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Over the past thirty-five years, anthropogenic disturbances around Bayou Lacombe have altered its fish assemblage. In 2005, the impact of Hurricane Katrina on southeast Louisiana presented me with a unique opportunity to explore the effects of a catastrophic storm on the Bayou. I explored the effects of natural and human disturbances on the Bayou's fish assemblage by electrofishing six historically sampled stations. My research goals were to determine: 1) which Bayou Lacombe fish assemblages were most resilient to the multiple effects of Hurricane Katrina, 2) if there were significant differences in the Bayou's fish assemblages over the past 35 years based on historical fish assemblage data, and 3) what are the drivers of fish assemblage change in Bayou Lacombe. I found significant differences in upstream fish assemblages before and after Hurricane Katrina in the Bayou. I also documented the disappearance of nearly all cyprinid species over the past 35 years.

Submitted by PRICILA VIANA DE OLIVEIRA null (biologa.prila@gmail.com) on 2016-05-13T19:12:08Z No. of bitstreams: 1 Dissertação Versão Final Pricila.pdf: 8047719 bytes, checksum: 2522aa1eeab9bf3e055ddc9ca38d23c1 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-05-18T16:29:25Z (GMT) No. of bitstreams: 1 oliveira_pv_me_ilha.pdf: 8047719 bytes, checksum: 2522aa1eeab9bf3e055ddc9ca38d23c1 (MD5) / Made available in DSpace on 2016-05-18T16:29:25Z (GMT). No. of bitstreams: 1 oliveira_pv_me_ilha.pdf: 8047719 bytes, checksum: 2522aa1eeab9bf3e055ddc9ca38d23c1 (MD5) Previous issue date: 2016-03-03 / A influência antrópica vem crescendo continuamente no ambiente, legitimando a importância de se investigar as causas de estresse aos animais nos sistemas naturais no qual se encontram. Como resultados a essas ações houve um aquecimento linear de 0,85 ºC nas últimas décadas e, estudos demonstram que essa temperatura pode aumentar em 4,8 ºC até 2100. A compreensão da atuação da temperatura sobre os animais aquáticos contribui significativamente para as previsões dos efeitos na população e níveis de comunidades, já que poucos fatores ambientais têm maior influência sobre a atividade animal do que a temperatura, devido a exposição física externa. O objetivo deste trabalho foi investigar a influência de diferentes temperaturas sobre o ciclo testicular e a espermatogênese de D. aequipinnatus. Para o presente estudo foram utilizados exemplares machos de D. aequipinnatus. Foram testadas três temperaturas 18º, 24º e 30º e utilizados 20 exemplares para cada temperatura. As coletas foram realizadas em um intervalo de 4 dias, totalizando 10 coletas, onde cinco indivíduos de cada tratamento foram coletados. Após aferidos dados biométricos os testículos foram submetidos a procedimentos usuais de histologia. As lâminas obtidas foram analisadas sob microscopia de luz. Para a análise estereológica, utilizou-se um grid de intersecção de 475 pontos (Motic Image Plus 2.0), sobreposto a uma área da secção histológica; foram contados os seguintes componentes: espermatogônias, espermatócitos, espermátides, espermatozoides, células de Sertoli, células com núcleo picnótico, interstício e lúmen. Para análise estatística, aplicou-se o teste de homogeneidade de Bartlett e o teste de normalidade de Shapiro Wilk. Quando atenderam as premissas, aplicou-se ANOVA e teste de Tukey (P<0.05), quando não, foi aplicado o teste de Kruskal Wallis. As fases de maturação gonadal encontradas foram maturação intermediária, maturação final - dividida em maturação final 1, maturação final 2, maturação final 3 e maturação final 4 – e regressão (somente em 18º), que apresenta características de reabsorção e proliferação celular ocorrendo na mesma fase. Os maiores valores de IGS ocorreram em indivíduos em maturação final 1. A temperatura de 30º acelerou a espermatogênese, e 18 ºC proporcionou período reprodutivo mais longo; porém na temperatura de 30 ºC houveram danos a morfologia testicular, causando uma desorganização gonadal e a supressão da espermatogênese nas regiões lesadas. Em 18 ºC houve a formação de vacúolos no citoplasma e núcleo das de espermatogônias e espermatócitos. Além disso, o número de células picnóticas foi maior em 18 ºC e 30 ºC, em relação ao controle. Indivíduos mantidos a 30º apresentaram maior número de espermatogônias. / The human influence has been growing continuosly in the environment, legitimizing the importance of investigating the causes of stress to animals in natural systems in which they are. As a result of these actions there was a linear heating of 0.85 ° C in recent