Evaluation of a cohabitation challenge model in immunization trials for channel catfish Ictalurus punctatus against Ichthyophthirius multifiliis

Calcein marking and cohabitation challenges have not been investigated in fish parasite research. This study evaluated a cohabitation challenge method in immunization trials against Ichthyophthirius multifiliis (Ich) using calcein, a fluorescent dye, to mark channel catfish Ictalurus punctatus (Rafinesque). Fish were marked by calcein immersion at 0, 500, and 1500 mg l(-1), and then challenged with 15 000 theronts fish(-1). No difference was noted in fish infection levels, mortality, and mean days to death (MDD) caused by Ich between unmarked and marked fish or between fish marked with high (1500 mg l(-1)) and low (500 mg l(-1)) concentrations of calcein. After ensuring that calcein marking had no effect on the susceptibility of fish to Ich theronts, 2 immunization trials were conducted to evaluate the cohabitation challenge model using calcein-marked catfish. Fish mortality, relative percent survival (RPS), and MDD were compared between cohabitation-challenged fish and fish challenged by non-cohabitation. No significant difference was observed in RPS for cohabitation-challenged fish and fish challenged by non-cohabitation. A cohabitation challenge can be used as an alternative challenge method in parasite studies, since it closely mimics natural exposure.     

Relations between histopathology and parasitaemias in Oncorhynchus mykiss infected with Cryptobia salmositica, a pathogenic haemoflagellate

Ichthyophthirius multifiliis is a ciliated protozoan parasite that infects the skin and gills of freshwater fish. This report describes the unusual finding of I. multifiliis within the peritoneal cavities of experimentally infected channel catfish Ictalurus punctatus. Twenty catfish fingerlings were exposed to I. multifiliis theronts using a standardized protocol. Five infected fish and 2 control fish were killed at various time points after infection and their tissues examined. Formalin-fixed, paraffin embedded sections were processed for light microscopy and immunohistochemical detection of I. multifiliis immobilization antigen. Trophonts were observed in skin and gill sections of all exposed fish. Parasites were associated with epithelial hyperplasia, focal areas of cellular disruption and necrosis. In addition to these usual sites of infection, individual trophonts were unexpectedly found within the peritoneal cavities of 4 fish. Staining for parasite antigen facilitated their detection within abdominal adipose tissue or adjacent to intestines. This discovery is interesting as it suggests I. multifiliis may be found in tissues other than the skin and gills during the course of a normal infection.     

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15-25 °C, 20-25 °C and 20-30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15-25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20-25 °C and 20-30 °C compared to fish at 15 °C, 20 °C and 15-25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0-10.7% mortalities). At cycling water temperature, fish vaccinated at 15-25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20-25 °C and 20-30 °C (P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20-30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15-25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.     

抗菌肽HBβ-C在全雄和杂交黄颡鱼对多子小瓜虫抗性差异中的作用

为研究全雄黄颡鱼(Pelteobagrus fulvidraco)、瓦式黄颡鱼(Pelteobagrus vachelli)和杂交黄颡鱼(黄颡鱼P.fulvidraco♀×瓦氏黄颡鱼P. vachelli♂)对多子小瓜虫(Ichthyophthirius multifiliis)的抗性差异,通过生物信息学分析黄颡鱼皮肤黏液蛋白质组,发现其血红蛋白源抗菌肽(HBβ-C)位于血红蛋白β链HBβ的碳端,共33个氨基酸。利用化学合成的不同浓度的HBβ-C肽段进行体外抗虫实验,研究发现其能有效杀死滋养体、包囊体和掠食体阶段的多子小瓜虫,其中15μg/mL的HBβ-C能在3min内杀死所有滋养体。基因表达量分析显示,在杂交黄颡鱼的鳃和皮肤组织中, HBβ的mRNA表达量高于全雄黄颡鱼;但在应对小瓜虫感染的过程中,全雄黄颡鱼的HBβmRNA转录水平快速提升,其表达水平和上升倍率显著高于杂交黄颡鱼。蛋白表达量分析显示,HBβ在全雄黄颡鱼鳃组织中的蛋白表达量明显高于杂交黄颡鱼。免疫荧光定位结果显示,抗菌肽HBβ-C特异地在红细胞中表达,可以分泌并附着在滋养体上。综上所述,相对于杂交黄颡鱼,全雄黄颡鱼中H...     

