Histopathological changes in Trypanosoma danilewskyi Laveran & Mesnil, 1904 and Trypanoplasma borelli Laveran & Mesnil, 1902 infections of goldfish, Carassiw auratus (L.)

Abstract. Syringe passaged stocks of Trypanosoma danilewskyi and Trypanoplasma borelli derived from carp were used for experimental infections of goldfish. Infections of T. danilewskyi and T . borelli produced a heavy parasitaemia in the peripheral blood of goldfish and extensive tissue damage, mainly in haemopoietic organs. There was no inflammatory reaction or haemorrhage in trypanosome infected goldfish. Infection with trypanoplasms provoked more varied changes including vascular lesions and inflammatory changes. The extent of pathological changes occurring at rising and peak levels of parasitaemia could account for the high mortality (up to 100%) of the infected fish.     

Cultivation of Trypanosoma danilewskyi (Laveran & Mesnil, 1904) in serum-free medium and assessment of the course of infection in goldfish, Carassius auratus (L.)

Abstract. Purification and in vitro cultivation techniques were developed for the fish haemoflagellate, Trypanosoma danilewskyi. The parasites were isolated and purified from the peripheral blood of experimentally infected goldfish, using a combination of Ficolt-paque gradient centrifugation to remove fish red blood cells and in vitro incubation to remove the remaining fish leucocytes. A serum-free culture medium for T. danilewskyi supported both short- and long-term cultivation of the haemoflagellates. The serum-free medium is a mixture of reagents available commercially: Leibovitz's L-15 medium, Dulbecco's Modified Eagle Medium and Hank's balanced salt solution. The doubling time was calculated to be 44.4 × 7.8h. Typically, a two- to five-fold increase in the number of cultured parasites was observed on day 7 after subculture with 1 × 10⁶ and 5 × 10⁵ trypanosomes ml⁻¹, respectively. When administered to fish, the in vitro -derived parasites caused an infection and pathology whose characteristics were similar to those observed following infection with trypanosomes obtained from infected goldfish. The freshly isolated and in vitro -grown parasites were successfully cryoprescrvcd in the culture medium containing 10% glycerine at −80°C for at least 3 months. Although the viability of the parasite decreased by 40–50% after thawing, cryoprcserved parasites retained the ability to infect goldfish.
Correspondence: Dr M. Belosevic, Associate Professor, Department of Zoology, CW-312 Biological Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2E9.     

China rose (Hibiscus rosasinensis) petals: a potent natural carotenoid source for goldfish (Carassius auratus L.)

Goldfish (Carassius auratus) are in demand in world markets due to their attractive golden colour. Carotenoids are the primary source of colour in the skin of fish. To optimize the colour in captivity, fish must obtain an adequate level of carotenoids in their feed. With this objective, four natural colour enhancers were tested. A common batch of feed was divided into five equal portions and colour ingredients, spirulina (D‐S), china rose petals (D‐C), marigold petals (D‐M) and Lactobacil, a commercial probiotic (D‐L), were added at 5 mg kg⁻¹ to four portions of feed; one portion (D‐O) was kept as a control without any additive. A feeding trial was conducted for 8 weeks. Each 70 L aquarium was stocked with 10 fish (average weight 1.6 g) and feed was given at 5% of the body weight. Growth rate, survival, biochemical composition and pigmentation in the skin of fish were measured. Histological studies of gonads were also conducted. Growth of fish in different treatments was significantly different. There was no difference in the proximate composition of the fish at the start of the experiment but after 8 weeks of feeding, fish fed the diet supplemented with china rose petals had a lower moisture content (70.48%) and higher protein (17.7%) and lipid (5.25%) levels than the group fed the control diet. Pigmentation was the highest (4.01 μg g⁻¹) in D‐C, followed by D‐M (3.16 μg g⁻¹), D‐S (2.92 μg g⁻¹) and D‐L (2.84 μg g⁻¹) and the lowest (0.24 μg g⁻¹) in D‐O. Gonad development of fish fed with the D‐C diet was better compared with the gonads of control (D‐O) fish, followed by D‐M‐, D‐L‐ and D‐S‐fed fish. Gonads of fish, fed D‐C, showed well‐marked changes in the testis where a large number of seminiferous tubules bound together by means of a thin layer of connective tissue were observed. These tubules were highly convoluted and were separated from each other by thin connective tissue stroma. The intra space contained connective tissue, blood capillaries and interstitial cells. The spermatogonia could be seen as a large spherical cell containing a large central nucleus with a distinct nucleolus. The study shows that the china rose (Hibiscus rosasinensis) petal is a potent natural carotenoid source for goldfish to enhance its colour and also accelerate gonadal development.     

