Protozoan parasites of gilthead sea bream, Sparus aurata L., from different culture systems in Spain

Abstract. In a parasitic survey of gilthead sea bream, Sparus aurata L., cultured in different systems in Spain, eight protozoan species were found. Ectoparasites included two ciliates ( Trichodina sp. and a Cryptocaryon -like species), one dinoflagellate ( Amyloodinium sp.) and the zooflagellate Ichthyobodo sp. Endoparasites were represented by members of the Apicomplexa ( Eimeria sp.) and Myxosporea ( Polysporoplasma sparis. Ceratomyxa sp. and Leptotheca sp.). Ichthyobodo sp., Eimeria sp., Ceratomyxa sp. and Leptotheca sp. are reported for the first time in S. aurata. Amyloodinium sp. and Ichthyobodo sp. were associated with outbreaks of disease in larval and juvenile fish. Data on prevalence and intensity of infection and the location in the host are provided. The potential importance of these protozoan parasites for gilthead sea bream culture is discussed.     

Experimental amoebic gill disease of Atlantic salmon, Salmo salar L.: further evidence for the primary pathogenic role of Neoparamoeba sp. (Page, 1987)

Amoebic gill disease (AGD) has been attributed to infection by Neoparamoeba sp. The causal mechanisms for AGD lesion development and the primary pathogenic role of Neoparamoeba sp. require elucidation. Three groups of Atlantic salmon were exposed to viable gill isolated amoebae, to sonicated amoebae, or to sea water containing viable amoebae without direct contact with gill epithelia. Fish were removed 8 days post-exposure and the gills assessed histologically for AGD. AGD occurred only when fish were exposed to viable trophozoites. Consequently, in an accompanying experiment, infection was evaluated histologically at 12, 24 and 48 h post-exposure in three groups of salmon, one group being mechanically injured 12 h prior to exposure. A progressive host response and significant increase (P < 0.001) in the numbers of attached amoebae was apparent over the 48-h duration in undamaged hemibranchs in both treatment groups. There were no significant differences to mucous cell populations. Attachment of Neoparamoeba sp. to damaged gill filaments was significantly reduced (P < 0.05) by 48 h post-exposure. These data further confirm and describe the primary pathogenic role of Neoparamoeba sp. and the early host response in AGD. Preliminary evidence suggests that lesions resulting from physical gill damage are not preferentially colonized by Neoparamoeba sp.     

欧洲鳗两极虫病的流行与防治

欧洲鳗鲡两极虫主要寄生在欧鳗的肾脏、鳃和皮肤，造成寄生部位的组织溃疡、发炎，影响鳗鱼摄食，容易诱发严重的细菌性疾病，导致严重的经济损失。欧洲鳗两极虫的感染为春夏高峰型，感染率和感染强度有明显的季节变化，感染高峰期在2～6月份，主要感染当年的欧洲鳗鳗苗。     

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.     

盐度对寄主暗纹东方鲀(Takifugu obscurus)存活影响及其对车轮虫的杀灭作用

为防治河鲀车轮虫病,分别设置0、2.5、5.0、7.5、10.0、12.5、15.0、17.5、20.0、25.0、30.0、35.0和40.0共13个盐度梯度,对暗纹东方鲀进行了浸浴实验。结果显示,盐度为15以下,对暗纹东方鲀的存活没有影响;盐度为0、2.5、5.0时,4 d内车轮虫的数量没有减少反而有增加趋势;盐度7.5作用4 d,盐度10.0作用3.5 d,盐度12.5作用3 d,杀虫率可达95%以上;盐度15.0作用32 h,盐度17.5作用24 h,盐度20.0作用10 h,盐度25.0作用4 h,盐度30.0作用1.5 h,盐度35.0作用1 h,盐度40.0作用0.5 h,对车轮虫的杀虫率均可达100%。应用SPSS软件,对杀虫盐度和时间进行拟合曲线方程分析,其数学模型为对数曲线方程y=34.19-5.55lnx。另外,车轮虫在暗纹东方鲀体表的复活试验显示,用提高盐度的方法杀灭暗纹东方鲀体表车轮虫后,再恢复到正常养殖盐度5.0的情况下,暗纹东方鲀的体表车轮虫病不再复发,说明盐度7.5以上能治疗暗纹东方鲀的车轮虫病。结合提高盐度治疗暗纹东方鲀车轮虫病的生产性试验结果,建议以盐度为7.5–15.0作为治疗暗纹东方鲀体表车轮虫的适宜盐度。     

