Sean J. Monaghan

Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA)

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.

Construction of a Vibrio alginolyticus hopPmaJ (hop) mutant and evaluation of its potential as a live attenuated vaccine in orange-spotted grouper (Epinephelus coioides)

&NA; Vibrio alginolyticus, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. However, little is known about the associated effectors (T3SEs) and their physiological role. In this study, the T3SE gene hopPmaJ (hop) was cloned from V. alginolyticus wild‐type strain HY9901 and the mutant strain HY9901&Dgr;hop was constructed by the in‐frame deletion method. The results showed that the deduced amino acid sequence of V. alginolyticus HopPmaJ shared 78–98% homology with other Vibrio spp. In addition, the HY9901&Dgr;hop mutant showed an attenuated swarming phenotype and a 2600‐fold decrease in the virulence to grouper. However, the HY9901&Dgr;hop mutant showed no difference in morphology, growth, biofilm formation and ECPase activity. Finally, grouper vaccinated via intraperitoneal (IP) injection with HY9901&Dgr;hop induced a high antibody titer with a relative percent survival (RPS) value of 84% after challenging with the wild‐type HY9901. Real‐time PCR assays showed that vaccination with HY9901&Dgr;hop enhanced the expression of immune‐related genes, including MHC‐I&agr;, MHC‐II&agr;, IgM, and IL‐1&bgr; after vaccination, indicating that it is able to induce humoral and cell‐mediated immune response in grouper. These results demonstrate that the HY9901&Dgr;hop mutant could be used as an effective live vaccine to combat V. alginolyticus in grouper. HighlightsThe biological functions of HopPmaJ in alginolyticus were investigated.HY9901&Dgr;hop suppressed swarming motility, adhesion and virulence.The RPS of grouper vaccinated with HY9901&Dgr;hop was 84%.HY9901&Dgr;hop could stimulate innate and acquired immune responses in E. coioides.

Potential of DIVA Vaccines for Fish

The expanding aquaculture industry continues to encounter major challenges from highly contagious viruses. Control and eradication measures for lethal and economically damaging notifiable viral diseases involve ‘stamping out’ policies and surveillance strategies. Mass-culling of stock and restricted movement of fish and fish products, used to control the spread of notifiable diseases, has considerable impacts on the trade of fish products. Although effective, these measures are expensive and ethically complex and could possibly be reduced by emulating innovative vaccination strategies used by the terrestrial livestock industry. DIVA (differentiating infected from vaccinated animal) strategies provide a basis to vaccinate and contain disease outbreaks without compromising ‘disease-free’ status, as antibodies induced during infection can be used to distinguish from those induced by vaccination. The potential and feasibility of DIVA vaccination in aquaculture is explored here with reference to DIVA strategies applied in higher vertebrates. Three economically important notifiable viruses, causing major problems in three different cultured fish industries, are considered. The increased availability and application of sophisticated biotechnology tools has enabled improved prophylaxis and serological diagnosis for control of viral haemorrhagic septicaemia in rainbow trout, infectious salmon anaemia in Atlantic salmon and koi herpesvirus disease in carp. Improving the specificity of serological diagnostics in aquaculture in conjunction with suitable vaccines could enable the application of DIVA strategies, but the immunological variation between different fish species and contrasting pathobiological characteristics of different viruses determines the feasibility and potential of such DIVA approaches for aquaculture industries.

Is There Any Species Specificity in Infections with Aquatic Animal Herpesviruses?-The Koi Herpesvirus (KHV): An Alloherpesvirus Model

Most diseases induced by herpesviruses are host-specific; however, exceptions exist within the family Alloherpesviridae. Most members of the Alloherpesviridae are detected in at least two different species, with and without clinical signs of a disease. In the current study the Koi herpesvirus (KHV) was used as a model member of the Alloherpesviridae and rainbow trout as a model salmonid host, which were infected with KHV by immersion. KHV was detected using direct methods (qPCR and semi-nested PCR) and indirect (enzyme-linked immunosorbant assay; ELISA, serum neutralization test; SNT). The non-koi herpesvirus disease (KHVD)-susceptible salmonid fish were demonstrated to transfer KHV to naive carp at two different temperatures including a temperature most suitable for the salmonid (15°C) and cyprinid (20°C). At 20°C KHVD was induced in carp cohabitated with infected trout. KHV was also detected virologically and serologically at the end of the experiment in both rainbow trout and carp.

Characterisation of proteins in excretory/secretory products collected from salmon lice, Lepeophtheirus salmonis

BackgroundThe salmon louse, Lepeophtheirus salmonis, is an ectoparasitic copepod which feeds on the mucus, skin and blood of salmonid fish species. The parasite can persist on the surface of the fish without any effective control being exerted by the host immune system. Other ectoparasitic invertebrates produce compounds in their saliva, excretions and/or secretions which modulate the host immune responses allowing them to remain on or in the host during development. Similarly, compounds are produced in secretions of L. salmonis which are thought to be responsible for immunomodulation of the host responses as well as other aspects of crucial host-parasite interactions.MethodsIn this study we have identified and characterised the proteins in the excretory/secretory (E/S) products of L. salmonis using LC-ESI-MS/MS.ResultsIn total 187 individual proteins were identified in the E/S collected from adult lice and pre-adult sea lice. Fifty-three proteins, including 13 serine-type endopeptidases, 1 peroxidase and 5 vitellogenin-like proteins were common to both adult and pre-adult E/S products. One hundred and seven proteins were identified in the adult E/S but not in the pre-adult E/S and these included serine and cysteine-type endopeptidases, vitellogenins, sphingomyelinase and calreticulin. A total of 27 proteins were identified in pre-adult E/S products but not in adult E/S.ConclusionsThe assigned functions of these E/S products and the potential roles they play in host-parasite interaction is discussed.

Characterization of the outer membrane proteome of Francisella noatunensis subsp. orientalis

The aims of the current study were to characterize the outer membrane proteins (OMPs) of Francisella noatunensis subsp. orientalis (Fno) STIR‐GUS‐F2f7, and identify proteins recognized by sera from tilapia, Oreochromis niloticus, (L) that survived experimental challenge with Fno.