Jakob Skov

Seroprevalence of Human Toxocariasis in Denmark

ABSTRACT The seroprevalence of Toxocara in the Danish population was assessed from 3,247 sera from individuals originally screened for toxoplasmosis. Of 87 enzyme-linked immunosorbent assay-positive sera, 79 were confirmed by Western blotting, yielding a crude seroprevalence of 2.4%. This indicates that the seroprevalence of toxocariasis in Denmark is low compared to those in other European countries.

Life cycle stages of heterophyid trematodes in Vietnamese freshwater fishes traced by molecular and morphometric methods.

A survey of digenean zoonotic trematodes infecting snails and fishes in a North Vietnamese freshwater fish culture system revealed shedding of three types of parapleurolophocercous cercariae from the snail host Melanoides tuberculata and the presence of metacercariae within the genus Haplorchis (H. pumilio and H. taichui) and Procerovum sp. in tissues of cultured fishes (silver carp, Indian carp and climbing perch). No morphological characters were able to link the different cercariae specifically to any of the metacercariae. Subsequent molecular work including PCR and sequencing of ribosomal DNA (the ITS2 region) in cercariae and metacercariae associated only one type of the cercariae to the recovered H. pumilio metacercariae. Further, full identity (100%) was found with regard to the ITS sequence of adult H. pumilio obtained from the same North Vietnamese region. None of the cercariae showed sequence identities with H. taichui but more than 99% identity was found between one cercaria type and the Procerovum sp. metacercaria. It was indicated that trematode parasites of farmed fishes may originate from sources outside the fish ponds and may be introduced as free-swimming cercariae when pond water is being replenished by river water. Likewise, cercariae from the ponds may not always result in metacercarial infections of the farmed fishes. The present study frames the needs for including molecular techniques as auxiliary tools when conducting ecological studies of cercariae in complex ecosystems. The parasites recorded in the fish ponds are not only known to affect the health of aquacultured fishes but also the documented zoonotic potential of the diagnosed metacercaria calls for alerts regarding human consumption of raw or inadequately processed fish dishes.

Occurrence of zoonotic nematodes Pseudoterranova decipiens, Contracaecum osculatum and Anisakis simplex in cod (Gadus morhua) from the Baltic Sea.

Baltic cod Gadus morhua (a total of total 224 specimens) captured east of the island of Bornholm in the southern Baltic Sea were subjected to a parasitological investigation between March 2013 and April 2014. Full artificial digestion of fillets from 188 cod and additional investigation of livers from 36 cod were performed. Cod or seal worm Pseudoterranova decipiens was recorded in musculature (prevalences up to 55% and intensities up to 56 worms per fish) associated with a negative correlation between worm intensity and condition factor. Liver worm Contracaecum osculatum (100% prevalence with intensities up to 320 worms per fish) in liver tissue were recorded but only a slight negative correlation between intensity and condition factor was noted. Seals act as final host for both worm species and the increased occurrence during recent years is associated with the increasing grey seal population in the area. Infection with Anisakis simplex (the herring or whale worm) in Baltic cod was found at a low level corresponding to previous studies.

Insight from Molecular, Pathological, and Immunohistochemical Studies on Cellular and Humoral Mechanisms Responsible for Vaccine-Induced Protection of Rainbow Trout against Yersinia ruckeri

