Immunofluorescence of the epizootic ulcerative syndrome pathogen,Aphanomyces invadans,using a monoclonal antibody

A monoclonal antibody (MAb), designated 3gJC9, was raised against a protein antigen of Aphanomyces invadans, the oomycete pathogen that causes epizootic ulcerative syndrome (EUS). The antigen was expressed on the surface of hyphae and secreted extracellularly. MAb 3gJC9 did not cross-react with other oomycete or fungal pathogens of fish, although it did react to the crayfish plague pathogen A. astaci. The MAb was used for immunofluorescent staining on histological sections of fish infected with EUS, and was found to be more sensitive than conventional staining methods for detecting A. invadans. It thus has utility in confirming the case definition of EUS. It also revealed very small filamentous structures, the significance of which is unclear, but they may represent an early stage of infection, thus allowing earlier detection of the disease, since they are not detected using conventional staining methods.     

Environmental influence on the occurrence of epizootic ulcerative syndrome (EUS) in freshwater fish in the Bellanwila–Attidiya wetlands, Sri Lanka

Water quality and rainfall conditions were analysed from November 1996 to September 1997 in selected sampling sites in the Bellanwila–Attidiya wetlands of Sri Lanka, an area where the epizootic ulcerative syndrome (EUS) has occurred, in order to examine the environmental factors that may be associated with EUS. Quantitative sampling of fish populations was carried out over the study period and the recurrence of EUS was monitored by gross clinical signs and histopathology. The occurrence of EUS from the sampling sites was recorded from January 1997 to March 1997. The outbreak was preceded by heavy rainfall. Diurnal variations in air temperature were relatively high during the outbreak period. Declining dissolved oxygen concentrations in water coincided with initiation of the disease outbreak. There were significantly low levels of dissolved oxygen in the water and significantly low rainfall in the area during the period of EUS in comparison with non-EUS periods. The biochemical oxygen demand of the water was significantly higher during the outbreak period than that in the period prior to the outbreak. Of the fish species sampled, the prevalence of EUS was highest in Trichogaster pectoralis (snake skin gourami). Histopathological studies on ulcerative lesions of fish confirmed that diffuse proliferative mycotic granulomatosis is a consistent feature of EUS. The results indicate that interaction between rainfall, deteriorating water quality and presence of pathogens could provide stressful conditions for fish, thereby inducing EUS lesions in susceptible fish populations.     

Epizootic ulcerative syndrome (EUS) in fish: history and current status of understanding

Epizootic ulcerative syndrome (EUS) is one of the most important diseases affecting more than 100 species of wild and cultured finfish. EUS was first reported in farmed ayu (Plecoglossus altivelis) from Japan in 1971 and has since then spread across different countries of four continents including Asia, Australia, North America and Africa. The spread of the disease, especially in Asia–Pacific region and Africa has led to substantial damage to the fish resources and livelihood of the fish farmers. No reports are available confirming the outbreak of the disease from Europe and South America. The latest outbreak of EUS has been reported from Canada in a new susceptible species brown bullhead, Ameiurus nebulosus. It seems that the disease has potential to spread further, owing to the epizootic nature of the disease and broad susceptible host range. Considering the global importance of this disease, this review provides the current status of understanding about the etiology, process of diseases development, species affected, diagnostic methods as well as control and preventive measures, in light of the historical developments in those areas.     

Characterization of a virus obtained from snakeheads Ophicephalus striatus with epizootic ulcerative syndrome (EUS) in the Philippines

