Challenge studies of European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), with epizootic haematopoietic necrosis virus

A challenge model for comparison of the virulence of epizootic haematopoietic necrosis virus (EHNV) to European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), was tested. The model investigated intraperitoneal (IP), bath and cohabitation routes at 10, 15 and 20 °C for 5–6 g fish and 15 °C for 20 g perch. In the IP challenges of perch, significant mortality occurred at 15 °C and 20 °C. In challenge trials for rainbow trout, significant mortalities were observed in IP and bath challenges at 20 °C. The mortality observed in IP challenged 20 g perch was not significantly different from that recorded for 6 g fish challenged IP. No significant mortality was observed in any other treatment groups. Re-isolation of ranavirus was confirmed by IFAT and was consistently associated with dead or moribund fish in the trial groups challenged with EHNV. The findings indicate that EHNV does not pose a high risk for wild perch and trout populations in Europe by natural exposure. Mortality appears to be primarily a function of environmental factors, with temperature playing an important role, and not just the presence of the virus in the fish.     

Characteristics of monoclonal antibodies against Piscirickettsia salmonis

A panel of 28 monoclonal antibodies against Piscirickettsia salmonis was produced using a purified fraction of the bacterium. To determine their specificity to the pathogen, the antibodies were assayed by ELISA and indirect immunofluorescence microscopy. Six monoclonal antibodies were selected based on their strong reaction against P. salmonis and absence of cross-reactivity with other common fish pathogens. Western blot analysis showed that the antibodies reacted to several antigens of P. salmonis . Immunofluorescence assays revealed that these antibodies reacted with the same specificity to different isolates of P. salmonis obtained from the south of Chile. This panel of monoclonal antibodies represents an important tool to develop simple, rapid, sensitive and highly specific methods for the detection of the pathogen and diagnosis of the disease.     

Piscirickettsia salmonis: a Gram-negative intracellular bacterial pathogen of fish

Piscirickettsia salmonis is the first Gram-negative, intracellular bacterial pathogen isolated from fish and is a significant cause of mortality in salmonid fish. Recent reports of P. salmonis or P. salmonis-like organisms from new fish hosts and geographic regions have increased the interest in the bacterium. In this review, the important characteristics of the bacterium including recent taxonomic changes, features of the disease caused by the bacterium including transmission, hosts, reservoirs, diagnostic procedures, and current approaches for prevention and treatment have been discussed. The reader is also directed to other reviews concerning the bacterium and the disease it causes (Fryer & Lannan 1994, 1996; Almendras & Fuentealba 1997; Lannan, Bartholomew & Fryer 1999; House & Fryer 2002; Mauel & Miller 2002).     

Real time PCR detection of Piscirickettsia salmonis from formalin-fixed paraffin-embedded tissues

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a transmissible disease of salmonid fish. Diagnosis of piscirickettsiosis has traditionally been based upon identification of typical pathological changes by histological investigation, with confirmation by immunohistochemistry on formalin-fixed, paraffin-embedded tissues. However, implementation of more rapid confirmatory techniques, preferably with higher levels of sensitivity and possibilities for quantification, is desirable. A real-time polymerase chain reaction (PCR) assay was designed for specific detection of P. salmonis and tested on samples extracted from formalin-fixed paraffin-embedded material. Construction of a PCR-target mimic allowed determination of detection limits, linearity of the real-time PCR and quantitative detection of P. salmonis. The present study demonstrates the capability of the described real time PCR assay for detection of P. salmonis from paraffin-embedded material with a high degree of sensitivity and specificity. Implementation of this assay constitutes an important development for a rapid and secure diagnosis of piscirickettsiosis.     

Experimental study of the susceptibility of Atlantic cod, Gadus morhua (L.), to infection with an IPNV strain pathogenic for Atlantic salmon, Salmo salar L

Intraperitoneal (IP) injection, cohabitation and immersion routes of infection were used to determine if Atlantic cod, Gadus morhua (L.), of 1 and 3 g are susceptible to infectious pancreatic necrosis (IPN). Mortalities of cod injected IP were significantly higher when challenged with infectious pancreatic necrosis virus (IPNV) than with phosphate buffered saline. This is the first report of Atlantic cod mortalities caused by IPNV. Fish challenged by cohabitation had significantly higher mortalities than the controls, but mortalities of Atlantic cod challenged with IPNV by immersion were not significantly different from controls. Titres of IPNV in the tissues of infected fish were sometimes very high (range 10²–10¹⁰ infectious units per gram of tissue) suggesting virus replication and titres of fish that died were generally higher than those of fish which survived. However, the relatively low mortality rates when challenged by cohabitation and immersion (20% and 17%, respectively), compared to the IP injection challenge (100%) suggest that 1 and 3 g cod have low susceptibility to IPN when challenged by more natural routes. These data strongly suggest that the cause of death of experimentally challenged cod was IPNV and further histological evidence for this came from 1 g cod challenged IP with IPNV in which the pancreas showed severe necrosis and heavy immunostaining for IPNV coincidentally with the peak of mortalities.     