decades, and studies show that this temperature could increase in 4.8 ° C until 2100. Understanding the temperature acting on aquatic animals contributes significantly to the predictions of the effects on population and levels of communities, as few environmental factors have the greatest influence on animal activity than the temperature due to external physical exposure. The aim of this study was to investigate the influence of different temperatures on testicular cycle and spermatogenesis of D. aequipinnatus. For this study were used copies males D. aequipinnatus. Three temperatures were tested 18 ºC, 24 ºC and 30 ºC, and used 20 specimens for each temperature. Samples were collected in a four-day intervals, totaling 10 collections, in which five individuals from each treatment were collected. After measured biometric data testes underwent usual histology procedures. The obtained slides were examined under light microscopy. For stereological analysis, we used a 475 grid intersection points (Motic Image Plus 2.0) overlapping an area of the histological section; were counted the following components: spermatogonia, spermatocytes, spermatids, spermatozoa, Sertoli cells, cells with pyknotic nucleus, interstitium and lumen. Statistical analysis was applied Bartlett's homogeneity test and the Shapiro-Wilk normality test. When met the assumptions applied ANOVA and Tukey test (P <0.05), if not, we applied the Kruskal Wallis test. It was found of intermediate maturation, final maturation - divided into: final maturation 1, final maturing 2, final maturation 3 and final maturation 4 - and regression (only 18ºC) having cell resorption and proliferation characteristics occurring in the same phase. The highest GSI values occurred in individuals in final maturation 1. Temperature of 30 ºC accelerated spermatogenesis, and 18 ° C provided a longer reproductive period; but at the temperature of 30 ° C there were damage to testicular morphology, causing gonadal disruption and suppression of spermatogenesis in the damaged regions. At 18 ° C was the vacuole formation in the cytoplasm and nucleus of spermatogonia and spermatocytes. Furthermore, the number of cells was higher in picnotic 18 ° C and 30 ° C, compared to the control. Animals kept at 30 ºC showed greater number of spermatogonia.

Orientador: Rosicleire Veríssimo-Silveira / Resumo: A influência antrópica vem crescendo continuamente no ambiente, legitimando a importância de se investigar as causas de estresse aos animais nos sistemas naturais no qual se encontram. Como resultados a essas ações houve um aquecimento linear de 0,85 ºC nas últimas décadas e, estudos demonstram que essa temperatura pode aumentar em 4,8 ºC até 2100. A compreensão da atuação da temperatura sobre os animais aquáticos contribui significativamente para as previsões dos efeitos na população e níveis de comunidades, já que poucos fatores ambientais têm maior influência sobre a atividade animal do que a temperatura, devido a exposição física externa. O objetivo deste trabalho foi investigar a influência de diferentes temperaturas sobre o ciclo testicular e a espermatogênese de D. aequipinnatus. Para o presente estudo foram utilizados exemplares machos de D. aequipinnatus. Foram testadas três temperaturas 18º, 24º e 30º e utilizados 20 exemplares para cada temperatura. As coletas foram realizadas em um intervalo de 4 dias, totalizando 10 coletas, onde cinco indivíduos de cada tratamento foram coletados. Após aferidos dados biométricos os testículos foram submetidos a procedimentos usuais de histologia. As lâminas obtidas foram analisadas sob microscopia de luz. Para a análise estereológica, utilizou-se um grid de intersecção de 475 pontos (Motic Image Plus 2.0), sobreposto a uma área da secção histológica; foram contados os seguintes componentes: espermatogônias, espermatócitos, esper... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The human influence has been growing continuosly in the environment, legitimizing the importance of investigating the causes of stress to animals in natural systems in which they are. As a result of these actions there was a linear heating of 0.85 ° C in recent decades, and studies show that this temperature could increase in 4.8 ° C until 2100. Understanding the temperature acting on aquatic animals contributes significantly to the predictions of the effects on population and levels of communities, as few environmental factors have the greatest influence on animal activity than the temperature due to external physical exposure. The aim of this study was to investigate the influence of different temperatures