Mycotic aspects of epizootic ulcerative syndrome (EUS) of Asian fishes

Abstract A survey of fish affected with epizootic ulcerative syndrome taken from outbreaks in countries throughout South and South-East Asia showed that a morphologically typical fungus was consistently present within lesions. Although the majority of the fungal mycelium was dead in most lesions it proved possible to isolate a very delicate and culturally demanding Aphanomyces from such lesions in a few cases. It also proved relatively easy to isolate other members of the Saprolegniaceae including Aphanomyces from the surface of lesions, but these were considered saprophytes derived from background spore burdens in the water. Sporangium morphology of the putatively pathogenic isolates of Aphanomyces was different from that of saprophytic Aphanomyces strains and they also had a lower thermal tolerance. When a mycelium from these strains was placed below the dermis of healthy fish, it caused an inflammatory response and proceeded to migrate down into the tissues of the fish, inducing severe myonecrosis with chronic epithelial reaction. The saprophytic isolates induced a local host response followed by healing of the induced lesion, and destruction or expulsion of the mycelium. It is considered that the specific slow-growing, thermo-labile Aphanomyces is the pathogenic fungus which causes so much tissue damage in this disease, although it may not be a primary pathogen in its own right.     

Immunodiagnosis of virulent strains of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) using a monoclonal antibody

Abstract. A virulent strain of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) was used to produce monoclonal antibodies that identified virulent strains of A. hydrophila. Antibodies from a clone, designated as F26P5C8, were found to identify the A. hydrophila serotype I isolates associated with EUS fish, and which were found to be virulent after subsequent inoculation studies. Immunodiagnosis of a large number of A. hydrophila from Australia and Japan showed some additional isolates to be identified by F26P5C8, but the status of their virulence is presently unknown.     

Histopathology of snakehead, Channa striatus (Bloch), experimentally infected with the specific Aphanomyces fungus associated with epizootic ulcerative syndrome (EUS) at different temperatures

Abstract Snakeheads, Channa striatus (Bloch), were inoculated with a spore suspension of the specific pathogenic Aphanomyces , isolated from fish affected by epizootic ulcerative syndrome (EUS), in South East Asia. Fish were held at three different temperatures: 19, 26 and 31 °C. Histological changes induced by the infection are described. In the early stages of the disease, degenerative changes were observed in all samples, but inflammatory infiltrate was much more marked in fish kept at 26 and 31 °C. By 8 days post-injection, extensive mycotic granulomatosis was observed in the samples kept at 26 and 31 °C. The fish kept at 19 °C developed a severe invasive myonecrosis with limited macrophage response. From 14 to 28 days post-injection, healing became well established at 26 and 31 °C and surviving fish kept at these temperatures recovered completely by 28 days. The lesion was still progressing at 21 days post-injection in fish kept at 19 °C, and all such fish succumbed by this time. Thus, mortalities in the fish kept at 19 °C were considerably higher than in the groups of fish kept at 26 and 31 °C. The findings help to explain why mortalities from EUS occur when water temperatures are low.     

Pathogenicity and culture studies comparing the Aphanomyces involved in epizootic ulcerative syndrome (EUS) with other similar fungi

Pathogenicity and cultural experiments described here provide futher evidence that a distinct species of Aphanomyces is responsible for much of the characteristic pathology of epizootic ulceration syndrome (EUS). Zoospores from 58 fungal isolates were injected intramuscularly in snakehead fish, Channa striata (Bloch). These fungi comprised: Aphanomyces strains isolated from EUS-affected fish; saprophytic Aphanomyces , Achlya and Saprolegnia spp. from infected waters; and further saprolegniaceous fungi involved in other diseases of aquatic animals. Only the Aphanomyces strains isolated from fish affected by EUS, Australian red spot disease (already considered synonymous with EUS) or mycotic granulomatosis described from Japan were able to grow invasively through the fish muscle and produce the distinctive EUS lesions. In contrast to Aphanomyces astaci Schikora, the EUS- Aphanomyces was shown to be unable to infect noble crayfish, Astacus astacus L. The snakehead-pathogenic strains were further distinguished from all the other fungi under comparison by their characteristic temperature-growth profile and inability to grow on certain selective fungal media.     