Systematic granulomas caused by a rickettsia-like organism in Nile tilapia, Oreochronuis niloticus (L.), from southern Taiwan

Abstract. A rickettsia-like organism was observed in diseased Nile tilapia, Oreochronuis niloticus (L.), from southern Taiwan. Most affected fish were pale and lethargic with haemorrhages and ulcers on the skin. The most significant gross pathological changes were varying degrees of ascites, and enlargement of the spleen, kidney and liver. Marked white nodules (as ring-shaped foci), varying in size, were found in these organs. A Gram-negative, rickettsia-like organism (RLO) frequently appeared as inclusions or within host cell intra-cytoplasmic vacuoles. Fibrin thrombi, perivascular necrosis, chronic inflammatory cells with hypertrophy and RLO-laden cells were characteristic of the disease. White nodules induced in experimental fish were similar to naturally infected cases and RLOs were reisolated in tissue culture using a CHSE-214 cell line. This provides evidence that the systemic granulomas in Nile tilapia were caused by RLOs.     

Metabolomic investigation of pathogenesis of myxosporean emaciation disease of tiger puffer fish Takifugu rubripes

Serum biochemical analysis was undertaken to study the pathophysiological details of emaciation disease of the tiger puffer fish Takifugu rubripes (Temminck and Schlegel). Serum parameters were measured by biochemical analysis using automated dry chemistry and gas chromatography/mass spectrometry (GC/MS). Serum concentrations of albumin, amylase, calcium, creatinine, glucose and total protein were significantly lower in the emaciated fish when compared with those of normal fish. Regression analyses found close correlation between concentrations of total protein, albumin, amylase, glucose and progress of the disease. In contrast, serum alanine aminotransferase increased significantly in emaciated fish indicating liver function disorder. Further, GC/MS metabolic profiling of the puffer serum showed that the profile of the emaciated fish was distinct to that of non‐infected control. The serum content of amino acids including glycine, 5‐oxo‐proline and proline, and ascorbic acid, fumaric acid and glycerol increased significantly in serum in moderately emaciated fish. The serum glucose, linolenic acid and tyrosine level decreased significantly in the late phase of the disease. Our results clearly show that prolonged intestinal damage caused by myxosporean infection impairs absorption of nutrients, resulting in extreme emaciation.     

The occurrence and distribution of Myxidium giardi Cépède, 1906 in wild and cultured European eels, Anguilla anguilla L., in England

Abstract. Myxidium giardi was found in wild and cultured eels. The prevalence rate in elvers was 77% in the River Torridge (Devon), 80% in the River Parrett (Somerset) and 90% in the River Ouse (Yorkshire). Adult eels from the River Ouse had a prevalence rate of 53%, Cultured fingerlings and adults had a prevalence rate of 60% and 1% , respectively. There was an apparent age-related pattern in the organ location of the histozoic trophozoite: an initially high prevalence of kidney trophozoites which gradually decreased with age; in the gills of elvers there was a low prevalence of histozoic trophozoites which increased in the adult stage. Histozoic trophozoites were also found in the stomach and intestine of elvers and fingerlings. A qualitative estimate of trophozoite numbers in each organ of elvers is given. The coelozoic trophozoite of M. giardi in the gall bladder of elvers and fingerlings is also described for the first time.     

