Herve Bercovier

A Herpesvirus Associated with Mass Mortality of Juvenile and Adult Koi, a Strain of Common Carp

A herpesvirus was isolated from adult koi, a strain of common carp Cyprinus carpio, suffering mass mortality in two outbreaks-one in the mid-Atlantic region of the United States and the second in Israel. The principal external signs of dying fish were pale and irregularly colored gills. There were few consistent internal signs in either outbreak. The most prominent microscopic lesions were in the gills, where hyperplasia and necrosis of the epithelium were severe. Other lesions included interstitial nephritis, splenitis, and enteritis. Affected cells often contained nuclei with marginated chromatin and faint intranuclear inclusions. Typical herpesvirus particles were present in branchial epithelial cells, hepatocytes, and among circulating leukocytes. Inoculations of the koi fin (KF-1) cell line with tissue extracts from the gill and kidney-spleen resulted in cytopathic effects characterized by severe vacuolation first detected after 7 d incubation at 20°C. Exposures of adult koi to the herpesvirus as propagated in KF-1 cells by bath or intraperitoneal injections resulted in 80-100% mortality during a 26-d period, and the virus was reisolated from the gill, kidney, liver, spleen, intestine, and brain of dead fish. The viral agents from koi in Israel and the United States appear to be similar if not identical; both could be distinguished from Herpesvirus cyprini by indirect fluorescent antibody tests with rabbit anti-H. cyprini serum. Other factors should be examined but we strongly suspect that this newly recognized koi herpesvirus (KHV) has the potential to be a significant cause of mortality among koi and presumably common carp.

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.

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.

The Mycobacterium tuberculosis Recombinant 27-Kilodalton Lipoprotein Induces a Strong Th1-Type Immune Response Deleterious to Protection

ABSTRACT Th1 immune response is essential in the protection against mycobacterial intracellular pathogens. Lipoproteins trigger both humoral and cellular immune responses and may be candidate protective antigens. We studied in BALB/c mice the immunogenicity and the protection offered by the recombinant 27-kDa Mycobacterium tuberculosis lipoprotein and the corresponding DNA vaccine. Immunization with the 27-kDa antigen resulted in high titers of immunoglobulin G1 (IgG1) and IgG2a with a typical Th1 profile and a strong delayed hypersensitivity response. A strong proliferation response was observed in splenocytes, and significant nitric oxide production and gamma interferon secretion but not interleukin 10 secretion were measured. Based on these criteria, the 27-kDa antigen induced a typical Th1-type immune response thought to be necessary for protection. Surprisingly, in 27-kDa-vaccinated mice (protein or DNA vaccines) challenged by M. tuberculosis H37Rv or BCG strains, there was a significant increase in the numbers of CFU in the spleen compared to that for control groups. Furthermore, the protection provided by BCG or other mycobacterial antigens was completely abolished once the 27-kDa antigen was added to the vaccine preparations. This study indicates that the 27-kDa antigen has an adverse effect on the protection afforded by recognized vaccines. We are currently studying how the 27-kDa antigen modulates the mouse immune response.

Yersinia aldovae (formerly Yersinia enterocolitica-like group X2): a new species of Enterobacteriaceae isolated from aquatic ecosystems

Previously, a group of 40 Yersinia enterocolitica-like strains that were isolated from water and fish were called group X2. These strains produced acid from L-rhamnose, did not ferment sorbose, cellobiose, melibiose, or raffinose, and rarely fermented sucrose (5% in 48 h. 10% in 7 days). This pattern of reactions separated group X2 strains from Yersinia enterocolitica, Yersinia intermedia, Yersinia frederiksenii, and Yersinia kristensenii. Positive reactions for acetoin production (Voges-Proskauer test), ornithine decarbox-ylase, and lack of acid production from melibiose distinguished group X2 strains from both Yersinia pseudotuberculosis and Yersinia pestis. Group X2 strains exhibited variable reactions only in tests for citrate utilization, hydrolysis of Tween 80, and acid production from maltose. Genetically, group X2 strains formed a single deoxyribonucleic acid hybridization group with an average level of relatedness of 86% or more (86% as determined by the S1 method at 60°C or by the hydroxyapatite method at 75°C and 92% as determined by the hydroxyapatite method at 60°C). The level of divergence among related sequences in 60°C reactions was 0.5%, as determined by the hydroxyapatite method. The relatedness of group X2 strains to other Yersinia species was 42 to 73% in 60°C reactions. The divergence in these reactions was 11.0 to 15.5%, and the relatedness to other yersiniae in 75°C reactions was 21 to 38%. Group X2 strains were 11 to 32% related to 67 species of the Enterobacteriaceae that belonged to genera other than Yersinia. On the basis of these biochemical and genetic data, we believe that group X2 represents a single new species in the genus Yersinia. The name Yersinia aldovae sp. nov. is proposed for this species. The type strain of Y. aldovae is strain CNY 6005 (= CDC 669-83 = ATCC 35236).