ABSTRACT The immunological mechanisms associated with protection of vaccinated rainbow trout, Oncorhynchus mykiss, against enteric redmouth disease (ERM), caused by Yersinia ruckeri, were previously elucidated by the use of gene expression methodology and immunochemical methods. That approach pointed indirectly to both humoral and cellular elements being involved in protection. The present study correlates the level of protection in rainbow trout to cellular reactions in spleen and head kidney and visualizes the processes by applying histopathological, immunohistochemical, and in situ hybridization techniques. It was shown that these cellular reactions, which were more prominent in spleen than in head kidney, were associated with the expression of immune-related genes, suggesting a Th2-like response. Y. ruckeri, as shown by in situ hybridization (ISH), was eliminated within a few days in vaccinated fish, whereas nonprotected fish still harbored bacteria for a week after infection. Vaccinated fish reestablished normal organ structure within a few days, whereas nonprotected fish showed abnormalities up to 1 month postinfection. Protection in the early phase of infection was mainly associated with the expression of genes encoding innate factors (complement factors, lysozyme, and acute phase proteins), but in the later phase of infection, increased expression of adaptive immune genes dominated. The histological approach used has shown that the cellular changes correlated with protection of vaccinated fish. They comprised transformation of resident cells into macrophage-like cells and increased occurrence of CD8α and IgM cells, suggesting these cells as main players in protection. Future studies should investigate the causality between these factors and protection.

Effects of adjuvant Montanide™ ISA 763 A VG in rainbow trout injection vaccinated against Yersinia ruckeri

Enteric redmouth disease (ERM) caused by the fish pathogen Yersinia ruckeri is a major threat to freshwater production of rainbow trout (Oncorhynchus mykiss) throughout all life stages. Injection vaccination of rainbow trout against Y. ruckeri infection has been shown to confer better protection compared to the traditionally applied immersion vaccination. It may be hypothesized, based on experience from other vaccines, that adjuvants may increase the protective level of ERM injection vaccines even more. Controlled comparative vaccination studies have been performed to investigate effects of the oil adjuvant Montanide™ ISA 763 A VG (Seppic) when added to an experimental Y. ruckeri bacterin (containing both biotype 1 and 2 of serotype O1). A total of 1000 fish with mean weight 19 g was divided into five different groups (in duplicated tanks 2 × 100 fish per group) 1) non-vaccinated control fish (NonVac), 2) fish injected with a commercial vaccine (AquaVac(®) Relera™) (ComVac), 3) fish injected with an experimental vaccine (ExpVac), 4) fish injected with an experimental vaccine + adjuvant (ExpVacAdj) and 5) fish injected with adjuvant alone (Adj). Injection of the experimental vaccine (both adjuvanted and non-adjuvanted) induced a significantly higher antibody (IgM) level, increased occurrence of IgM(+) cells in spleen tissue and significant up-regulation of several immune genes. Additional experiments using a higher challenge dosage suggested an immune enhancing effect of the adjuvant as the challenge produced 100% mortality in the NonVac group, 60% mortality in both of ComVac and Adj groups and only 13 and 2.5% mortalities in the ExpVac and the ExpVacAdj groups, respectively.

Comparative evaluation of infection methods and environmental factors on challenge success: Aeromonas salmonicida infection in vaccinated rainbow trout

When testing vaccine-induced protection an effective and reliable challenge method is a basic requirement and we here present a comparative study on different challenge methods used for infection of rainbow trout Oncorhynchus mykiss with Aeromonas salmonicida, a bacterial pathogen eliciting furunculosis. Fish were vaccinated with three different adjuvanted trivalent vaccines containing formalin killed A. salmonicida, Vibrio anguillarum O1 and O2a. These were 1) the commercial vaccine Alpha Ject 3000, 2) an experimental vaccine with water in paraffin oil adjuvant, 3) an experimental vaccine with water in paraffin oil in water adjuvant. Fish were then exposed to A. salmonicida challenge using i.p. injection, cohabitation in freshwater, cohabitation in saltwater (15 ppt) or combined fresh/saltwater cohabitation. Cohabitation reflects a more natural infection mode and was shown to give better differentiation of vaccine types compared to i.p. injection of live bacteria. The latter infection mode is less successful probably due to the intra-abdominal inflammatory reactions (characterized in this study according to the Speilberg scale) induced by i.p. vaccination whereby injected live bacteria more effectively become inactivated at the site of injection. Compared to cohabitation in freshwater, cohabitation in saltwater was less efficient probably due to reduced survivability of A. salmonicida in saltwater, which was also experimentally verified in vitro.