Effects of temperature on disease progression and swimming stamina in Ichthyophonus-infected rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss , were infected with Ichthyophonus sp. and held at 10 °C, 15 °C and 20 °C for 28 days to monitor mortality and disease progression. Infected fish demonstrated more rapid onset of disease, higher parasite load, more severe host tissue reaction and reduced mean-day-to-death at higher temperature. In a second experiment, Ichthyophonus -infected fish were reared at 15 °C for 16 weeks then subjected to forced swimming at 10 °C, 15 °C and 20 °C. Stamina improved significantly with increased temperature in uninfected fish; however, this was not observed for infected fish. The difference in performance between infected and uninfected fish became significant at 15 °C ( P  =   0.02) and highly significant at 20 °C ( P  =   0.005). These results have implications for changes in the ecology of fish diseases in the face of global warming and demonstrate the effects of higher temperature on the progression and severity of ichthyophoniasis as well as on swimming stamina, a critical fitness trait of salmonids. This study helps explain field observations showing the recent emergence of clinical ichthyophoniasis in Yukon River Chinook salmon later in their spawning migration when water temperatures were high, as well as the apparent failure of a substantial percentage of infected fish to successfully reach their natal spawning areas.     

Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel)

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate‐buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.     

Development of proliferative kidney disease in rainbow trout, Oncorhynchus mykiss (Walbaum), following short-term exposure to Tetracapsula bryosalmonae infected bryozoans

The initial site of infection in the fish host for Tetracapsula bryosalmonae , causative agent of proliferative kidney disease (PKD) is poorly understood. Following the recent recognition that freshwater bryozoans harbour the infective stages to salmonid fish, experimental transmission studies were undertaken to investigate (1) the route of entry of the parasite into the fish host and (2) the minimum exposure time required to induce clinical signs of PKD. In-situ hybridization (ISH) studies were carried out on naïve rainbow trout exposed to the naturally infected bryozoan Fredericella sultana for up to 90 min. The sporoplasm of T. bryosalmonae was detected entering the fish via mucous cells in the skin epithelium within the first minute of exposure. In addition, T. bryosalmonae cells were infrequently detected in the skeletal musculature of exposed experimental fish up to 72 h post-exposure. The route of migration through the fish to the kidney and spleen was not determined. All fish exposed to infected, disrupted bryozoans for 10, 30 and 90 min and maintained for up to 8 weeks developed clinical PKD.     

Effect of marine Eubothrium sp. (Cestoda: Pseudophyllidea) on the growth of Atlantic salmon, Salmo salar L.

Atlantic Salmon, Salmo salar L., experimentally infected with marine Eubothrium sp. were kept together with uninfected salmon in the laboratory for 11 months in two tanks, 80 infected and 80 uninfected in each tank. The infected fish had a reduced growth rate compared with the uninfected fish. Significant differences in growth between infected and uninfected fish were not observed until several months post-infection. There was no correlation between the number of Eubothrium sp. and fish weight, indicating that even low intensity alters the growth rate of the salmon. The cestode had the same effect on both sexes of salmon. Haematocrit level was found to be significantly lower in infected compared with uninfected salmon in one of the samples during the experiment.     

The impact of nutritional factors on the immune system in Atlantic salmon, Salmo salar L.

Abstract. Pancreas disease (PD) was induced by inoculating freshwater parr, Salmo salar L., with a homogenate of pooled tissues collected from four fish in the acute phase of disease. Pathological changes were observed in the liver and pancreas on day 4 post inoculation (PI) and in the heart on day 8 PI. Regeneration of necrotic acinar tissue was evident by day 8 PI. The infectious nature of PD was manifested by fish cohabiting with experimentally infected fish succumbing to PD after 13 days. The PD agent/agents retained its infectivity after filtration through a 0-2-μm filter.     