鳢流行性溃疡综合征病原分离鉴定与病理形态学观察

四川地区养殖的白乌鳢 (Channa argus) 在2021年秋冬阴雨季节频繁发生溃疡性死亡事件。为确定白乌鳢溃疡性疾病的原因,通过临床观察、分离培养、组织学和分子生物学等技术开展了研究。大体病理研究发现,患病鱼主要表现为皮肤脱落并暴露皮下肌肉层,肌肉出现深层溃疡的情况;在光镜下,发现肌肉触片中有直径为10~20 μm的无隔膜分枝状菌丝,通过霉菌分离纯化和ITS基因序列分析,鉴定该病原菌为侵入性丝囊霉菌 (Aphanomyces invadans)。组织病理学显示,在皮肤真皮层和肌肉中的溃疡边缘包围着成熟的肉芽肿,而这些肉芽肿可侵袭至肉眼所观察到的正常肌肉处,对病鱼造成进一步的影响。研究结果表明,此次白乌鳢暴发性死亡的原因是侵入性丝囊霉菌引起的流行性溃疡综合征 (Epizootic Ulcerative Syndrome, EUS),这是关于白乌鳢流行性溃疡综合征的首次报告。研究结果为侵入性丝囊霉菌的鉴定和临床诊断提供了理论参考,也是侵入性丝囊霉菌宿主进一步扩大的证明。     

鳢流行性溃疡综合征病原分离鉴定与病理形态学观察

为寻找冬春季鳢(Channa maculata)烂身病的病原,应用组织压片、组织切片、扫描电镜技术、HE 常规染色法和 Grocott 六胺银染色法、霉菌分离纯化及 ITS 的序列分析等对冬春季患疑似溃疡综合征的烂身病鳢进行了病原学与病理学研究。病变组织压片观察到大量直径 10~20 μm,分枝较少、纤细菌丝。扫描电镜观察到肌肉组织中延伸出大量的纤细的丝状真菌。患病鳢皮肤和肌肉表现为变性、坏死与炎性细胞浸润,溃疡灶肌肉内可见大量的慢性肉芽肿结节和炎性细胞浸润。结节基本结构由类上皮细胞和多核巨细胞、炎性细胞、霉菌菌丝(横断面呈圆形,斜断或纵断呈丝状)组成,霉菌位于结节中央。Grocott 六胺银染色观察到结节中有大量棕色菌体。无菌分离培养可观察到典型丝状霉菌;霉菌在灭菌池塘水 20℃过夜, 12 h 后可观察原代孢子群形成,真菌 ITS 序列分析表明其与侵袭丝囊菌(Aphanomyces invadans)同源性为 100%。侵袭丝囊霉菌(A. invadans)是杂交鳢溃疡综合症病的主要病原,为鱼类流行病学调查和疾病防治的深入研究奠定基础。     

Oomycete fungi associated with ulcerative mycosis in menhaden, Brevoortia tyrannus (Latrobe)

Abstract. Deep necrotic ulcers in Atlantic menhaden, Brevoortia tyrannus (Latrobe), were consistently associated with the presence of oomycete fungi. These organisms were found in over 95% of all lesions both early and late. More than one species of fungus appears to be involved, indicating that these organisms are secondary to some other stressor. However, all characteristically elicit an atypical intense granulomatous inflammatory response and do not behave pathologically as typical oomycetes. Our observation that these fungi appear to be the only organisms consistently present in the earliest stages of the disease indicates that some non-infectious stressor may be responsible for initiating the lesions and subsequently allowing the fungi to induce this atypical response.     

Epidemiology of epizootic haematopoietic necrosis virus (EHNV) infection in farmed rainbow trout, Oncorhynchus mykiss (Walbaum): findings based on virus isolation, antigen capture ELISA and serology

Abstract. The epidemiology of epizootic haematopoietic necrosis virus (EHNV) infection was studied in farmed rainbow trout, Oncorhynchus mykiss (Walbaum). Estimates of mortality during five outbreaks on a commercial farm from 1986 to 1992 ranged from 0033 to 0.2% per day and total mortality did not exceed 3–4% in any outbreak. Affected fish were 0+ and less than 125 mm forklength. Clinical signs were non-specific, and laboratory examination was required to confirm the diagnosis. At the height of the outbreak in 1992, EHNV was demonstrated by virus isolation and antigen capture ELISA in 89% of clinically affected fish and 51% of dead fish, while the prevalence of infection in apparently healthy in-contact fish was 4%. Two and 4 months later the virus was not detected in a group of apparently healthy fish that had been affected earlier. Antibodies specific for EHNV were not found in rainbow trout from the infected farm; however, strong humoral responses were detected by ELISA in two immunized fish, indicating that the virus was immunogenic. These data suggested that EHNV was poorly infective but highly virulent in rainbow trout. Clinical EHNV infection was positively correlated with high rearing density and a low rate of water exchange, and therefore, with presumed poor water quality. Water temperature, which ranged from 11 to 17°C during outbreaks, did not appear to determine the incidence of clinical infection. EHNV infection in farmed rainbow trout was preceded by infection in free-living redfin perch, Perca fluviatilis L., in the water catchment, but it was uncertain whether this represented the source of infection for rainbow trout.     