on testicular cycle and spermatogenesis of D. aequipinnatus. For this study were used copies males D. aequipinnatus. Three temperatures were tested 18 ºC, 24 ºC and 30 ºC, and used 20 specimens for each temperature. Samples were collected in a four-day intervals, totaling 10 collections, in which five individuals from each treatment were collected. After measured biometric data testes underwent usual histology procedures. The obtained slides were examined under light microscopy. For stereological analysis, we used a 475 grid intersection points (Motic Image Plus 2.0) overlapping an area of the histological section; were counted the following components: spermatogonia, spermatocytes, spermatids, spermatozoa, Sertoli cells, cells with pyknotic nucleus, interstitium and lume... (Complete abstract click electronic access below) / Mestre

Cyprinid herpesvirus 3 (CyHV-3) also known as koi herpesvirus (KHV) is a causative agent of highly contagious disease (koi herpesvirus disease) and can cause significant losses in fish stocks. The disease is restricted to koi and common carp, but recent investigations have shown that other cyprinids as well as non-cyprinid species may be asymptomatically susceptible to this virus and might play roles as potential carriers or can contribute to biological conservation leading to persistence of this virus in environment. Therefore, it seems to be important to verify not only the susceptibility of other cyprinid and non-cyprinid species, but also their ability to transmit KHV infection to susceptible species. We investigated the susceptibility of stone loach (Barbatula barbatula) and sterbel - a hybrid between sterlet (Acipenser ruthenus) and beluga (Huso huso) to KHV. The investigation was performed by means of their cohabitation together with na?ve koi and intraperitoneally KHV-infected koi (primary challenge). This part of investigation is followed by secondary challenge, when a portion of the surviving stone loach and sterbel was cohabited with health na?ve koi (testing of ability to carry KHV). All samples of fish both from primary challenge and secondary challenge were tested for the presence of KHV DNA by nested PCR. In the primary challenge, results of PCR revealed the presence of KHV DNA in 95% of cohabited na?ve koi samples. Furthermore, PCR analysis of fish samples surviving primary challenge revealed the presence of viral DNA in 77.8% (7/9) of stone loach and in 22.2% (2/9) of sterbel. In case of samples of fish coming from secondary challenge, nested PCR did not reveal any of them to be positive for KHV DNA. Next investigation was focused on assessment of the susceptibility of topmouth gudgeon (Pseudorasbora parva). In this case, we performed cohabitation based on two different conditions. All experiments consisted of primary and secondary challenges as well as in all previous cases. Firstly, we tested topmouth gudgeon under standard conditions (no-stress experiment). After the primary challenge, nested PCR did not reveal the presence of KHV DNA in any specimen of cohabited topmouth gudgeon, but all specimens of dead koi were KHV DNA positive. Nested PCR of fish tissues subjected to the secondary challenge did not show the transfer of virus to naive fish. After that, we changed the experimental conditions and we applied two stress factors (scaring by net and removal of skin mucus) to imitate the stress most commonly encountered in the wild. In this case, all samples were tested for the presence of KHV DNA using real-time PCR. After exposure to stress (removal of skin mucus), real-time PCR revealed four out of five samples (80%) of topmouth gudgeon to be positive for KHV DNA. Two out of five samples (40%) of topmouth gudgeon treated by scaring were found to be positive for the presence of viral DNA. Real-time PCR after the secondary challenge did not reveal any viral DNA positivity in specimens of topmouth gudgeon from groups previously exposed to stress. The stress experiments showed that removal of skin mucus might potentially lead to susceptibility of topmouth gudgeon to CyHV-3 infection, but the transmission of the virus to koi carp was not observed. Even though PCRs positive findings of KHV DNA in tissues of fish were relatively low, the presented results of cohabitation assays of cyprinid and non-cyprinid fish species indicate other species showing slight asymptomatic susceptibility to CyHV-3. On the other hand, on the base of our results coming from "virus-carrier" assays, we could not prove that hybrids between sterlet and beluga, stone loach and topmouth gudgeon can transfer this virus to naive koi.