Differences in virulence between two serotypes of Ichthyophthirius multifiliis

Naive channel catfish Ictalurus punctatus were infected by 2 isolates of the parasitic ciliate Ichthyophthirius multifiliis that differed in virulence. The isolates, NY1 and G5, Serotypes A and D, respectively, express different surface immobilization-antigens. The virulence of the 2 isolates was compared using tail-fin infections to quantitate parasite numbers and by analysis of the survival of infected fish. Although NY1 infected fish at a lower level than G5, all NY1-infected fish died, but 51% of G5-infected fish survived. The greater virulence of NY1 is apparently a consequence of its shorter life cycle, which results in overwhelming reinfection of fish before they can develop a protective immune response. This report represents the first experimental evidence for differences in virulence between serotypes of I. multifiliis.     

Effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis

Two trials were conducted to determine the effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis Fouquet (Ich). In trial I, catfish were immunized intraperitoneally (IP) with: 1) 1% formalin-inactivated trophonts, 2) 3% formalin-inactivated trophonts and 3) freeze-thawed trophonts. Positive and negative control catfish were immunized with live theronts and 5% bovine serum albumin (BSA), respectively. At day 14, 28 and 50 post-immunizations, no statistical difference was noted in serum or cutaneous anti-Ich antibody titers to formalin-inactivated trophonts or freeze-thawed trophonts. The survival of catfish challenged with live theronts ranged from 33.3% to 43.3% for the formalin-inactivated or freeze-thawed trophonts at 50 d post-immunization. The survival of catfish immunized with live theront and BSA was 93.3 and 0%, respectively. In trial II, catfish were IP immunized with sonicated trophonts at doses of 1) 5 trophonts or 10.2 microg protein g(-1) fish, 2) 10 trophonts or 20.4 microg protein g(-1) fish, 3) 20 trophonts or 40.8 microg protein g(-1) fish, and 4) 5% BSA as the control. Fish immunized with 10 or 20 trophonts g(-1) fish showed highest serum (1/210 to 1/480) and cutaneous antibody titers (1/48 to 1/52), respectively, at 22 d post-immunization and survival (63.3-60.0%). The fish immunized with 5 trophonts g(-1) fish had titers of 1/52 and 1/12 for serum and cutaneous antibody and survival of 23.3%. BSA immunized catfish had background titers and a survival of 6.7%. There was a significant correlation between doses of sonicated trophonts used to immunize and catfish survival (correlation coefficient = 0.859, p < 0.01). These results indicate that doses of sonicated trophonts, but not formalin-inactivated or freeze-thawed trophonts provided both serum and cutaneous antibody responses and survival to live trophont challenge.     

Ichthyophthirius multifiliis as a potential vector of Edwardsiella ictaluri in channel catfish

There is limited information on whether parasites act as vectors to transmit bacteria in fish. In this trial, we used Ichthyophthirius multifiliis and fluorescent Edwardsiella ictaluri as a model to study the interaction between parasite, bacterium, and fish. The percentage (23-39%) of theronts fluorescing after exposure to E.?ictaluri was significantly higher than control theronts (~?6%) using flow cytometry. Theronts exposed to E.?ictaluri at 4?×?10(7) CFU?mL(-1) showed a higher percentage (~?60%) of fluorescent theronts compared to those (42%) exposed to 4?×?10(3) CFU?mL(-1) at 4?h. All tomonts (100%) carried the bacterium after exposure to E.?ictaluri. Edwardsiella ictaluri survived and replicated during tomont division. Confocal microscopy demonstrated that E.?ictaluri was associated with the tomont surface. Among theronts released from tomonts exposed to E.?ictaluri, 31-66% were observed with attached E.?ictaluri. Sixty percent of fish exposed to theronts treated with 5?×?10(7) E.?ictaluri?mL(-1) were positive for E.?ictaluri at 4?h as determined by qPCR or fluorescent microscopy. Fluorescent E.?ictaluri were observed on trophonts in skin and gill wet mounts of dead fish. This study demonstrated that Ich could vector E.?ictaluri to channel catfish.     