First report of viral erythrocytic necrosis in Alaska, USA, associated with epizootic mortality in Pacific herring, Clupea harengus pallasi (Valenciennes)

Abstract. Viral erythrocytic necrosis (VEN) has been associated with large epizootics and high mortality in Pacific herring, Clupea harengus pallasi (Valenciennes), from two different locations in south-eastern Alaska, USA. Clinical signs of disease included whirling, pale gills, watery colourless blood, discoloured livers, pathog-nomonic magenta erythrocytic inclusion bodies and histopathological changes consistent with other infectious haemolytic anaemias in higher animals. Transmission electron microscopy confirmed the presence of iridovirus-type particles associated with the cytoplasmic erythrocytic inclusion bodies. Other apparently healthy herring of various age classes from four additional areas in south-east Alaska also had clinical signs of VEN suggesting a wide distribution of the virus in Alaskan Pacific herring populations. Evidence regarding the two herring epizootics indicated that osmoregulatory stress may have precipitated mortalities in fish having severe anaemia caused by the VEN virus. This is the first reported occurrence of VEN in Alaska and the first natural epizootic known to be associated with the disease.     

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter,Scortum barcoo (McCulloch & Waite)

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70–90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus‐specific PCR. The full‐length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV‐like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.     

Serological studies of the rhabdovirus of penaeid shrimp (RPS) and its relationship to three other fish rhabdoviruses

Abstract. The serological relationship of rhabdovirus of penaeid shrimp (RPS) to three fish rhabdoviruses was determined by plaque-reduction and neutralization kinetics techniques using polyclonal antiserum against RPS. Although RPS was found to be serologically related to Rhabdovirus carpio (RC), a Vesiculovirus type, the two viruses were clearly distinguishable from one another, RPS was unrelated to two other fish rhabdoviruses of the Lyssavirus type, infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). Under single cycle growth conditions, immunofluorescence studies showed the appearance of RPS viral antigens as early as 3h post-infection (pi). The degree of fluorescence and the number of fluorescing cells progressively increased until 24 h pi when more than 90% of the cells showed viral antigens.     

Variations in fatty acids composition of pikeperch (Sander lucioperca) liver with respect to gonad maturation

Abstract –  We examined seasonal variations in liver condition factor, total lipid and fatty acid composition of maturing pikeperch ( Sander lucioperca) from Lake Eğirdir, Turkey. The spawning of pikeperch in Eğirdir Lake started in March, continued very fast during May and gradually ended in July as determined by measuring the gonadosomatic index. The total lipid content of male and female liver reached its highest level in September and November (the end of dense feeding period during which temperature decreased sharply), but declined to the lowest level in May (just after spawning). The highest level of liver condition factor was observed in January and March during which gonads mature rapidly. Palmitic acid was the predominant saturated fatty acid (SFA) in female and male liver. The ratio of monounsaturated fatty acids (MUFA) was higher than that of polyunsaturated fatty acids (PUFA) in both sexes. Docosahexaenoic acid (22:6 n -3), eicosopentaenoic acid (20:5 n -3) and arachidonic acid (20:4 n -6) were the predominant PUFA. Seasonal variations in PUFA were more evident than that in other fatty acids. PUFA, especially the n -3 fatty acids ratio, increased to the maximum level during the sharp decreases in temperature (in November). However, the amount of PUFA decreased to the lowest level in reproductive period. The results suggest that the ratio of pikeperch liver fatty acids is influenced by gonad maturation and temperature variations and that pikeperch requires a large amount of PUFA and the n -3 fatty acids for the development of gonads.     

White spot syndrome virus (WSSV) prevalence associated with disease resistance among wild populations of black tiger shrimp,Penaeus monodon (Fabricius)

White spot disease caused by white spot syndrome virus (WSSV) is the major issue of huge economic destruction globally in the shrimp aquaculture industry. In the present investigation, WSSV prevalence associated with disease resistance was studied among wild black tiger shrimp, Penaeus monodon (Fabricius) from four distant geographic locations along the East coast of India during 2009–2010. Results suggested that the WSSV prevalence in wild P. monodon was the highest (56.2%) in Chennai, Tamil Nadu followed by Digha, West Bengal (10.9%), Visakhapatnam, Andhra Pradesh (0.6%) and Chilika, Orissa (0%). Quantitative data suggested that the mean copy number of WSSV among these four places was 1.4 × 10⁶, 4.6 × 10⁴, 1.6 × 10² and 2.3 × 10² copies μg^?1 shrimp genomic DNA respectively. The disease resistant prevalence using the 71 bp microsatellite DNA marker was the highest among Chilika, Orissa (63.6%) and Visakhapatnam, Andhra Pradesh (63.5%). Higher WSSV prevalence in Chennai, Tamil Nadu and Digha, West Bengal corresponded to lower disease resistant prevalence (24% and 40.2%). Conclusively, probably collection of broodstock of P. monodon from places like Chilika and Visakhapatnam would be a much safer approach for the development of specific pathogen‐resistant shrimp aquaculture.     