The life history of Haplorchis pumilio (Looss, 1896) from cultured tilapias

Abstract. The metacercaria of Haplorchis pumilio (Looss, 1896) (Digenea: Hetero-phyidae) was found to occur commonly in intensively reared tilapias ( Tilapia, Sarotherodon ) in an East African fish farm. The life cycle on the farm was found to involve the gastropod Melanoides tuberculata (Müller) as the first intermediate host. The infection was experimentally transmitted to fish in fresh water from naturally infected molluscs. The salinity of the water on the farm varied with season from 0 to 3%₀, but cercariae were shown to remain infective up to 10%₀ although longevity was reduced at 7.5%₀. Piscivorous predators, Ardea cinerea L., Phalocrocorax sp. and Varanus sp., which were resident or regular visitors to the farm were heavily infected with the parasite. Mice, hamsters and pigeons were found to be convenient laboratory hosts. H. pumilio shows a lack of specificity at the second intermediate and final host level as well as a wide geographic range and has been reported to infect man. Its potential as a possible cause of zoonoses, and control methods are discussed.     

The pathology of Saprolegnia infections of Anguilla anguilla L. elvers

Abstract. The clinical signs and histopathology of Saprolegnia sp. [related to S. diclina Humphrey (syn. S . parasitica Coker)] are described in cultured Anguilla anguilla L. elvers under an intensive production system. The main lesions observed were loss of epithelium leading to ulceration, oedema and myofibrillar degenerative changes of the muscle mass. The oedema resulted in swelling of the inter-myotomal connective tissue, loss of nuclei and minor host reaction. Traumatic lesions with subsequent bacterial infections had a mononuclear inflammatory response. It is suggested that the rapid extension of the lesion is due to the loss of integrity of the integument and the widespread oedema, which altered the viability of tissues and assisted the Saprolegnia infection. The condition was fatal for infected elvers.     

Water temperature,time of exposure and population density are key parameters in Enteromyxum leei fish-to-fish experimental transmission

Enteromyxum leei is a myxozoan histozoic parasite that infects the intestine of several teleost fish species. In gilthead sea bream (Sparus aurata), it provokes a chronic disease, entailing anorexia, delayed growth, reduced marketability and mortality. Direct fish-to-fish transmission, relevant in aquaculture conditions, has been demonstrated for E. leei via effluent, cohabitation, and oral and anal routes. However, the minimum time of exposure for infection has not been established, nor the possible effect on the fish immune response. Two effluent trials were performed at different temperatures (high: average of 25.6°C; and low: constant at 18°C), different times of exposure to the effluent (1, 3, 5 and 7 weeks) and different population densities. The results showed that 1 week was enough to infect 100% of fish at high temperature and 58.3% at low temperature. High temperature not only increased the prevalence of infection in posterior intestine, but also induced a higher production of specific antibodies, limiting the progression of the infection along the intestine. Longer time of exposure to the parasite and higher fish densities facilitated E. leei infection. These results show that effective diagnosis, lowering animal density and removal of infected fish are key aspects to manage this disease in aquaculture facilities.     

The occurrence of proliferative kidney disease (PKD) in cultured and wild fish: further investigations

Abstract. In an investigation of the occurrence of proliferative kidney disease (PKD) in freshwater fish other than rainbow trout, 18 species of wild fish and seven species of fish raised in cultivation wore sampled from waters where the disease occurred annually in rainbow trout, Oncorhynchus mykiss (Richardson). Results revealed that certain wild stocks of brown trout. Salmo trutta L., grayling, Thymallus thymallus L., and pike, Esox lucius L., were infected with PKD, as were cultivated Atlantic salmon, Salmo salar L., parr, brown trout and char, Salvelinus alpinus (L.). Microscopical examination revealed the presence of the PKX cell in these species and also intraluminal protozoa possibly related to the PKX cell, which were not found in the rainbow trout. Other species of freshwater fish had myxosporidan infections but, unlike PKD infection, there was little host/parasite tissue response. The PKX cell as a myxosporidan stage is discussed and the presence of the disease in wild fish is reported.     