应用间接荧光抗体技术快速检测花鲈病原菌——鳗弧菌

以花鲈（Lateolabrax japonicus)弧菌病的病原菌-鳗弧菌（Vibrio anguillarum)W-1为抗原，制备兔抗血清；利用异硫氰酸荧光素标记的羊抗兔免疫球蛋白（FITC-IgG）为荧光标记二抗，并以罗丹明标记的牛血清白蛋白为背景染色，建立检测鳗弧菌的间接荧光抗体快速检测技术。应用该技术对人工感染后的花鲈组织（肌肉、鳃、肠、肾）样品和养殖水体样品进行了鳗弧菌检测，结果显示间接荧光抗体技术不仅可以用于诊断发病的感染花鲈，也可用于检测带菌状态或未发病的感染花鲈。     

Induction of infectious pancreatic necrosis (IPN) in covertly infected Atlantic salmon, Salmo salar L., post-smolts by stress exposure, by injection of IPN virus (IPNV) and by cohabitation

Atlantic salmon post-smolts were given an intraperitoneal (ip) injection of tissue homogenate of Atlantic salmon fry from an outbreak of infectious pancreatic necrosis (IPN), and cohabitants were given an ip injection of Earle's balanced salt solution (EBSS). Parallel treatment groups were exposed to recurrent episodes of environmental stress by water drainage twice a week. Fish injected with EBSS and non-injected fish were exposed to water drainage. The control fish were left untreated. Mortality due to IPN started 3 weeks after challenge in non-injected and EBSS-injected fish that had been exposed to water drainage. This showed that the fish used in the experiment were covertly infected with IPN virus (IPNV) prior to challenge, although no virus was detected in the fish sampled before the experiment. In fish that received an injection of IPNV, mortality started 5-6 days after challenge, regardless of the presence or absence of stress exposure. The EBSS-injected cohabitants started to die after an additional 5-6 days, also regardless of the presence or absence of stress exposure. The final cumulative mortality in the IPNV-injected fish was significantly lower than in the EBSS-injected cohabitants, thus suggesting that the secondary immune response after injection of IPNV provided more protection than the response after a water-borne infection. No disease outbreak was observed in the control fish.     

鱼立克次氏体的研究进展

立克次氏体是鱼类及其他水生生物的重要病原之一，有较高的感染率和死亡率。目前主要采用Fryer法和依照《贝格恩细菌分类手册（第二版）》标准的方法对立克次氏体进行分类，细胞培养法、组织切片技术、PCR、核酸探针及ELISA等方法是广泛应用的检测手段，研究成果表明此种病害主要通过水平和垂直方式进行传播，而抗立克次氏体疫苗的研制也进入了商品化生产。本研究对该领域的研究成果作一综述，旨为此种病害的研究提供借鉴及参考。     

Evidence of exotoxin secretion of Piscirickettsia salmonis,the causative agent of piscirickettsiosis

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial‐free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE‐214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.     

Ca. Branchiomonas cysticola,Ca. Piscichlamydia salmonis and Salmon Gill Pox Virus transmit horizontally in Atlantic salmon held in fresh water

Elucidation of the role of infectious agents putatively involved in gill disease is commonly hampered by the lack of culture systems for these organisms. In this study, a farmed population of Atlantic salmon pre‐smolts, displaying proliferative gill disease with associated Candidatus Branchiomonas cysticola, Ca. Piscichlamydia salmonis and Atlantic salmon gill pox virus (SGPV) infections, was identified. A subpopulation of the diseased fish was used as a source of waterborne infection towards a population of naïve Atlantic salmon pre‐smolts. Ca. B. cysticola infection became established in exposed naïve fish at high prevalence within the first month of exposure and the bacterial load increased over the study period. Ca. P. salmonis and SGPV infections were identified only at low prevalence in exposed fish during the trial. Although clinically healthy, at termination of the trial the exposed, naïve fish displayed histologically visible pathological changes typified by epithelial hyperplasia and subepithelial inflammation with associated bacterial inclusions, confirmed by fluorescent in situ hybridization to contain Ca. B. cysticola. The results strongly suggest that Ca. B. cysticola infections transmit directly from fish to fish and that the bacterium is directly associated with the pathological changes observed in the exposed, previously naïve fish.     

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.     

A review of infectious gill disease in marine salmonid fish

Infectious gill diseases of marine salmonid fish present a significant challenge in salmon-farming regions. Infectious syndromes or disease conditions affecting marine-farmed salmonids include amoebic gill disease (AGD), proliferative gill inflammation (PGI) and tenacibaculosis. Pathogens involved include parasites, such as Neoparamoeba perurans, bacteria, such as Piscichlamydia salmonis and Tenacibaculum maritimum, and viruses, such as the Atlantic salmon paramyxovirus (ASPV). The present level of understanding of these is reviewed with regard to risk factors, potential impacting factors, methods of best practice to mitigate infectious gill disease, as well as knowledge gaps and avenues for future research.     