Nucleotide sequence variations of the major structural proteins (VP15, VP19, VP26 and VP28) of white spot syndrome virus (WSSV), a pathogen of cultured Litopenaeus vannamei in Mexico

White spot syndrome virus (WSSV) was first reported in farmed Litopenaeus vannamei stocks in Sinaloa and Sonora, Mexico during 1999 and continues to cause severe shrimp losses. WSSV genes encoding nucleocapsid (VP26 and VP15) and envelope proteins (VP19 and VP28) of a Mexican isolate were cloned in the pMosBlue vector. The nucleotide sequences of these genes were compared with WSSV isolates in GenBank. VP15 is highly conserved, and VP26 showed 99% homology to a Chinese isolate. The VP28 fragment demonstrated 100% homology to the majority of the isolates analysed (UniProt accession no. Q91CB7), differing from two Indian WSSV and one Chinese WSSV isolates by two non-conserved and one conserved replacements, respectively. Because of their highly conserved nature, these three structural proteins are good candidates for the development of antibody-based WSSV diagnostic tools or for the production of recombinant protein vaccines to stimulate the quasi-immune response of shrimp. In contrast, VP19 of the Mexican isolate was distinguishable from almost all isolates tested, including an American strain of WSSV (US98/South Carolina, GenBank accession no. AAP14086). Although homology was found with isolates from Taiwan (GenBank accession no. AAL89341) and India (GenBank accession no. AAW67477), VP19 may have application as a genetic marker.     

Variation in early growth and survival of pink salmon (Oncorhynchus gorbuscha) families with respect to temperature and transfer to salt water

Five pairs of paternal half-sib families of pink salmon (Oncorhynchus gorbuscha) were transferred from fresh to 28‰ salt water 1, 7, and 28 days after button-up of the fry and reared for 51 days at 13°C. In another experiment, pink salmon from three populations were also reared in fresh and salt water at 9.5°C for 49 days after fry button-up. Juvenile growth and survival rates were greater when juveniles were reared for a period of time in fresh water after button-up. Juveniles transferred to salt water 1 day after button-up had the lowest growth and survival rates. Juveniles reared at 13°C grew up to five times faster than those reared at 9.5°C. There was evidence of genetic differences in juvenile growth and survival among half-sib families.     

Morphopathological features of a severe ulcerative disease outbreak associated with Tenacibaculum maritimum in cultivated sole, Solea senegalensis (L.)

This study describes morphopathologic changes in naturally infected farmed Senegalese sole affected by tenacibaculosis caused by Tenacibaculum maritimum. Macroscopic observation, in addition to light microscopy and scanning electron microscopy, was used to study the lesions. Main lesions were characterized by complete loss of epidermis and dermis, as well as extensive necrosis of muscle layers. Mild-to-moderate inflammatory response with the presence of macrophages was noted around hyaline degenerated muscle cells. Gram-negative filamentous bacteria could be detected only at the dermis. Under scanning electron microscopy, filamentous bacteria located over the scales without epithelium could be observed. These findings together with the isolation and PCR detection of the bacteria in kidney and skin tissues suggest that once the bacteria reach the dermis, probably through eroded epidermis, they are able to proliferate and produce enzymes that are responsible for the damage in the underlying tissues.     

A comparison of RNA concentrations and ornithine decarboxylase activity in Atlantic salmon (Salmo salar) muscle tissue,with respect to specific growth rates and diel variations

RNA concentrations and enzyme activities are often used as indices of recent growth in fish, but few studies have used both methods to assess the same fish. This study measured RNA concentrations and ornithine decarboxylase (ODC) activity in muscle tissue of juvenile Atlantic salmon (Salmo salar) to compare their usefulness for reflecting specific growth rates, and to determine whether either growth index was influenced by diel variations or time of feeding. Three groups (n = 54 in total) were fed 1.5% of body weight in commercial pellets in four feedings per day. One group was fed only in the morning (0830–1230h), one in the afternoon (1430–1830h), and one in the morning and afternoon (0830–1830h). At the end of ten days, fish were sampled at three times (0130h, 1030h, 1630h) over a single 24h period. Correlations to specific growth rate were slightly higher for RNA concentrations than for ODC activity, but both were highly significant. RNA and ODC activity were also correlated to each other. These results suggest that RNA concentration and ODC activity, taken together, can be used to monitor changes in both the numbers and activity of ribosomes. For RNA concentrations, there was no evidence of an effect of diel variations or the time of feeding. For ODC activity, a significant diel effect (all feed schedules combined) was detected if one non-growing fish was excluded from the analysis; activity of the enzyme was slightly higher in the sample taken at night (0130h) than in the two daytime samples. Contribution no. 8, Catamaran Brook Habitat Research Project