Thesis (Ph.D. (Zoology)) --University of Limpopo, 2021 / The present study was conducted to evaluate the morphological and molecular characterisation of species of Dactylogyrus parasitising Enteromius spp. from the Limpopo River System, South Africa. In addition, the study was intended to establish host-parasite associations from this system. A total of 95 host specimens were collected from eight localities between 2015 and 2016 within the Limpopo River System. Fish hosts were collected using gill nets, seine nets, fyke nets, and an electric shocker. From these, three host species were identified, E. afrohamiltoni, E. unitaeniatus, and E. trimaculatus where after monogenean parasites were retrieved from the gills using stereo microscopes. Morphometric analysis of the haptoral hard parts and male copulatory organs were studied for species identification, supported by nuclear ribosomal DNA sequences of the partial 18S rDNA region and the entire ITS-1 and partial 5.8S rDNA region, and the partial 28S rDNA region. Examination of E. afrohamiltoni revealed the presence of D. afrohamiltonii which is the first record of a monogenean parasite from this host. In addition, E. unitaeniatus revealed the presence of two species of Dactylogyrus: D. letabaensis and D. limpopoensis which are the first record of monogenean parasites from this host. The remaining Dactylogyrus species were retrieved from E. trimaculatus, namely, D. afrolongicornis, D. allolongionchus, and D. myersi. Enteromius trimaculatus harboured five of the species retrieved. The two species, D. afrolongicornis and D. allolongionchus were the most abundant from six of the eight localities studied, followed by D. myersi abundant in five of the eight localities. Dactylogyrus afrohamiltonii was considered a strict specialist, while the remaining species were considered to be intermediate specialists. Forty-one sequences of the partial 18S rDNA and the entire ITS-1 and partial 5.8S rDNA region and 19 sequences of the partial 28S rDNA region of Dactylogyrus species, including Pseudodactylogyrus anguillae were included to reconstruct the phylogenetic relationships. Based on this, molecular analysis of D. afrolongicornis from Enteromius hosts were recorded for the first time for the combined 18S rDNA and the entire ITS-1 and partial 5.8S rDNA region. The analysis revealed several groupings of Dactylogyrus species inferred largely from European cyprinoids and corresponded to host specificity. From the partial 28S rDNA, three clades were revealed linked to their biogeographical regions. Phylogenetic analysis from the 28S rDNA suggests that D. aspili from E. macrops and D. afrolongicornis are closely related. / National Research Foundation (NRF) and DST Innovation Scholarship and the Vlaamse Interuniversitaire RaadUniversity Development Corporation (VLIR-UOS)

Since the late 1990s, common carp and koi production enterprises around the world have suffered enormous losses due to a viral disease caused by cyprinid herpesvirus-3 (CyHV-3). Genetic variation in resistance to CyHV-3 infection was observed in different common carp strains, indicating that disease resistance can be improved by selective breeding. Marker-assisted selection is a breeding strategy that can accelerate genetic gain; however, this approach requires genetic markers and a genetic linkage map. To develop molecular tools for breeding CyHV-3-resistant aquaculture stock, several candidate genes for antiviral innate immune response from common carp were isolated, and single nucleotide polymorphisms (SNPs) were identified. SNP markers for common carp immune response genes were developed for testing their linkage to disease resistance and for generating a genetic linkage map. Common carp immune response genes were isolated using degenerate primers developed from conserved peptide regions among other fish species for polymerase chain reaction (PCR) amplification. The amplified products were cloned and sequenced. Gene-specific primers were designed based on the isolated carp gene sequences to amplify gene fragments from genomic DNA of three carp strains and koi. The amplified products were cloned and sequenced to identify SNPs. For the genes that are duplicated, locus-specific primers were used for PCR amplification. SNPs were identified in several genes, including TLR2, TLR3a, TLR3b, TLR4a, TLR4b, TLR7a, TLR7b, TLR9, TLR21, TLR22, MyD88a, MyD88b, TRAF6a, TRAF6b, type I IFN, IL-1β, IL10a and IL10b. Putative SNPs were genotyped in a SNP discovery panel consisting of different common carp strains and koi to evaluate their allele frequencies and in a full-sib family to validate their segregation patterns using the SNaPshot method. Validated SNPs were used to genotype a mapping family. Twenty-three SNPs (19 exonic and 4 intronic SNPs) were informative in a mapping family. Among these genes, polymorphisms in IL10a suggested a possible association with resistant and susceptible phenotypes of CyHV-3-challenged fish. These SNPs will be analyzed with a set of approximately 300 microsatellites to generate a second-generation genetic map and to identify quantitative trait loci (QTLs) affecting resistance to CyHV-3. Among the common carp genes that were isolated and sequenced, TLR9 is known for its ability to detect viral DNA and requires adaptor molecules MyD88 and TRAF6 for signal transduction. Therefore TLR9, MyD88 and TRAF6 may be important candidate genes for mediating host antiviral response to CyHV-3. To elucidate possible functions of these genes, full-length cDNAs of common carp TLR9, MyD88 and TRAF6 were isolated and tissue-specific mRNA expression was determined. cDNA sequences of MyD88 and TRAF6 revealed that these genes are duplicated. These findings were the first report of MyD88 and TRAF6 duplications in a vertebrate. Protein domain characterization demonstrated that structural characteristics of these genes are conserved and resemble those of other vertebrates, indicating that common carp TLR9, MyD88 and TRAF6 genes may have identical functions with their mammalian orthologs. The mRNA expression of TLR9, MyD88a and b, and TRAF6a and b varied among tissues. Differential expression of the MyD88 and TRAF6 paralogous transcripts were observed in muscle tissues, suggesting that one paralog has evolved and attained a non-immune function. This genomic information will facilitate further research to better understand the ligand specificity of TLR9 and the role of TLR9, MyD88 and TRAF6 in the common carp immune response. / Ph. D.