Molecular and morphometric study of metacercariae and adults of Pseudamphistomum truncatum (Opisthorchiidae) from roach (Rutilus rutilus) and wild American mink (Mustela vison)

The presence of metacercariae and adults of the trematode Pseudamphistomum truncatum in roach and mink, respectively, was recorded in Lake Fure North of Copenhagen, Denmark. This zoonotic digenean opisthorchiid represents a threat to humans due to its ability to infect the biliary system following ingestion of inadequately processed infected fish. Therefore precise species identification of infective metacercariae in fish used for human consumption is essential. Due to the relatively limited information on metacercarial identity obtained by morphometric studies a series of molecular techniques were used to link the larval parasite in fish with the un-equivocally diagnosed adults in the biliary system of the mink. By the use of carefully selected polymerase chain reaction (PCR) primers and subsequent sequencing of the ITS region from both metacercariae and adults full sequence identity of both metacercariae and adults were confirmed. The presence of this parasitosis in fish from a lake used for both commercial and recreational fisheries call for hygienic alerts in order to prevent accidental human infection with this opisthorchiid.

Dose Dependent Effects of Dietary Immunostimulants on Rainbow Trout Immune Parameters and Susceptibility to the Parasite Ichthyophthirius

Immunostimulants offered to fish in feed are considered to confer protection against various bacterial diseases but the effects on the antiparasitic response are largely unknown. Therefore effects of dietary β-1,3-glucan on innate immune parameters of juvenile rainbow trout Oncorhynchus mykiss and on susceptibility to the skin-parasitic cili- ate Ichthyophthirius multifiliis (Ich) have been investigated. A basal diet (dry pelleted feed) was supplemented with 0% (control), 0.2% (low), 2.0% (medium), and 5.0% (high) of the β-1,3-glucan particulate insoluble algae glucan, paramylon, from Euglena gracilis. Fish (total 440) were divided into four groups each with 110 fish (kept in duplicate tanks of 55) and each diet was fed to two replicate groups at a daily feeding rate of 1.5% of fish biomass for 56 consecutive days. Liver and plasma sampling was performed at day 0 and after feeding with β-1,3-glucans for 14, 28, 42, and 56 days and subsamples of fish were exposed to Ich at day 14 and 45. Gene expression in trout liver was investigated by real-time qPCR and genes encoding immune molecules including acute phase proteins (SAA, hepcidin, and precerebellin), immunoglobulins (IgM and IgT), cytokine (IL-1β), and lysozyme were investigated. In addition plasma lysozyme activity was recorded. At the start of the experiment the 5.0% glucan supplemented fish became more infected by parasites compared to control fish (0.0%) but after 45 days feeding they obtained signifi - cantly fewer trophonts. Plasma lysozyme activity of fish fed low (0.2%) and medium (2.0%) glucan supplementation fluctuated, while high (5.0%) glucan was associated with an elevation of lysozyme activity. Plasma lysozyme activity was positively correlated to expression of the lysozyme gene and to body mass of fish. Groups fed low (0.2%) and medium (2%) glucan diets showed a trend for down-regulation of immune relevant genes whereas the group fed with high (5%) glucan showed a trend for up-regulation of genes especially the acute phase reactant SAA.

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin-killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin-killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710-1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60-s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.

Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis and a major fish health problem in salmonid aquaculture worldwide. Injection vaccination with commercial mineral oil-adjuvanted bacterin vaccines has been partly successful in preventing the disease but in Danish rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i.p.) injection. The fish were exposed to virulent A. salmonicida 7 weeks after immunization. To assess the efficacy of the subunit vaccines we evaluated the immune response in fish after immunization and challenge infection by measuring the antibody levels and monitoring the survival of fish in different groups. The survival of fish at 3 weeks after challenge infection showed that all 3 groups of fish immunized with 3 different protein combinations exhibited significantly lower mortalities (17–30%) compared to the control groups (48% and 56%). The ELISA results revealed significantly elevated antibody levels in fish against several protein antigens, which in some cases were positively correlated to the survival.