Avi Eldar

Genome Sequences of Three Koi Herpesvirus Isolates Representing the Expanding Distribution of an Emerging Disease Threatening Koi and Common Carp Worldwide

ABSTRACT Since the mid-1990s, lethal infections of koi herpesvirus (KHV) have been spreading, threatening the worldwide production of common carp and koi (both Cyprinus carpio). The complete genome sequences of three KHV strains from Japan, the United States, and Israel revealed a 295-kbp genome containing a 22-kbp terminal direct repeat. The finding that 15 KHV genes have clear homologs in the distantly related channel catfish virus (ictalurid herpesvirus 1) confirms the proposed place of KHV in the family Herpesviridae, specifically in the branch with fish and amphibian hosts. KHV thus has the largest genome reported to date for this family. The three strains were interpreted as having arisen from a wild-type parent encoding 156 unique protein-coding genes, 8 of which are duplicated in the terminal repeat. In each strain, four to seven genes from among a set of nine are fragmented by frameshifts likely to render the encoded proteins nonfunctional. Six of the affected genes encode predicted membrane glycoproteins. Frameshifts or other mutations close to the 3′ ends of coding sequences were identified in a further six genes. The conclusion that at least some of these mutations occurred in vivo prompts the hypothesis that loss of gene functions might be associated with emergence of the disease and provides a basis for further investigations into the molecular epidemiology of the virus.

Experimental streptococcal meningo-encephalitis in cultured fish

In 1984 a disease of fish appeared in Israel which spread rapidly in cultured fishponds. The disease affected tilapia (Oreochromis aura x Oreochromis nilotica hybrids) and trout (Oncorhynchus mykiss). Common carp (Cyprinus carpus), although reared in community with tilapia were not susceptible to the disease. Various species of ornamental cyprinids and cichlids were also affected. Morbidity was high and mortality ranged between 50% (in trout) and 30% (in tilapia). Clinical and pathological findings indicated that the tilapia and trout suffered from meningitis and menigo-encephalitis. Two new streptococcal species, Streptococcus shiloi and Streptococcus difficile were isolated from diseased fish. The disease was reproduced experimentally in both trout and tilapia with the two streptococcal species. The LD50s of S. shiloi and S. difficile strains cultured in vitro (two to three passages on BHI medium) were 10(7)-10(8) cfu. The virulence of these strains was increased (LD50:10(2)-10(5) cfu) after three passages in vivo (brain to brain passage in fish without culture on agar plates). Highly virulent strains did not differ from low virulent strains by any identifiable extrachromosomal elements.

Streptococcus shiloi andStreptococcus difficile: Two new streptococcal species causing a meningoencephalitis in fish

A bacterial meningoencephalitis in St. Peters fish (Tilapia spp.) and trout (Oncorhynchus mykiss) appeared in Israel in 1986 and rapidly spread throughout the country, causing considerable economic losses. We isolated and identified the agents of this disease. They were Gram-positive, nonsporulating, facultatively anaerobic chain-forming cocci, catalase negative. They were able to grow at pH 9.6 but not at 10°C nor at 45°C nor in the presence of 40% (vol/vol) bile salts or in the presence of 6.5% NaCl (wt/vol). DNA base composition (G+C%=37%), DNA-DNA hybridizations, biochemical and serological studies indicated that these strains constitute two new distinct species of streptococci that we propose to nameStreptococcus shiloi (type strain ND 2-16) andStreptococcus difficile (type strain ND 2-22). Unclassified isolates from Japan and from Taiwan belonged toStreptococcus shiloi orStreptococcus difficile, showing the cosmopolitan distribution of these two newly described species.

Enterococcus seriolicida is a junior synonym of Lactococcus garvieae, a causative agent of septicemia and meningoencephalitis in fish.

Abstract. The reference strains of Enterococcus seriolicida (ATCC 49156T) (T = type strain) and of Lactococcus garvieae (ATCC 43921T) and 30 field strains of Gram-positive cocci isolated from diseased rainbow trout in Italy were found to be phenotypically (API 20 STREPT and API 50 CH) and genetically (DNA-DNA hybridization) similar. The high DNA-DNA homologies (70–100%) and the low ΔTm(e) (less than 1.1°C) among these strains showed that Enterococcus seriolicida and Lactococcus garvieae are synonyms, describing a single bacterial species. E. seriolicida strains should be classified as L. garvieae, which must be considered as a major pathogen of freshwater and salt water fish with a world-wide distribution.

Development and efficacy of a vaccine against Streptococcus iniae infection in farmed rainbow trout

Formalin killed bacteria were used as a vaccine against Streptococcus iniae infections in farmed rainbow trout. A single intraperitoneal injection of this vaccine in trout resulted in specific antibody production detectable for 6 months. Trout vaccinated at 50 g were protected under laboratory (experimental disease) and field conditions (natural disease) for at least 4 months against S. iniae infection. Passive transfer of S. iniae specific antibodies conferred protection. Under field conditions, mortality of non vaccinated trout exceeded 50%, whereas mortality of vaccinated trout did not reach 5%. In addition, vaccinated trout under field conditions gained 20% weight when compared with non vaccinated fish.

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

BackgroundOutbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.ResultsA robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection.ConclusionThe TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

Recovery of Streptococcus iniae from diseased fish previously vaccinated with a streptococcus vaccine

ABSTRACT Streptococcus iniae was recovered from diseased rainbow trout (Oncorhynchus mykiss, Walbaum) previously vaccinated against streptococcosis. PCR and serological methods indicate the presence of a new serotype in the diseased fish.

Streptococcus shiloi, the name for an agent causing septicemic infection in fish, is a junior synonym of Streptococcus iniae.

Streptococcus shiloi strains, including the type strain, which were isolated in Israel and the United States, and Streptococcus iniae ATCC 29178(T) (T = type strain) are phenotypically identical (as determined with API 20STREP and API 50CH kits; beta-hemolytic on sheep blood agar). DNA-DNA hybridization experiments revealed levels of homology of 77 to 100%. Thus, S. shiloi should be considered a junior synonym of S. iniae. This bacterium is a major fish pathogen that is distributed worldwide.

Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish.

ABSTRACT The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that ∼70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.

Clonality and Diversity of the Fish Pathogen Lactococcus garvieae in Mediterranean Countries

ABSTRACT Infection with Lactococcus garvieae is considered the most important risk factor for the European trout industry, and the losses are approximately 50% of the total production. To improve our understanding of the genetic links among strains originating from different countries, we examined the population structure of L. garvieae by comparing 81 strains isolated from different sources and ecosystems (41 farms in six countries) in which the bacterium is commonly found. Genetic similarities (as assessed with molecular tools, including restriction fragment length polymorphism ribotyping with two endonucleases) were compared with serological data. The combined results reveal that in endemic sites the bacterial population displays a clonal structure, whereas bacterial diversity characterizes sites where the infection is sporadic.