Exposure of Wildlife to the Schmallenberg Virus in France (2011-2014): Higher, Faster, Stronger (than Bluetongue)!

The Schmallenberg virus (SBV) has recently emerged in Europe, causing losses to the domestic livestock. A retrospective analysis of serodata was conducted in France for estimating seroprevalence of SBV among six wildlife species from 2011-2012 to 2013-2014, that is during the three vector seasons after the emergence of the SBV in France. Our objective was to quantify the exposure of wildlife to SBV and the potential protective effect of elevation such as previously observed for bluetongue. We also compared the spatiotemporal trends between domestic and wild animals at the level of the departments. We tested 2050 sera using competitive ELISA tests. Individual and population risk factors were further tested using general linear models among 1934 individuals. All populations but one exhibited positive results, seroprevalence up to 30% being observed for all species. The average seroprevalence did not differ between species but ranged from 0 to 90% according to the area and period, due to the dynamic pattern of infection. Seroprevalence was on average higher in the lowlands compared to areas located up to 800 m. Nevertheless, seroprevalence above 50% occurred in areas located up to 1500 m. Thus, contrary to what had been observed for bluetongue during the late 2000s in the same areas, SBV could spread to high altitudes and infect all the studied species. The spatial spread of SBV in wildlife did not fully match with SBV outbreaks reported in the domestic livestock. The mismatch was most obvious in mountainous areas where outbreaks in wildlife occurred on average one year after the peak of congenital cases in livestock. These results suggest a much larger spread and vector capacity for SBV than for bluetongue virus in natural areas. Potential consequences for wildlife dynamics are discussed.

Differentiation between Cyprinid herpesvirus type-3 lineages using duplex PCR.

To date, all the isolates of Cyprinid herpesvirus type-3 (CyHV3) responsible for serious outbreaks in carps Cyprinus carpio have been found to be very similar or identical on the basis of DNA sequences of a few reference genes. However, two genetic lineages (U/I and J) are distinguished by full-length genome sequencing. Two molecular markers presenting genetic variations were targeted for developing a duplex PCR assay able to distinguish CyHV3-U/I from CyHV3-J while avoiding DNA sequencing. The method was validated on a series of 42 samples of infected carps from France, The Netherlands and Poland collected from 2001 to 2008. Among these samples, both the U/I and J genotypes were identified, but also a third genotype representing a genetic intermediate between U/I and J for one of the two molecular markers. A classification of CyHV3 genotypes, based on the alleles of the two molecular markers, is proposed. The assay is easy to perform and provides a genotype information with samples moderately or highly concentrated. This tool should improve our knowledge regarding the present distribution and future diversification of this emerging virus.

In vitro immunomodulating influence of methisoprinol on the head kidney phagocyte and lymphocyte activity after suppression induced by VHSV in rainbow trout (Oncorhynchus mykiss).

The aim of this study was to evaluate the in vitro influence of methisoprinol on head kidney phagocyte and lymphocyte activity after suppression induced by VHSV. The VHSV statistically significantly decreased the respiratory burst activity of head kidney phagocytes and the proliferative response of lymphocytes stimulated by ConA. The results of our study showed that methisoprinol at a concentration of 100 microl/ml modulated and restored the phagocyte and lymphocyte activity suppressed by VHSV.

Influence of methisoprinol on the replication of rhabdoviruses isolated from carp (Cyprinus carpio) and catfish (Ictalurus melas): in vitro study.

Rhabdoviruses constitute one of the most pathogenic viruses isolated from rainbow trout and carp culture. Several viruses were also isolated from other species of fish. These viruses are mostly associated with epizootics and heavy losses. Spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV) have been the most extensively studied, due to their significant economic impact. Significant progress has been made towards controlling the major bacterial fish diseases using vaccines, but this approach has not yet been successful in preventing viral diseases in fish culture. However, for an effective therapeutic approach, specific drugs should be developed to selectively inhibit virus replication and/or stimulate antiviral protection. In this investigation we examined the in vitro influence of methisoprinol on the SVCV and virus isolated from catfish (Ictalurus melas) replication by measuring their RNA synthesis. The viruses were propagated in EPC cells and cell cultures containing methisoprinol were followed by infection with SVCV or catfish rhabdovirus suspension containing 10(7) TCID50/ml. Methisoprinol (Polfa, Poland) at concentrations of 0, 100, 200, 300, 400 and 500 microg/ml of medium (Glasgow MEM) was used in this study. The results of this study show the strong inhibition of incorporation (cpm) of [3H]-uridine into SVCV and catfish rhabdovirus RNA in cell culture exposed to methisoprinol at various concentrations. The highest percent of inhibition of viral RNA at 72 h after infection with two rhabdoviruses were observed in doses of 400 and 500 microg/ml of methisoprinol in medium. The results of this in vitro study showed that methisoprinol inhibits the rhabdoviruses isolated from carp and catfish.