Susceptibility of European sheatfish Silurus glanis to a panel of ranaviruses

The current study was undertaken in order to assess the risk that different ranaviruses might impose on European sheatfish aquaculture. As the European sheatfish virus (ESV) is a known pathogen causing losses in European sheatfish aquaculture, it was assumed that closely related exotic ranaviruses might also be able to infect European sheatfish and probably cause disease and mortality in this species. The differential susceptibility of European sheatfish (Silurus glanis) to various ranavirus isolates was assessed at two different temperatures (15°C and 25°C) in a recirculation system. Fish were infected experimentally with a panel of ranavirus isolates including ESV, European catfish virus (ECV), European catfish virus isolate 24 (ECV‐24), Epizootic haematopoietic necrosis virus (EHNV), Rana esculenta virus isolate Italy 282/ I02 (REV), short‐finned eel virus (SERV), Bohle iridovirus (BIV), guppy virus 6 (GV6), doctor fish virus (DFV) and Frog virus 3 (FV3). Significant mortalities were observed, as expected, in fish infected with ESV at 15°C (100%) as well as at 25°C (86/83%). Fish infected with ECV at 15°C showed no clinical signs of disease (8% mortality), whereas those fish infected at 25°C exhibited a cumulative mortality of 54%. Fatal disease was also induced by Italian isolate ECV‐24 at 25°C (81%). Virus isolates ESV, ECV and ECV‐24, generally the most genetically closely related viruses, were successfully isolated from dead fish by cell culture with subsequent identification by polymerase chain reaction (PCR) and sequence analysis. However, no mortality or clinical signs of disease were observed in the groups of sheatfish infected with the other ranaviruses investigated in the study, and none of those viruses were re‐isolated in cell culture or identified by PCR. It was concluded that European sheatfish are susceptible to infection with ESV, ECV and ECV‐24 under laboratory conditions, but not to infection with EHNV, REV, SERV, BIV, GV6, DFV or FV3. For ESV, the incubation period was shorter at 25°C compared to 15°C water temperature, but whereas all fish died after ESV infection at 15°C, some fish survived the infection at 25°C. Futhermore, the very young sheatfish were susceptible to ECV and ECV‐24 at 25°C, whereas there was no significant mortality in the group of older sheatfish challenged with ECV at 15°C. Therefore, the clinical characteristics of the disease seem to depend on the age of the fish as well as on the water temperature.     

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.     

Treatment of ichthyophthiriasis after malachite green. II. Earth ponds at salmonid farms

We tested formalin, chloramine-T-formalin and Desirox-formalin, for use against white spot disease (ichthyophthiriasis) caused by Ichthyophthirius multifiliis at 3 salmonid farms (Salmo salar and S. trutta smolt reared in earth ponds). I. multifiliis disappeared from most individuals 4 to 5 wk after the first treatment (and after the first I. multifiliis were found) with all chemicals, indicating that combinations of these chemicals, and even formalin alone, can be used to lower the parasite burden in earth ponds to such a level that no mortality occurs. This was the case when the fish were treated frequently at the beginning of the infection. Treatment can be stopped once the fish have achieved immunity to ichthyophthiriasis. The developing immunity was also revealed by the distribution of ciliates in the course of the disease. At the beginning of the infection I. multifiliis individuals were randomly distributed among the fish, but after 2 to 3 wk, when all the fish were infected, ciliates had increased in numbers and were aggregated in such a way that some fish carried quite heavy burdens. However, over 60% of the fish were free of the parasites after 4 to 5 wk, and had few or no ciliates, meaning that the distribution was even more aggregated. Sea trout had fewer parasites than salmon, and they also recovered from infection earlier even though the treatments and ponds were similar, indicating variation in resistance to I. multifiliis between fish species. It was also evident that the chemicals and their concentrations must be planned carefully to suit the conditions at each farm.     

Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout.

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliis infections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128-256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.     

草鱼外周血细胞数量及其在胁迫或感染下的变化

为了提供可反映草鱼(Ctenopharyngodonidellus)健康状况的血细胞指标的基础数据,研究了4种质量规格组的正常草鱼的外周单位体积血细胞数量,以及分别在硫酸铜或敌百虫处理下和多子小瓜虫感染后的变化。结果表明,1000g组草鱼的单位体积红细胞数显著高于其他3种质量规格组;60g组草鱼的单位体积白细胞数显著低于其他3种质量规格组。硫酸铜或敌百虫处理组的单位体积红、白细胞数显著高于对照组;多子小瓜虫感染组的单位体积白细胞数极显著高于对照组。这里提出一个初步参考指标,60～1000g健康草鱼的红细胞值为1.15×10⁶～3.33×10⁶cells/μL,白细胞值为2.20×10⁵～4.40×10⁵cells/μL。     

Comparison of serum antibody responses and host protection against parasite Ichthyophthirius multifiliis between channel catfish and channel × blue hybrid catfish