This is the first report of the isolation and characterization of a fish virus from the Philippines. The virus was isolated using snakehead spleen cells (SHS) from severely lesioned epizootic ulcerative syndrome (EUS)-affected snakehead Ophicephalus striatus from Laguna de Bay, in January 1991. The virus induced cytopathic effects (CPE) in SHS cells yielding a titer of 3.02 x 10(6) TCID50 ml(-1) at 25 degrees C within 2 to 3 d. Other susceptible cell lines included bluegill fry (BF-2), catfish spleen (CFS) and channel catfish ovary (CCO) cells. Replication in chinook salmon embryo cells (CHSE-214) was minimal while Epithelioma papulosum cyprini cells (EPC) and rainbow trout gonad cells (RTG-2) were refractory. Temperatures of 15 to 25 degrees C were optimum for virus replication but the virus did not replicate at 37 degrees C. The virus can be stored at -10 and 8 degrees C for 30 and 10 d, respectively, without significant loss of infectivity. Viral replication was logarithmic with a 2 h lag phase; viral assembly in the host cells occurred in 4 h and release of virus occurred 8 h after viral infection. A 1-log difference in TCID50 titer between the cell-free virus and the total virus was noted. Freezing and thawing the virus caused a half-log drop in titer. Viral exposure to chloroform or heating to 56 degrees C for 30 min inactivated the virus. Exposure to pH 3 medium for 30 min resulted in a more than 100-fold loss of viral infectivity. The 5-iododeoxyuridine (IUdR) did not affect virus replication, indicating a RNA genome. Neutralization tests using the Philippine virus, the ulcerative disease rhabdovirus (UDRV) and the infectious hematopoietic necrosis virus (IHNV) polyvalent antisera showed slight cross-reaction between the Philippine virus antiserum and UDRV but established no serological relationship with SHRV and IHN virus. Transmission electron microscopy (TEM) of SHS cells infected with the virus showed virus particles with typical bullet morphology and an estimated size of 65 x 175 nm. The Philippine virus was therefore a rhabdovirus, but the present study did not establish its role in the epizootiology of EUS.     

Horizontal transmission of epizootic ulcerative syndrome (EUS)-associated virus in the snakehead Ophicephalus striatus under simulated natural conditions

Natural transmission of the epizootic ulcerative syndrome (EUS) was conducted on na?ve snakeheads Ophicephalus striatus (also known as Channa striata) kept (A) in aquifer water, (B) in lakewater, (C) cohabiting with EUS snakeheads in lakewater, and (D) cohabiting with apparently healthy snakeheads in lakewater during the 1994 to 1995 EUS season. The results showed that EUS-like lesions developed in 6 to 14 d among na?ve snakeheads cohabiting with EUS snakeheads and with apparently healthy snakeheads in lakewater (Treatments C and D). Among na?ve fish exposed to lakewater (Treatment B), similar lesions developed in 16 to 21 d, while na?ve fish in aquifer water (Treatment A) did not develop EUS-like lesions. EUS signs began as Grade I (slight) lesions that gradually progressed to Grades III-IV (severe) 3 to 5 d from lesion onset, similar to the naturally affected EUS fish. The virus was recovered from some but not all naturally EUS-affected snakeheads, snakeheads with healing lesions and apparently healthy snakeheads, but not from na?ve snakeheads. The results provide evidence of a waterborne horizontal transmission of the EUS-associated virus. This is the first report of a successful horizontal transmission of the EUS-associated virus from apparently healthy snakeheads to na?ve fish under natural conditions and of virus recovery in tissue culture from naturally exposed experimental fish.     

Aphanomyces invadans,the causal agent of Epizootic Ulcerative Syndrome,is a global threat to wild and farmed fish

Aphanomyces invadans is a eukaryotic pathogen and the causative agent of Epizootic Ulcerative Syndrome (EUS) in fish and is responsible for mortalities of up to 100% in aquaculture. A. invadans was first discovered in Japan in 1971, and since then it has been found in Australia, North America, Southern African countries and Asia. Methods for the correct identification of A. invadans are well established now and involve PCR-based detection and microscopy. However, the pathogenesis of A. invadans is poorly understood. Environmental stress (mainly temperature) and the associated immunocompromised fish seem to induce infections of A. invadans and outbreaks of EUS. Understanding the process of infection in more depth is fundamental for the discovery of novel effective treatments to combat the disease. In this review, we discuss morphological characteristics of A. invadans and its pathogenicity as well as various approaches of treatment.     

Factors influencing the sporulation and cyst formation of Aphanomyces invadans, etiological agent of ulcerative mycosis in Atlantic menhaden, Brevoortia tyrannus