Mycobacteria isolated from Chesapeake Bay fish

Mycobacteriosis in fish can result in ulcers, emaciation, and in some cases death. Mycobacteria have been previously isolated from a variety of Chesapeake Bay fish species, and the current study was designed to identify potential host specificity and location fidelity of mycobacterial isolates. Mycobacteria were isolated from wild fish of the Chesapeake Bay collected from the Upper Bay, the Choptank River, Herring Bay, the Chicamacomico River, the Pocomoke River and the Potomac River in 2003–2006. Mycobacterial isolates were recovered from striped bass, Morone saxatilis , Atlantic menhaden, Brevoortia tyrannus , white perch, Morone americana , summer flounder, Paralichthys dentatus , spot, Leiostomus xanthurus , largemouth bass, Micropterus salmoides , channel catfish, Ictalurus punctatus , common carp, Cyprinus carpio carpio , spotted seatrout, Cynoscion nebulosus , killifish, Fundulus sp., blueback herring, Alosa aestivalis , American gizzard shad, Dorosoma cepedianum and American silver perch, Bairdiella chrysoura . Twenty-nine well-defined mycobacterial groups resulted from gas chromatography dendrogram clustering of isolates. The majority of groups included more than one host species and more than one site of collection. However, four groups contained only striped bass isolates, three of which were similar to M. shottsii . Therefore, multiple Chesapeake Bay fish species are colonized with multiple mycobacterial isolates, of which few appear to be host or location specific.     

Infection of the epaulette shark, Hemiscyllium ocellatum (Bonnaterre), by the nematode parasite Proleptus australis Bayliss (Spirurida: Physalopteridae)

Seventy-two epaulette sharks, Hemiscyllium ocellatum (Bonnaterre), were infected with the nematode parasite Proleptus australis Bayliss, 1933. The parasite population was overdispersed. Infection intensity ranged from 3 to 1002 worms per fish stomach, and there was a positive correlation between shark length and number of parasites present. The majority of worms were attached to the stomach wall, and scanning electron microscopy and histological examination showed that worms penetrated the stomach lining. Worms were observed within the lamina propria of the stomach and occasionally penetrated the muscularis mucosa. Little to no inflammatory or cellular immune reaction to the presence of the parasites was observed, except in one case where a worm was being degraded by a host tissue response. There was a large amount of connective tissue proliferation as a result of nematode attachment, but no obvious effects on the overall health of the sharks were seen. Three sharks were also found to be infected by the cestode Callitetrarhynchus sp.     

Early stages of Margaritifera margaritifera glochidiosis in Atlantic salmon: Morphopathological characterization

Freshwater mussels of the order Unionida encyst into the fish mucosa to metamorphose and complete their life cycle, causing a parasitic disease known as glochidiosis. This parasitic stage represents a bottleneck for the survival of naiads, particularly for critically endangered species as Margaritifera margaritifera; however, little is known about the events occurring during this critical stage. Therefore, this study aimed to histologically characterize the development of M. margaritifera glochidiosis in Atlantic salmon to get insight into the pathogenesis of this interaction. Fish exposed to glochidia were sampled during the first 44 days post-exposure, and organs were observed by stereomicroscopy and light microscopy. Glochidia attached to the gills by pinching the lamellar epithelium, whereupon an acute proliferative branchitis engulfed most of the larvae. However, during the first 14 days, a severe detachment of unviable glochidia occurred, associated with the presence of pleomorphic inflammatory infiltrate and epithelial degeneration. In the cases where larvae remained attached, a chronification of the lesions with none to scarce inflammation was observed. These results provide key information to better understand the complex host–parasite interaction during the early stages of glochidiosis and provide valuable information to optimize artificial rearing of naiads in conservation of threatened freshwater mussel populations.