Persistence of cyprinid herpesvirus 2 in asymptomatic goldfish Carassius auratus (L.) that survived an experimental infection

Cyprinid herpesvirus 2 (CyHV‐2) is the causative agent of herpesviral haematopoietic necrosis (HVHN) in goldfish, Carassius auratus, and Prussian carp, C. auratus gibelio. In this study, we investigated virus persistence in goldfish experimentally infected with CyHV‐2. Virus DNA presence in organs was monitored in survivors reared at a virus permissive temperature and also in survivors treated with a non‐permissive temperature for 4 days, initiated at three different time points post‐infection in order to obtain fish with different virus loads. We detected virus DNA in all organs tested at 51 days post‐infection (dpi) and in the spleen, trunk kidney and gills of survivors at 81 dpi, although the virus load in fish influenced the subsequent number of organs that tested positive for virus DNA. In addition, some organs dissected from four out of five asymptomatic survivors tested positive by PCR following incubation in vitro in a medium for 5 days. Following inoculation with the homogenate of PCR‐positive kidney incubated in vitro, one of the three inoculated fish died, showing that the detected virus by PCR produced infectious particles. This study suggests that CyHV‐2 can establish a persistent infection in some organs, especially the spleen and trunk kidney, and that asymptomatic surviving fish can be a source of infection.     

Use of canola oil in the feed of larval and juvenile goldfish, Carassius auratus (L.)

Goldfish were used as a model for the evaluation of canola oil as a lipid source in the feeds of larval and juvenile cyprinids. Goldfish larvae were raised from hatching until 24 weeks of age on diets containing cod liver oil. canola oil or a mixture of the two oils as the lipid source. Survival, weight gain and weight-length relationship did not differ among groups of fish fed the three diets. Carcass fatty acid profiles largely reflected those of the diets except that carcasses of fish fed canola oil contained long chain (n-3) and (n-6) polyunsaturated fatty acids (PUFA) that are not found in canola oil. This indicates that goldfish are capable of producing these fatty acids from 18-carbon precursors. The flesh of fish fed canola oil would be inferior for human nutrition to that offish fed marine oils, due to lower (n-3) PUFA levels. However, the results do indicate that canola oil has good potential as a lipid source in larval cyprinid diets.     

Aetiology of an ulcerative disease in goldfish, Carassius auratus (L.): characterization of the causative agent

Abstract. A bacterium resembling Aeromonas salmonicida and determined to be the aetiologic agent of a cutaneous ulcerative disease in goldfish Carassius auratus (L.) was further characterized in this study.
Forty-five isolates of the bacterium (43 from the United States and one each position (moles % guanine plus cytosine) and DNA homology. The bacteriological atypical A. salmonicida previously described. Several important biochemical characteristics distinguished the goldfish isolates from typical A. salmanicida , but the DNA binding experiments indicated a high degree of relatedness between the goldfish isolates and typical A. salmonicida strains.     

Aetiology of an ulcerative disease in goldfish, Carassius auratus (L.): experimental induction of the disease

Abstract. Infection experiments were conducted to determine the primary aetiology of an ulcerative disease in goldish, Carassius auratus (L.). Goldfish were exposed to atypical Aeromonas salmonicida and A. hydrophila , previousl y isolated from cutaneous ulcers, and to 04 5 μm filtrates of cutaneous ulcers and kidneys from diseased fish. Fish were exposed to each preparation by intraperitoneal, intramuscular or subcutaneous injection and by a method in which a small patch of scales was removed from each side of the fish and the inoculums applied. Most of the fish injected with A. salmonicida died without developing coetaneous ulcers; however, ulcers were induced in five of the ten fish exposed by the scale removal technique. Exposure to ultra filtrates or A. hydrophila did not result in consistent ulcer formation o r death. Additional experiments showed that a 30-min exposure of goldfish, without prior treatment, to water containing 3 × 10⁵ colony forming units (cfu/ml) of A. salnumicida was sufficient to produce cutaneous lesions in nine often fish exposed. Multiple lesions were produced in most fish and A. salmonicida was consistently recovered. Fish exposed by similar waterborne challenge s to 6·2 × 10⁶ cfu/ml of A. hydrophila or to 7·2 × 10⁶ cfu/ml of another lesion isolate identified as a member of the A. hydrophila complex produced no lesions, eve n when scales were removed. The studies demonstrate that atypical A. salmonicida can initiate cutaneous lesion s characteristic of ulcerative disease, while A. hydrophila and an A. hydrophila complex organism cannot.     