Oomycete infections caused by Aphanomyces invadans occur in freshwater and estuarine fishes around the world. Along the east coast of the USA, skin ulcers caused by A. invadans are prevalent in Atlantic menhaden, Brevoortia tyrannus. From laboratory observations low salinities appear crucial to transmission of the pathogen. To better understand aspects of transmission, we characterized sporulation and cyst formation of secondary zoospores of two isolates of A. invadans at different salinities and temperatures. Sporulation occurred only at low salinities. At room temperature (ca. 20-22 C), using "pond water" augmented with artificial sea salts, the endemic strain WIC and the Thailand strain PA7 of A. invadans produced free-swimming secondary zoospores at salinities of 0, 1 and 2 psu (practical salinity unit = per thousand), but not at 4 psu or higher. Secondary zoospores of another species, ATCC-62427 (Aphanomyces sp.), were observed at 1, 2, 4 and 8 psu but not at 0 and 12 psu. Secondary zoospores of all three isolates, especially WIC, were abundant and motile 1-2 d postsporulation. Sporulation was temperature dependent and occurred over a relatively narrow range. No sporulation occurred at 4, 30 or 35 C for either WIC or PA7. For both strains zoospore production within 1-3 d after the initiation of sporulation was more prolific at 25 C than at 20 and 15 C. At 15 C production of zoospores was sustained over 11 d for WIC and 5 d for PA7. At room temperature single WIC secondary zoospores remained motile 12-18 h. Salinities exceeding 4 psu or vigorous shaking caused immediate cyst formation of WIC secondary zoospores. Exposure to menhaden tissue, but not tissues of other fishes to secondary zoospores (WIC), caused rapid (2 h) cyst formation. Cysts were capable of excysting when transferred to 1 psu water within 2-3 h of cyst formation. Cysts that had remained encysted in 6.5 psu for 24 h did not excyst when transferred to 1 psu water. Salinity and temperature requirements for sporulation indicate that juvenile menhaden must acquire infections during rain or in low salinity oligohaline waters.     

Emergence of epizootic ulcerative syndrome in native fish of the Murray-Darling River System, Australia: hosts, distribution and possible vectors

Epizootic ulcerative syndrome (EUS) is a fish disease of international significance and reportable to the Office International des Epizootics. In June 2010, bony herring Nematalosa erebi, golden perch Macquaria ambigua, Murray cod Maccullochella peelii and spangled perch Leiopotherapon unicolor with severe ulcers were sampled from the Murray-Darling River System (MDRS) between Bourke and Brewarrina, New South Wales Australia. Histopathology and polymerase chain reaction identified the fungus-like oomycete Aphanomyces invadans, the causative agent of EUS. Apart from one previous record in N. erebi, EUS has been recorded in the wild only from coastal drainages in Australia. This study is the first published account of A. invadans in the wild fish populations of the MDRS, and is the first confirmed record of EUS in M. ambigua, M. peelii and L. unicolor. Ulcerated carp Cyprinus carpio collected at the time of the same epizootic were not found to be infected by EUS, supporting previous accounts of resistance against the disease by this species. The lack of previous clinical evidence, the large number of new hosts (n = 3), the geographic extent (200 km) of this epizootic, the severity of ulceration and apparent high pathogenicity suggest a relatively recent invasion by A. invadans. The epizootic and associated environmental factors are documented and discussed within the context of possible vectors for its entry into the MDRS and recommendations regarding continued surveillance, research and biosecurity are made.     

Immune responses and protection in catla (Catla catla) vaccinated against epizootic ulcerative syndrome

Three different antigenic preparations from the epizootic ulcerative syndrome (EUS) pathogen Aphanomyces invadans were evaluated as vaccine candidate in catla (Catla catla). Anti-catla enzyme immunoconjugate was prepared after isolating catla immunoglobulin and raising hyperimmune sera against it, in rabbit. Three antigens namely, fungal extract (FE), fungal extract mixed with Freund’s incomplete adjuvant (FIA) in a 1:1 (v/v) ratio (FE + A) and extra cellular product (ECP) were prepared and three groups of catla were vaccinated intramuscularly with all these antigens (200 μg/fish). Different cellular and humoral immune responses were measured for the entire vaccinated and control group on 0th, 5th, 15th and 25th day post vaccination. Thirty days after the vaccination, the fish were challenged with an A. invadans zoospore dose of 1 × 10⁵ ml⁻¹ and mortality and relative percent of survival (RPS) were recorded. Study of cellular immunological parameters including antigen-specific leukocyte proliferation, antigen-specific nitric oxide production and superoxide anion production showed significantly higher (p < 0.05) values, in general, on 5th and 15th day post vaccination than the 0th day. Among all the antigenic groups, FE + A showed most significant response compared to the other groups. Among the humoral immune responses, lysozyme activity showed almost similar trend like cellular parameters. Anti-Aphanomyces antibody production was measured by enzyme-linked immunosorbent assay (ELISA) and it was increased with increasing days of vaccination in all the vaccinated groups with the highest observed on 25th day. Among the antigens, FE + A showed the highest antibody production following vaccination. The result of the homologous pathogen challenge study showed reduction in mortality in all the vaccinated groups. However, this reduction was not statistically significant (p > 0.05). Increased immune responses and protection have important implications with regard to the control of EUS by vaccination.     

Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.     

In vitro investigations on extracellular proteins secreted by <Emphasis Type="Italic">Aphanomyces invadans</Emphasis>, the causative agent of epizootic ulcerative syndrome

Background

Proteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated.

Results

BLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively.

Conclusions

The identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.

     

Aphid infestation induces PR-proteins differently in barley susceptible or resistant to the birdcherry-oat aphid (Rhopalosiphum padi)

The effect of infestation by the birdcherry-oat aphid ( Rhopalosiphum padi L.), on induction of PR-proteins was investigated in barley ( Hordeum vulgare L.), using barley lines susceptible or resistant to R. padi. The PR-proteins PR-1a (unknown function), PR-5a (acidic thaumatin) and peroxidase (EC 1.11.1.7) were not affected, whereas one chitinase (EC 3.2.1.14) and 4 β -1,3-glucanases (EC 3.2.1.39) were induced by the aphid treatment. In the resistant breeding line CI 16145, but not in the susceptible cultivar Golf, accumulation of one basic chitinase and two acidic β -1,3-glucanases increased with time from 2 until 11 days after infestation, as determined by western blots, with antibodies raised against purified chitinase (PR-3a) and β -1,3-glucanase (PR-2a) from barley. By isoelectric focusing, two additional basic β -1,3-glucanases were detected, which increased after infestation in both the resistant and the susceptible barley. The basic chitinase was only detected at days 7 and 11 in the susceptible cultivar, but already at day 2 in the resistant line. The induction was localized to the infested leaf. The PR-proteins PR-3a and PR-2a were also induced by the fungal pathogen ( Blumeria [syn. Erysiphe ] graminis f. sp. hordei ), methyl salicylate and, to a lower extent, by wounding with tweezers and methyl jasmonate (MeJA). Needle wounding performed to mimic aphid stylet penetration did not induce chitinase or β -1,3-glucanase. It is concluded that the fungal pathogen and the aphid infestation induce both similar and different responses, and that the aphid induction is not due to wounding only. The different responses in resistant and susceptible lines indicate that the induced enzymes may play a role in the resistance against aphid infestation.     

Comparative transcriptome profiling of resistant and susceptible sugarcane genotypes in response to the airborne pathogen Fusarium verticillioides

Molecular Biology Reports - Fusarium verticillioides is the pathogen associated with pokkah boeng disease (PBD), the most significant airborne disease of sugarcane. The molecular mechanisms that...     

Involvement of reactive oxygen species in the response of resistant (hypersensitive) or susceptible cowpeas to the cowpea rust fungus

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea ( Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus ( Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.     

Responses of Nasonovia ribisnigri (Homoptera: Aphididae) to susceptible and resistant lettuce

Nymphs and alates of aphid Nasonovia ribisnigri (Mosley) (Homoptera: Aphididae) were tested on 10 lettuce cultivars with N. ribisnigri resistance gene Nr and 18 cultivars without the resistance gene in various bioassays. Bioassays used whole plants, leaf discs, or leaf cages to determine susceptibility of commercial lettuce cultivars to N. ribisnigri infestation and to evaluate screening methods for breeding lettuce resistance to N. ribisnigri. Resistant and susceptible plants were separated in 3 d when using whole plant bioassays. Long-term (> or =7 d) no-choice tests using leaf cages or whole plants resulted in no survival of N. ribisnigri on resistant plants, indicating great promise of the Nr gene for management of N. ribisnigri. Effective screening was achieved in both no-choice tests where resistant or susceptible intact plants were tested separately in groups or individually and in choice tests where susceptible and resistant plants were intermixed. Leaf discs bioassays were not suitable for resistance screening. All lettuce cultivars without the resistance gene were suitable hosts for N. ribisnigri, indicating the great importance of this pest to lettuce production and the urgency in developing resistant lettuce cultivars to manage N. ribisnigri.