Uncovering and understanding the processes that have led to the biological diversity we observe today are of fundamental interest in biology. Since direct observation of speciation is usually impossible, knowledge about the processes behind species formation can be gathered by studying mutations, natural/sexual selection, and genetic drift. In this thesis I aim to identify evolutionary processes that cause species divergence and, ultimately, speciation using Swedish cyprinid fish as a model system. Assuming that the demographic history of a population is mirrored in the genome, I studied the effects of a bottleneck on genetic variability in populations of roach. As expected, I found that a decrease in population size caused a decrease in genetic variability, a pattern that was obtained from both microsatellite and mitochondrial data. The importance of hybridization for speciation is debated, however, by analyzing morphology and microsatellites I could show that common bream and white bream and their interspecific hybrids are phenotypically and genetically differentiated and that ongoing geneflow is mainly unidirectional. Ongoing geneflow antagonizes the effect of genetic drift, but by studying isolated populations (= no gene flow) the impact of genetic drift can be assessed. Long-term isolated populations of roach and perch surprisingly showed stable levels of genetic diversity over time despite decreasing effective population size. However, each population genetically diverged during the period of investigation, a finding that is consistent with the effect of drift. An analysis of the systematic relationship of the 18 species of Swedish cyprinids revealed low congruence of phylogenies based on two different genetic markers. The position of the tench remains unresolved and the relationship of common bream and white bream as sister species cannot be confirmed. Within cyprinid fishes, diversification rates reveal a slowdown with time, a pattern that I found also in other fish clades and that is consistent with density-dependent cladogenesis. Overall, based on the findings presented in this thesis I emphasize that the maintenance of genetic variation in populations is essential since genetic variation is the key element for processes of divergence to act upon.

The composition and preferences of depth levels and habitats of pelagic fingerling was studied in the canyon-shaped Vír Reservoir during years 2009-2011. The perch (Perca fluviatilis) fingerling was dominant in all three years. The density of fingerling was above the mean in years 2009 and 2010. It was as many as 278 individuals in 100m3(mean is 52 individuals in 100m3). A marked fall on 1,9 individual in 100m3 was registered in 2011. Perch fingerling was mainly observed in metalimnion during the day and also at night, where concentration of oxygen was very low. The highest density of perch fingerling was observed in the middle part within longitudinal profile. A significant difference was found in food composition among the years with high density and low density of fingerling. A predation of carp (Cyprinus carpio) and bream (Abramis brama) was proved during the years with high density of fingerling.