A comparison of artificial and natural foods and their combinations in the rearing of goldfish, Carassius auratus (L.)

Intensive grow‐out of goldfish, Carassius auratus (L.), larvae and juveniles in closed systems requires the control of environmental conditions and feeding. This study investigates the use of different types of live food and combinations of live food and dry food in a series of four rearing experiments. Juvenile goldfish can be weaned from Artemia onto live food at about 24 days after the onset of feeding without causing a reduction in growth and survival. The replacement of Artemia by Daphnia at day 10 appears feasible, as growth and survival were not significantly affected. Fish fed decapsulated Artemia cysts grew better than fish fed live Artemia. Within the first 14 days, goldfish juveniles should be fed at least 155 cysts per fish per day to achieve fast growth and to minimize size variation.     

The susceptibility of salmonid fish to an atypical strain of Aeromonas salmonicida that infects goldfish, Carassius auratus (L.), in Australia

Abstract. An enzootic, Australian, atypical strain of Aeromonas salmonicida isolated from diseased goldfish, Carassius auratus (L.), was inoculated into Atlantic salmon, Salmo salar L., brown trout, S. trutta L., rainbow trout, S. gairdneri Richardson, and brook trout, Salvelinus fontinalis (Mitchill), fingerlings by intraperitoneal injection (i.p.) and by bath challenge, the latter with and without prior abrasion of skin. The 10-day LD₅₀ (i.p.) was estimated to be 7·4 × 10^-3 colony forming units (cfu) for Atlantic salmon, 3·0 × 10^-2 cfu for brown trout, 3·7 × 10² cfu for brook trout and 6·4 × 10³ cfu for rainbow trout. Brown, rainbow and brook trout succumbed to bath challenges with between 10⁵–10⁶ cfu/ml, developing ulcers of the skin and septicaemia. The organism was trasmitted from inoculated fish to five of 195 within-tank control fish via water and established a carrier state in one of 14 Atlantic salmon. It was concluded that the organism poses a significant threat to the salmonid farming industry and wild salmonid fisheries in Australia.     

Establishment, characterization and viral susceptibility of a new cell line derived from goldfish, Carassius auratus (L.), tail fin

A continuous cell line [goldfish tail fin (GFTF)] derived from a goldfish tail fin, Carassius auratus, was established and characterized. GFTF cells predominantly consist of fibroblast-like cells that were maintained and subcultured more than 50 times over a period of 15 months. Cells grew at temperatures between 15 and 37°C, with an optimum temperature of 25°C. The growth rate of GFTF cells increased proportionally with the foetal bovine serum (FBS) concentration (5-20%), with optimum growth at 20% FBS. The chromosome numbers were 88-112, with a modal peak of 104 chromosomes. Five known fish viruses were tested to determine susceptibility. Results demonstrated that GFTF is susceptible to snakehead rhabdovirus, spring viraemia of carp virus and channel catfish virus (CCV). In addition, GFTF demonstrated a higher sensitivity to, and increased viral production of, CCV than that observed in the control cell line, channel catfish ovary cells. This suggests that GFTF cells would be useful as a diagnostic tool for viral diseases in this fish species, as well as for the isolation and study of goldfish viruses in the future. Furthermore, these cells were transfected with pEGFP-N1 vector DNA and some fluorescent signals were observed, suggesting that GFTF cells could be a useful tool for transgenic and genetic manipulation studies.     