Standardization of experimental infection with Flavobacterium psychrophilum, the agent of rainbow trout Oncorhynchus mykiss fry syndrome.

Rainbow trout fry syndrome (RTFS) is a septicaemic infection of young rainbow trout Oncorhynchus mykiss occurring at low temperatures and responsible for severe economic losses in European fish farming. The causative agent, Flavobacterium psychrophilum, is a gliding bacterium, and difficulties in culturing it have long been an impediment to investigations on pathogenesis and immunity. Successful attempts at experimentally inducing the disease have been reported, but no experimental model resulting in well-controlled and quantitatively reproducible effects has been described. Recent improvements in F. psychrophilum cultivation made it possible to produce bacterial suspensions with nearly constant viability and to complete challenge injections in rainbow trout fingerlings, using accurately adjusted infective doses. Parenteral injection resulted in significant mortality, which was higher when administered intramuscularly (IM) than intraperitoneally (IP). Lethal doses 50 % lower than 10(3) colony forming units were consistently obtained in trout weighing 3 to 5 g, and the regular shape of the cumulative mortality curves appeared to lend itself to quantitative analyses. Bath experiments produced milder effects, although mortality ranging between 45 and 60 % was obtained in 6 g trout when skin lesions or stressors were induced along with bacterial exposure. Temperature, salinity and the process of preserving isolates (at least over short periods of time) did not seem to be associated with the severity of infection. Nevertheless, infection trials performed at 2 different locations differing both in water quality and in the system of fish maintenance resulted in different mortalities. These findings notwithstanding, the proposed IM model appears easy to apply under standardized experimental conditions and should contribute to effective advances in the study of the disease.

Effects of iridovirus-like agent on the cell-mediated immunity in sheatfish (Silurus glanis)--an in vitro study.

An iridovirus-like agent was tested for the first time in vitro on cell-mediated immunity in sheatfish (Silurus glanis). The influence of the iridovirus-like agent on pronephric macrophage metabolism was examined at two temperatures, 20 and 30 degrees C, by studying the respiratory burst activity stimulated by phorbol myristate acetate as well as the proliferative ability of lymphocytes stimulated by concanavalin A (ConA) and lipopolysaccharide (LPS) measured by MTT assay. The results showed that the iridovirus-like agent decreased the macrophage activity at incubation temperatures of 20 and 30 degrees C. The highest inhibitory effect was observed at 30 degrees C. The proliferative ability of pronephric lymphocytes had a similar pattern. The results showed that applying a virus at the same time or after the mitogen at 20 and 30 degrees C decreased the lymphocyte proliferation that was stimulated by either ConA or LPS. The highest suppressive effect was observed when virus was applied 14 h after the mitogen. This preliminary in vitro study demonstrated a strong suppressive influence of the iridovirus-like agent on pronephric macrophage and lymphocyte activity in sheatfish.

Effects of dimerized lysozyme (KLP-602) on the cellular and humoral defence mechanisms in sheatfish (Silurus glanis): in vitro and in vivo study.

This study examined the effects of the dimerized lysozyme (KLP-602) on the immunocompetence cell activity in sheatfish (Silurus glanis) and its influence in vivo on the non-specific defence mechanisms and protection against motile aeromonad septicaemia (MAS). The in vitro study showed that the lysozyme dimer (KLP-602), at concentrations between 5 and 50 micrograms/mL of medium significantly (P < 0.05) increased the respiratory burst activity and potential killing activity of pronephric macrophages, as well as the proliferative ability of pronephric lymphocytes stimulated by ConA and LPS. The in vivo study showed that injecting lysozyme dimer (Lydium-KLP) intraperitoneally at doses of 50 micrograms/kg bw stimulated cell-mediated and humoral-mediated imunity. On day 5, after application of Lydium-KLP in vivo, a statistically higher (P < 0.05) respiratory burst activity and potential killing activity of blood and pronephros phagocytes were observed. A higher proliferative ability of blood and pronephros lymphocytes stimulated by Concanavaline A (ConA) or lipopolysaccharide (LPS) was also observed. At the same time, the myeloperoxidase activity in the PMN cells and the lysozyme activity and total Ig levels in serum were significantly higher (P < 0.05), compared to the control group. A challenge test with Aeromonas hydrophila showed that dimerized lysozyme increased the protection against MAS. Dimerized lysozyme stimulates non-specific cellular and humoral mechanisms and protection against MAS in sheatfish.