Fallowing of marine Atlantic salmon, Salmo salar L., farms as a method for the control of sea lice, Lepeophtheirus salmonis (Kroyer, 1837)

Abstract. Infections of Lepeophtheirus salmonis (Krøyer, 1837) and Caligus elongates Nordmann, 1832 were monitored over a 20-month period on four marine Atlantic salmon, Salmo salar L., farms in Scotland. Three farms were fallowed between harvesting and restocking, and thus, contained only one year class of fish at a time. The fourth site was not fallowed and contained two year classes of fish. Results show that fallowing led to low numbers of L. salmonis on newly introduced fish for several months after stocking, with much less need to use chemotherapy. New fish in the non-fallowed site became very rapidly infected with L. salmonis and required chemotherapy within 2 months of stocking. Fallowing did not appear to be effective in restricting the abundance of C. elongatus. Longer periods of fallowing were more effective in controlling lice numbers.     

Biofouling as a reservoir of Neoparamoeba pemaquidensis (Page, 1970), the causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD) is currently the most important disease affecting the Tasmanian salmonid industry and is caused by a marine amoeba, Neoparameoba pemaquidensis (Page, 1970). In this study biofouling communities on salmon cages were surveyed for the presence of the disease agent over a period of 4 months. Malt–yeast–seawater (MYS) agar plates were used to culture N. pemaquidensis with its presence confirmed by immunofluorescent antibody test (IFAT). Positive percentages of categorised samples ranged from 0% to 55%. The survey detected the presence of N. pemaquidensis on a number of macrofouling species (in particular bryozoan Scrupocellaria bertholetti and solitary ascidian Ciona intestinalis), and in microfouling and water samples. High percentages of positive IFATs occurred in microfouling aggregates, the solitary ascidian, C. intestinalis, and centrifuged water samples. No positive IFATs occurred from samples of Caprella sp. The presence of N. pemaquidensis was sporadic and varied in species and over sampling month. Experimental exposure of Atlantic salmon, Salmo salar, to lightly fouled netting was conducted to assess the potential for microfouling to act as a source of infection. No signs of the disease were detected in fish exposed to lightly fouled netting treatments, while 100% of positive control fish were infected and had an average of 4.24±1.79 amoebae per field of view in IFAT of mucus smears. When combined with N. pemaquidensis loads in the water column, the loads of amoebae in biofouling communities may contribute to disease outbreaks. Thus, biofouling should be considered a risk factor for AGD outbreaks.     

Infection and pathology in Queensland grouper,Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery‐reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re‐isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high‐dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.     

Wild fish are not a significant reservoir for Neoparamoeba pemaquidensis (Page, 1987)

Amoebic gill disease (AGD), caused by the protozoan Neoparamoeba pemaquidensis (Page, 1987) is the most important disease affecting salmon farms in Tasmania. Reservoirs for this protozoan parasite are largely unknown. This study investigated wild fish as a potential reservoir of N. pemaquidensis . A total of 325 wild fish, comprising 12 different fish species, were caught from and around salmon farms and examined for the presence of AGD. None of the wild fish were infected with AGD. In a laboratory trial, seahorse, Hippocampus abdominalis , greenback flounder, Rhombosolea tapirina, and Atlantic salmon, Salmo salar, were challenged with N. pemaquidensis . Neoparamoeba pemaquidensis was detected on the gills on 10 of 15 (66.7%) flounder, nine of 24 (37.5%) seahorses, and six of six (100%) Atlantic salmon. However, paramoebae positive flounder and seahorse lacked the characteristic AGD gill pathology. It is concluded that AGD does not appear in wild fish and wild fish do not seem to be a reservoir of the pathogen.     

滇池金线鲃对椭圆背角无齿蚌寄生效果的研究

滇池金线鲃(Sinocyclocheilus grahami)为云南滇池特有鱼类,选用滇池金线鲃作为椭圆背角无齿蚌(Anodonta woodiana elliptica)的寄生鱼,并对第1次和第2次寄生效果进行分析。结果表明,钩介幼虫主要寄生在鳍条(背鳍、胸鳍、腹鳍和尾鳍)以及鳃丝上,也有少量个体寄生在颌须、鼻瓣膜、鳃盖及体侧。每1尾滇池金线鲃的鳃内大约可寄生140个钩介幼虫。在整个鱼体上的平均寄生量为194个/尾,单位体长的寄生数量为28.6个/cm。二次寄生时,在寄生部位上没有变化,但在寄生数量上,除鳃丝上的寄生数量没有减少外,其它部位上的寄生数量均有显著的减少。本研究选用土著鱼类作为贝类的寄生性鱼类,为研究土著鱼类在贝类繁殖过程中所起到的作用及珍稀物种的保育提供了科学依据。