Comparison of food selection and growth performance of koi carp, Cyprinus carpio L., and goldfish, Carassius auratus (L.) in mono- and polyculture rearing in tropical ponds

To compare the effect of polyculture against conventional monoculture on ornamental carp production, investigations on food selection and growth performance of koi carp (K), Cyprinus carpio L. and goldfish (G), Carassius auratus (L.) were conducted in a 11‐week rearing experiment in two monoculture (100% K and 100% G) and five polyculture (90% K–10% G, 70% K–30% G, 50% K–50% G, 30% K–70% G and 10% K–90% G) conditions in tropical ponds. There were three replicates for each treatment. Environmental conditions and food availability were similar in all the treatments. Ivlev's electivity index showed that both fish species avoided phytoplankton and preferred cladocerans to other zooplankton groups (copepods and rotifers) in monotypic conditions. However, in the polyculture treatments, the positive electivity of goldfish towards cladocerans reduced significantly (P<0.05), while the percentage of copepods, rotifers and phytoplankton in the gut content increased. No significant differences in weight gain, specific growth rate and deformities were recorded at harvest for koi carp between the different treatments (P>0.05). Even the survival rate of koi carp recorded above 90% in all the treatments. However, the goldfish recorded significantly better weight gain, specific growth rate and survival in monoculture (100% G), compared with the polyculture treatments (P<0.05). Goldfish deformities were lowest (P<0.05) in the monoculture treatment (2.42%). The number of marketable fish above a set size limit of 4 g total weight was significantly higher in the two monoculture treatments, compared with the five polyculture treatments (P<0.05). Keeping in view of the dietary similarities of koi carp and goldfish, and the aggressive nature of koi carp in polyculture, it is suggested to refrain from polyculture of goldfish and koi carp until further documentations relating to optimum stocking density and management of polyculture of ornamental carps are available.     

Cellular responses of goldfish, Carassius auratus (L.), to metacercariae of Ribeiroia marini (Faust & Hoffman, 1934)

Goldfish, Carassius auratus (L.), mobilized an acute, non-specific cellular inflammatory response against experimental infection with metacercariae of the digenean Ribeiroia marini in lateral line scale canals. The host cellular response to primary infection consisted of a rapid infiltration of neutrophilic granulocytes and macrophages that adhered to the metacercial cyst wall. Granulocytes released cytoplasmic granules, forming a necrotic zone around the cyst. Outside the necrotic zone, a fibrocytic zone and epithelioid cells formed a matrix upon which reactive leucocytes were attached in a progressive granulomatous encapsulation. Interdigitating epitheliod cells formed a syncytial border separating the necrotic zone from the outer fibrocytic region of the granuloma. Acute inflammatory response preceded the expulsion of most metacercariae from the scale canals. Few cysts remained encapsulated in epidermal tissue. Cellular response to challenge infections was more intense, but no major differences in leucocyte composition between primary and challenge infections suggested a continuum of inflammation and cell-mediated responses. In late primary and challenge infections, the predominant eosinophilic granular cell released cytoplasmic granules. We propose that the eosinophilic granular cell of the goldfish has an antiparasitic function, releasing bioactive granules that alter the environment of the scale canal and cause the expulsion of parasites.     

Isolation of a cyprinid herpesvirus 2 from goldfish, Carassius auratus (L.), in the UK

Haematopoietic necrosis virus [cyprinid herpesvirus 2 (CyHV-2)] was isolated during disease outbreaks in goldfish, Carassius auratus, at an ornamental fish retail site in southern England in 2004. Signs of disease included lethargy and inappetence and were first seen after water temperatures increased from 14-15 to 19-21 degrees C. External gross pathology included pale patches on the gills and skin and internally the spleen was enlarged, often with distinctive white nodules. The most prominent histopathological changes observed were necrotic lesions in the spleen and kidney and focal patches of necrosis in the gill lamellae. Necrotic cells often contained nuclei with marginated chromatin and pale intranuclear inclusions. Ultrastructural examination of the spleen tissue revealed typical herpesvirus-like particles measuring 100 nm in diameter. The virus was isolated from extracts of gill tissue in KF-1 cells at 20 degrees C and oligonucleotide primer sets were designed based on conserved gene sequences and used to amplify viral DNA by polymerase chain reaction (PCR). The PCR assays were then used to detect the virus in DNA extracted from tissues sampled during earlier disease investigations at the retail site owner's holding facility in 2002 and 2003 and stored at -70 degrees C since then. Polymerase gene-specific PCR amplification products obtained from tissue samples and from the virus isolated in cell culture shared 100% nucleotide sequence identity with the published sequence for CyHV-2.     