There is limited information available on the immune protection of channel catfish Ictalurus punctatus × blue catfish I. furcatus (CB) hybrid against the fish parasite Ichthyophthirius multifiliis (Ich). The objective of this study was to compare serum antibody response and host protection between channel catfish and CB hybrid catfish using a cohabitation model. Channel catfish and CB hybrid catfish were immunized with live theronts by immersion or by IP injection at the dose of 10,000–20,000 theronts per fish in two trials. The fish were then challenged with theronts to compare serum antibody response and protection against the parasite between channel catfish and CB hybrid catfish. The immunized channel catfish and CB hybrid catfish showed a significantly higher (p < 0.05) serum anti-Ich antibody (titer > 1120) compared to non-immunized controls (titer = 0). After being challenged with live theronts, the immunized channel catfish and CB hybrid catfish had none or a low number of the parasites (<50 trophonts per fish) and showed a significantly higher (p < 0.05) survival (90–100%) than non-immunized controls (0%). Overall results indicated that there was no statistical (p > 0.05) difference on serum anti-Ich antibody, parasite infection and fish survival between immunized channel catfish and CB hybrid catfish.     

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.     

Ichthyophthirius multifiliis (Fouquet) in the mirror carp, Cyprinus carpio L.

Mirror carp were infected with Ichthyophthirius multifiliis (Fouquet) under standardized conditions. The size and number of parasites at selected sites on the body were recorded during the course of the infection. Initial exposure to 40 mature parasites resulted in a mild infection with 100% recovery after 18 days. Recovered fish did not appear to be carriers of the parasite. Exposure to 400 parasites resulted in 100% mortality between 22–25 days. The growth rate of the parasite was linear. Parasites were more numerous in the dorsal surface of the fish than in the lateral or ventral surface. The increase in parasite numbers during the disease was greater in the gills than in the skin.     

Efficacy of in-feed probiotics against Aeromonas bestiarum and Ichthyophthirius multifiliis skin infections in rainbow trout (Oncorhynchus mykiss, Walbaum)

Aims:  The aim of this study was to assess the efficacy of in-feed probiotics as a preventive measure against skin infections caused by Aeromonas bestiarum and Ichthyophthirius multifiliis (Ich) in rainbow trout.
Methods and Results:  Fin rot was induced in fish by intradermal injection with 0·1 ml volumes containing 10⁵ cells per ml A. bestiarum at the base of the dorsal fin. Ich infections resulted from immersion in Ich-contaminated water. Each probiotic was administered orally [10⁸ cells per g feed for GC2 ( Aeromonas sobria ) and 10¹⁰ cells per g feed for BA211 ( Brochothrix thermosphacta )] for 14 days. Results showed that, after challenge with A. bestiarum , probiotics GC2 and BA211 led to 76% and 88% survival, respectively, in contrast to 22% survival for controls. Fish fed with probiotic GC2 had 100% survival after challenge with Ich compared with 2% for probiotic BA211 and 0% for controls. Analysis of innate immune responses revealed that probiotic GC2 promoted higher phagocytic activity, whereas probiotic BA211 led to enhanced respiratory burst activity.
Conclusion:  Of the two probiotics examined, GC2 was more effective in protecting against both fin rot and Ich. Each probiotic appeared to stimulate different pathways within the innate immune system.
Significance and Impact of the Study:  This is the first demonstration that probiotics can protect fish against surface infections. Furthermore, this is the first time a probiotic has been shown to protect against a eucaryotic pathogen, namely I. multifiliis .     

Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in west Alabama (USA) in 2009

Three isolates (AL09-71, AL09-72, and AL09-73) of Aeromonas hydrophila were cultured from infected channel catfish Ictalurus punctatus during a disease outbreak in west Alabama, USA, in August 2009. Sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR), cpn60, gyrB, and rpoD genes of the 3 strains revealed that the 3 strains were closely related to each other, sharing 97 to 99% nucleotide sequence similarities. However, ISR sequences of the 3 isolates from 2009 shared only 64% nucleotide sequences with AL98-C1B, a 1998 isolate of A. hydrophila cultured from diseased fish in Alabama. Sequences of cpn60, gyrB, and rpoD from the 3 isolates from 2009 shared 91 to 95% homologies with AL98-C1B. Based on both LD50 and LD95 values of intraperitoneal injection assays, the virulences of the 3 isolates from 2009 were not significantly different from each other, but were at least 200-fold more virulent than AL98-C1B, indicating that the 3 west Alabama isolates of A. hydrophila from 2009 were highly virulent to channel catfish.     