The expanding aquaculture industry continues to encounter major challenges in the form of highly contagious aquatic viruses. Control and eradication measures targeting the most lethal and economically damaging virus-induced diseases, some of which are notifiable, currently involve ‘stamping out’ policies and surveillance strategies. These approaches to disease control are performed through mass-culling followed by restriction in the movement of fish and fish products, resulting in considerable impacts on trade. Although effective, these expensive, ethically complex measures threaten the sustainability and reputation of the aquatic food sector, and could possibly be reduced by emulating innovative vaccination strategies that have proved pivotal in maintaining the success of 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 specifically to infection can be distinguished from those induced in vaccinated animals. Various approaches were carried out in this study to assess the feasibility of marker/DIVA vaccination for two of the most important disease threats to the global Atlantic salmon and common carp/koi industries, i.e. infectious salmon anaemia (ISA) and koi herpesvirus disease (KHVD), respectively. Antibody responses of Atlantic salmon (Salmo salar L.), following immunisation with an ISA vaccine, administered with foreign immunogenic marker antigens (tetanus toxoid (TT), fluorescein isothiocyanate (FITC) and keyhole limpet hemocyanin (KLH)) were assessed by antigen-specific enzyme linked immunosorbent assay (ELISA). Although antibodies were induced to some markers, these were unreliable and may have been affected by temperature and smoltification. Detectable antibodies to ISAV antigen were also largely inconsistent despite low serum dilutions of 1/20 being employed for serological analysis. The poor antibody responses of salmon to the inactivated ISA vaccine suggested that DIVA vaccination is not feasible for ISA. A similar approach for KHV, utilising green fluorescent protein (GFP) as the marker, similarly failed to induce sufficiently detectable antibody responses in vaccinated carp (Cyprinus carpio L.). However, as high anti-KHV antibody titres were obtained with an inactivated KHV vaccine (≥1/3200), alternative approaches were carried out to assess the feasibility of DIVA vaccination for carp. Investigations of early KHV pathogenesis in vivo and antigen expression kinetics in vitro (0-10 days post infection (dpi)) provided valuable data for the diagnostics necessary for DIVA surveillance strategies. Following viral infection, molecular methods were shown to be the most effective approach for early detection of KHV infected fish prior to sero-conversion, during which time antibodies are not detectable. An experimental immersion challenge with KHV, however, revealed complications in molecular detection during early infection. The KHV DNA was detected in external biopsies of skin and gills, but also internally in gut and peripheral blood leukocytes ≤ 6 hours post infection (hpi), suggesting rapid virus uptake by the host. The gills and gut appeared to be possible portals of entry, supported by detection of DNA in cells by in situ hybridisation (ISH). However, many false negative results using organ biopsies occurred during the first 4 dpi. The gills were the most reliable lethal biopsy for KHV detection by various polymerase chain reaction (PCR) assays, with a PCR targeting a glycoprotein-gene (ORF56) and a real-time PCR assay being the most sensitive of the 7 methods investigated. Importantly, non-lethal mucus samples reduced the number of false negative results obtained by all KHV PCR assays during the earliest infection stages with large levels of viral DNA being detected in mucus (up to 80,000 KHV DNA genomic equivalents 200 μL-1). KHV DNA was consistently detected in the mucus as a consequence of virus being shed from the skin. Determining the expression kinetics of different viral structural proteins can be useful for DIVA serological tests. Analysis of KHV antigen expression in tissues by immunohistochemistry and indirect fluorescent antibody test was inconclusive, therefore 2 novel semi-quantitative immunofluorescence techniques were developed for determining KHV antigen expression kinetics in susceptible cell lines. During the course of KHV infection in vitro, a greater abundance of capsid antigen was produced in infected cells compared to a glycoprotein antigen (ORF56), as determined by detection with antigen-specific monoclonal antibodies (MAbs). The capsid antigen was characterised as a ~100 kDa protein by SDS-PAGE and identified as a product of KHV ORF84 by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/TOF MS). This antigen was subsequently detected in the serum of >25% of KHV infected/exposed carp (6/17), as well as in carp vaccinated with a live attenuated vaccine (3/4), but not with an inactivated vaccine (0/7), by Western blot making it a potential DIVA target for an inactivated vaccine. Attempts were made to improve the sensitivity of KHV serological testing by taking advantage of recombinant proteins specific for KHV (CyHV-3), rORF62 and rORF68 and eliminating any interference by cross-reacting antibodies to carp pox (CyHV-1). These proteins successfully reacted with anti-KHV antibodies. The feasibility of DIVA strategies for KHVD was determined using these recombinant antigens to coat ELISA plates. Differential antibody responses were detected from carp sera to an internal virus tegument protein (rORF62) and external region of a transmembrane protein (rORF68). Fish vaccinated with an inactivated vaccine produced significantly lower antibody responses to rORF62 than to rORF68, whereas infected, exposed and live attenuated vaccinated fish recognised both proteins allowing differentiation between vaccinated and infected carp. However, the sensitivity of the assay was limited, possibly by high levels of natural antibodies detected at the relatively low serum dilutions (1/200) used. As the capsid antigen (ORF84) and tegument protein (ORF62) are derived from internal KHV structural proteins, they induce non-neutralising antibodies, which may be useful for DIVA strategies. Such antibodies are longer lasting than neutralising antibodies and often comprise the majority of fish anti-viral antibodies. This was noted in a fish surviving experimental challenge, which had an antibody titre of 1/10,000, but neutralising titre of 1/45. Such antigens may therefore hold potential for developing effective serological diagnostic tests for KHV and provide the potential for DIVA strategies against KHVD. Natural antibodies will, however, continue to present a challenge to the development of sensitive and reliable KHV serological tests, and hence the application of DIVA strategies.