The life cycle of Chloromyxum auratum (Myxozoa) from goldfish, Carassius auratus (L.), involves an antonactinomyxon actinospore

The myxozoan parasite Chloromyxum auratum Hallett, Atkinson, Holt, Banner & Bartholomew, 2006 , was shown experimentally to have a two‐host life cycle which involved a previously undescribed antonactinomyxon actinospore stage. Myxospores obtained from gall bladders of naturally infected feral goldfish, Carassius auratus (L.), were used to infect samples of mixed species of oligochaete worms obtained from the same locality as the fish: Fern Ridge Dam, Oregon, USA. After some 110 days post‐exposure, actinospores were detected from the water above the oligochaetes. The 18S rDNA sequence of these actinospores was identical to the original myxospores. Spore release was sporadic, of low intensity and short duration, which confounded efforts to identify the host oligochaete species and infect naïve fish. This is the first life cycle that incorporates an actinospore of the collective group Antonactinomyxon, and the first life cycle demonstrated in the laboratory for a species of Chloromyxum.     

Lutein as a natural carotenoid source: Effect on growth,survival and skin pigmentation of goldfish juveniles (Carassius auratus)

This study aimed to evaluate the effect of lutein supplementation on growth, survival and skin pigmentation for goldfish juveniles. Four diets enriched with different carotenoid sources (lutein, astaxanthin, canthaxanthin and a combination of lutein and canthaxanthin) were compared to a control diet without carotenoid supplementation. The carotenoid inclusion level was standardized at 50 mg kg‐1 in all treatments. 240 goldfish juveniles (1.07?0.57 g) were cultivated in 30 aquariums (30L) during 84 days. The experimental design was completely randomized with five treatments and six replicates. The dietary inclusion of carotenoid pigments did not affect the growth and feeding efficiency of goldfish juveniles. Supplementation with lutein presented higher survival values when compared to the other treatments. Astaxanthin and canthaxanthin supplementation increased the concentration of carotenoids on the skin of goldfish juveniles in relation to the control treatment. For the fish fed with the diet containing lutein, the skin pigmentation was as efficient as astaxanthin and canthaxanthin, but did not differ from the control and combined treatment (canthaxanthin + lutein). The lutein supplementation (50 mg kg‐1) improved survival and promoted efficient carotenoid pigmentation on the skin of goldfish juveniles.     

Variability in microsatellite DNA markers in gynogenetic and backcross progenies obtained from ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrid females

Inheritance and segregation at five microsatellite loci were studied in diploid gynogenetic and triploid backcross progenies obtained from koi × goldfish hybrid females, which produce diploid eggs. Gynogenetic and backcross progenies were obtained from three individual hybrid females by inseminating eggs with genetically inactivated and intact sperm of parental species respectively; no shock treatments were applied to the early embryos. Complete absence of paternally specific alleles at all investigated microsatellite loci has proven successful genetic inactivation of spermatozoa by irradiation and confirmed gynogenetic origin of progenies. Genotypic segregations at microsatellite loci showed almost complete homogeneity of gynogenetic progenies and their identity to female parents. These results correspond with previous cytogenetic data on the occurrence of premeiotic endomitosis in hybrid females producing diploid eggs. Fish from triploid backcross progenies had genotypes resulting from combination of entire diploid female genome and haploid genome from male.     

Influence of lupin and rapeseed meals on the integrity of digestive tract and organs in gilthead seabream (Sparus aurata L.) and goldfish (Carassius auratus L.) juveniles

Two rearing trials were conducted to evaluate the effects of dietary lupin (LM) and rapeseed (RM) meals in gilthead seabream (Sparus aurata L.) and goldfish (Carassius auratus L.) juveniles. Each plant meal was incorporated at the rate of 200 g kg^?1 in two distinct diets, which were compared with a fishmeal‐based diet as control. After 1 month, the two plant diets did not influence the whole body growth, but the digestive systems were affected. The splenosomatic index was reduced with two plant meals in goldfish and with RM in seabream. The hepatosomatic index was only reduced in LM‐fed seabream. Cellular characteristics were also affected. The largest liver cells were observed in RM‐fed goldfish suggesting changes in metabolic function. The LM and RM diets stimulated in seabream, especially the reaction in haematopoietic tissues with the proliferation of melano‐macrophages centres, and a tendency for elongated villus height in the anterior intestine thus that possibly compensated for a reduction in digestive function. Such adaptive structural modifications and the absence of degenerative signs allowed concluding that the integrity of the digestive system was maintained in fish fed plant meals.