combination of experimental data and functional analysis of morphological characters. For this purpose, laboratory-reared actinospores of Myxobolus cerebralis, Myxobolus parviformis, Henneguya nuesslini and Myxobolus pseudodispar were employed to exemplarily investigate aspects of host attachment and invasion. The process of polar filament discharge of M. cerebralis actinospores was analysed, showing that full discharge occurs in less than 10 msec. Additionally, a mechanism that rapidly contracts the discharged filament after discharge is described for the first time. Its purpose is most likely to bring the actinospore apex rapidly into intimate contact with the surface of the host. Unlike M. cerebralis, M. parviformis actinospores did not discharge polar filaments after mechanical and chemical stimulation, suggesting a different mode of triggering. For H. nuesslini actinospores, experimental results indicated that polar filament discharge is independent of external calcium-ion concentration but is influenced by osmolality. After attachment of an actinospore and prior to penetration into the host, an ensheathed unit ('endospore'), containing the sporoplasm, was emitted from the valves in a manner which varied from species to species. Experimentally induced sporoplasm emission was time-dependent and was found to be independent of polar filament discharge in H. nuesslini. Remarkably, it could be concluded that the sporoplasm is able to recognize host-stimuli while still within the intact spore. An updated summary of the sequential course of events during host recognition and invasion by actinospores is given.

Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae).

Neobenedenia girellae, a monogenean skin parasite, shows low host specificity. N. girellae is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. To clarify the mechanisms of host specificity, we purified and identified the attachment-inducing substances of oncomiracidia from tiger puffer fish. The attachment-inducing substances were mainly included in skin mucous extract. Skin mucous extract lost its ability to induce attachment after boiling and/or exposure to the reducing agent dithiothreitol, suggesting that attachment-inducing substances are of a proteinaceous nature. Since lectins such as Con A, WGA, PHA-L, and PSA inhibited the induction of attachment, attachment-inducing proteins were suspected to be glycoproteins. Glycoproteins specifically interacting with Con A were collected and purified by anion exchange chromatography, resulting in two active peaks (peaks 3-A and 6). The active component in peak 3-A was identified as Wap 65-2 by N-terminal amino acid sequencing, while the glycoprotein in peak 6 could not be identified. These results suggested that oncomiracidia recognised Wap 65-2 and another glycoprotein of their host.

Response of Neobenedenia girellae (Monogenea) oncomiracidia to brightness and black-and-white contrast.

Neobenedenia girellae, a capsalid monogenean, is a significant pathogen due to both its ability to cause high mortality in fishes and its low host specificity. Established control methods have both advantages and disadvantages. Biological control measures with no unfavourable effects on the environment should be incorporated into the control strategy. The response of N. girellae oncomiracidia to brightness and black-and-white contrast was investigated to search for an alternative approach of disease prevention or control. Japanese flounder, Paralichthys olivaceus (Paralichthyidae), were exposed to oncomiracidia in an aquarium divided into areas of different brightness ( approximately 1.3, 41.3 and 138.0 lux). The number of parasites on the fish group reared in 138.0 lux was significantly higher than on those reared in the lower brightness levels. Thus, the fish tended to be more vulnerable to infection by N. girellae under brighter conditions. Challenge trials using host fish mucus and whole live fish were established to detect the response by oncomiracidia to black-and-white contrast on a white versus a black background. Markedly more N. girellae oncomiracidia attached to black-painted areas and dark-coloured fish (normal spotted halibut, Verasper variegatus (Pleuronectidae) compared with white-painted areas and light-coloured fish (mal-coloured V. variegatus) on a white-coloured background. On a black-coloured background, more N. girellae oncomiracidia tended to attach to white-painted areas and light-coloured fish. Thus, black-and-white contrast is considered important for host finding by N. girellae oncomiracidia. The simplicity of the positive phototactic behaviour and the response to black-and-white contrast may lead to the development of a simple, practical and inexpensive method to control N. girellae outbreaks.

The tick salivary protein, Salp15, inhibits the development of experimental asthma.

Activation of Th2 CD4(+) T cells is necessary and sufficient to elicit allergic airway disease, a mouse model with many features of human allergic asthma. Effectively controlling the activities of these cells could be a panacea for asthma therapy. Blood-feeding parasites have devised remarkable strategies to effectively evade the immune response. For example, ticks such as Ixodes scapularis, which must remain on the host for up to 7 days to feed to repletion, secrete immunosuppressive proteins. Included among these proteins is the 15-kDa salivary protein Salp15, which inhibits T cell activation and IL-2 production. Our objective for these studies was to evaluate the T cell inhibitory properties of Salp15 in a mouse model of allergic asthma. BALB/cJ mice were Ag sensitized by i.p. injection of OVA in aluminum hydroxide, with or without 50 mug of Salp15, on days 0 and 7. All mice were challenged with aerosolized OVA on days 14-16 and were studied on day 18. Compared with control mice sensitized with Ag, mice sensitized with Ag and Salp15 displayed significantly reduced airway hyperresponsiveness, eosinophilia, Ag-specific IgG1 and IgE, mucus cell metaplasia, and Th2 cytokine secretion in vivo and by CD4(+) T cells restimulated with Ag in vitro. Our results demonstrate that Salp15 can effectively prevent the generation of a Th2 immune response and the development of experimental asthma. These studies, and those of others, support the notion that a lack of ectoparasitism may contribute to the increasing prevalence of allergic asthma.