Differences in initial and acquired resistance to Ichthyophthirius multifiliis between populations of rainbowfish

Wild-caught rainbowfish Melanotaenia spp. originating from three isolated populations were infected with a quantified dosage of parasites Ichthyophthirius multifiliis in a controlled environment. The Melanotaenia eachamensis from Dirran Creek were much more susceptible to ichthyophthiriasis than were M. splendida from the Lake Tinaroo or Bluewater Creek populations. When the highly susceptible Dirran Creek rainbowfish were crossed with rainbowfish from a fourth population, Lake Eacham M. eachamensis , they produced hybrids with significantly higher resistance than pure-bred Dirran Creek, but not higher than pure-bred Lake Eacham fish. Hence, intraspecific hybridization increased resistance to I. multifiliis infection in M. eachamensis. Hosts from all three populations were much less susceptible to infection on their second exposure to the parasite. However, the Bluewater Creek population was better able to acquire immunity to I. multifiliis than either the Dirran Creek or Lake Tinaroo populations. It is tentatively suggested that there may be a link between the heterozygosity of populations of rainbowfish and their initial ability to resist infection by Ichthyophthirius multifiliis.     

Infection dynamics of Centrocestus armatus cercariae (Digenea: Heterophyidae) to second intermediate fish hosts

The infection dynamics of Centrocestus armatus cercariae with respect to second intermediate fish hosts were investigated using cercariae collected from naturally infected Semisulcospira libertina. Cercariae survival and infectivity were recorded at 2-hr intervals. Survival remained constant but decreased abruptly at 30 hr of age. An age-dependent model presented the best-fit curve for the survival data (r2 0.936), implying that cercariae tended to allocate resources equally among themselves and then died once those resources were depleted. Cercariae infectivity remained constant over the first 10 hr of life and then declined; an age-dependent model also provided a better fit (r2 = 0.956). The transmission rate of C. armatus cercariae to the fish host was relatively low near the time of emergence from the snail host but peaked at 85% after 1 hr and then maintained a plateau period until 5 hr of exposure to fish. The pattern of transmission was also investigated initially at varying densities of cercariae and then by maintaining a constant cercariae density but varying the total number. Results revealed that the pattern of transmission was frequency-dependent.     

High permissivity of the fish cell line SSN-1 for piscine nodaviruses.

Seventeen isolates of piscine nodavirus from larvae or juveniles of 13 marine fish species affected with viral nervous necrosis (VNN) were examined for their infectivity to a fish cell line SSN-1. Based on cytopathic effects (CPE) and virus antigen detection by fluorescent antibody technique (FAT) after incubation at 25 degrees C, the infectivity of these virus isolates was divided into 4 groups. Group 1, including 9 virus isolates from 4 species of grouper, 2 species of sea bass, barramundi, rock porgy, and Japanese flounder showed CPE characterized by rounded, granular cells with heavy cytoplasmic vacuoles within 3 d post-incubation (p.i.), and the monolayer partially or completely disintegrated over 3 to 6 d p.i. Scattered FAT-positive cells appeared at 3 h p.i. and spread through the cell sheet with an increasing fluorescence signal over 24 h p.i. Group 2, consisting of 3 virus isolates from striped jack, induced CPE with thin or rounded, granular, refractile cells without conspicuous vacuole formation, and extensive FAT-positive reaction was observed in a time course similar to that of Group 1. Cells inoculated with Group 3 (1 isolate from tiger puffer) developed no distinct CPE but viral infection was evidenced by localized FAT-positive cells. There were no FAT-positive cells in Group 4, which included 4 isolates from Japanese flounder, Pacific cod and Atlantic halibut. However, when incubation was performed at 20 degrees C, the SSN-1 cells inoculated with the Group 3 isolate showed CPE similar to that of Group 1 and extensive FAT-positive reaction. Evidence of virus proliferation at 20 degrees C was also obtained in Group 4 isolates. The virus titers in the infected fish varied from 10(11) to 10(16) tissue culture infectious dose (TCID50) g(-1) of fish. There is a good correlation between these infectivities to the SSN-1 cells and the coat protein gene genotypes of the isolates. The present results indicate that SSN-1 cells are useful for propagating and differentiating genotypic variants of piscine nodavirus.