Immersion Route Research Articles

Administration of hot-water extract of Padina boergesenii via immersion route to enhance haemolymph-immune responses of Fenneropenaeus indicus (Edwards)

Various polysaccharides extract from marine algae to increase the non-specific immune system in crustacean. In this study, effects of hot-water extract on total haemocyte count, total plasma protein, Phagocytic activity, bacterial clearance efficiency and bactericidal activity while the shrimp Fenneropenaeus indicus (10.12±2.18 g) were immersed in seawater (40 g L−1 and 25±0.8 °C) containing hot-water extract of brown algae Padina boergesenii at 100, 300 and 500 mg L−1 for 1–4 h, were investigated. These parameters increased significantly (P<0.05) when the shrimp were immersed in seawater containing hot-water extract at 100 mg L−1 after 3 h and 300 and 500 mg L−1 after 2 h. Fenneropenaeus indicus that were immersed in hot-water extract at 300 and 500 mg L−1 had significantly increased phagocytic activity and increased clearance efficiency to Vibrio harveyi after 2 h. But bactericidal activity increased after 1 h immersed in 500 mg L−1 concentration.

Analysis of the vaccine potential of a natural avirulent Edwardsiella tarda isolate

Edwardsiella tarda is a severe aquaculture pathogen that can infect many fish species and cause a systematic disease called edwardsiellosis, which can lead to high mortality under certain conditions. Currently, most vaccine candidates against edwardsiellosis are based on pathogenic E. tarda strains, which can be a concern in some cases. In this study, the vaccine potential of a natural E. tarda isolate, ATCC 15947, was examined in a Japanese flounder model. ATCC 15947 was found to be relatively avirulent to flounder but able to disseminate into and survive transiently in fish tissues following intraperitoneal (i.p.) injection. Fish vaccinated with ATCC 15947 via i.p. injection exhibited a high level of survival rate, which was increased to 100% by a booster injection. Fish vaccinated with ATCC 15947 orally in the form of alginate microspheres showed a moderate survival rate, while fish vaccinated with ATCC 15947 via the immersion route exhibited a low rate of survival. Following oral vaccination, ATCC 15947 could colonize transiently in the gut, liver, and spleen of the vaccinated fish. Both injection and oral vaccination with ATCC 15947 induced production of specific serum antibodies, the levels of which at different time points following vaccination were generally higher in fish vaccinated with ATCC 15947 via injection than in fish vaccinated with ATCC 15947 orally. Compared to control fish, fish vaccinated with ATCC 15947 showed enhanced serum bactericidal activity and significantly increased expression in genes encoding a number of immune-related factors. These results indicated that ATCC 15947 possesses good immunoprotective potential, which may be exploited in the development of E. tarda vaccines.

Surface microstructurization of a sputtered magnesium thin film via a solution–immersion route

Surface microstructurization is one of the important procedures to fabricate a superhydrophobic surface of materials. Thus, this study was focused on the modification of the surface microstructure of magnesium materials to tailor its wettability. Bias magnetron sputtering was used to prepare magnesium thin films. In the deposition process, a relatively dense magnesium film was obtained using the bias voltage of 200 V. A solution–immersion method was subsequently applied to modify the surface of the magnesium thin film. A honeycomb-like layer, mainly composed of Mg(OH) 2, was formed on the film's surface after the surface modification. Accordingly, this route provides a basis for the following surface functionization of magnesium materials.

Oral and parenteral vaccines against Flavobacterium columnare: evaluation of humoral immune response by ELISA and in vivo efficiency in Nile tilapia (Oreochromis niloticus)

Flavobacterium columnare is a bacterial pathogen for many freshwater fish species. It is responsible for outbreaks in fish farms worldwide, causing high mortality rates. Fish vaccination is a potential approach for prevention and control of disease, with oral vaccines suitable for fish because of their easier application, low cost and minimum stress to fish. Alginate microparticles have been widely used as controlled release systems, including for fish vaccination. The aim of this study was to evaluate the capacity of oral and parenteral vaccines against F. columnare to induce a humoral response, as well as the in vivo efficiency in Nile tilapia fingerlings. The fingerlings were immunized with bacterin by intraperitoneal (i.p.), intramuscular (i.m.), oral and immersion routes, as well as orally with alginate microparticles containing formalin-killed bacteria. A sandwich ELISA was developed to detect specific antibodies against F. columnare. The animals were challenged with pathogenic strain BZ-1 to determine the relative percentage of survival. A significant humoral response was induced by bacterin administered by i.p. and i.m. routes (P < 0.05). However, none of the vaccine preparations were effective in protecting fish against F. columnare infection (P < 0.05). In spite of high antibody levels, there was no relation between immunoglobulin titers and resistance to columnaris for Nile tilapia fingerlings. These data suggest that use of serological analysis as the only method to determine vaccine efficiency against F. columnare infection in Nile tilapia can lead to imprecise results for the usefulness of these products in vivo.

Pathogenicity of Streptococcus ictaluri to Channel Catfish

The pathogenicity of a Streptococcus ictaluri isolate in channel catfish Ictalurus punctatus at the fry (0.5 g), fingerling (15 g), and juvenile (55 g) stages was determined by experimental bath immersion and injection experiments. Channel catfish were exposed in 1-L immersion baths containing 10(8), 10(9), 10(10), 10(11) or 10(12) colony-forming units (cfu) of S. ictaluri. Fish were also injected intraperitoneally with 0.1 mL of bacterial solution for final doses of 10(4), 10(5), 10(6), 10(7), or 10(8) cfu of S. ictaluri per fish. Streptococcus ictaluri caused mortality in fry, fingerling, and juvenile channel catfish within 21 d postinfection. When mortalities were calculated based on size and challenge route, the cumulative percent mortalities were 11% for fry and 0% for fingerlings by the bath immersion route and 14% for fingerlings and 6% for juveniles by the injection route. Isolation of S. ictaluri from moribund and dead catfish was confirmed by the newly established BIOLOG profile (MicroLog3 system). The results indicate that channel catfish were only susceptible to high concentrations of S. ictaluri and that juvenile channel catfish were less susceptible, possibly explaining why little mortality has been attributed to S. ictaluri infection in catfish aquaculture.

Genotyping of Streptococcus agalactiae strains isolated from fish, human and cattle and their virulence potential in Nile tilapia

Streptococcus agalactiae (Lancefield group B; GBS) is a pathogen that causes meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. The objective of this study was to characterize S. agalactiae isolated from fish (n=27), cows (n=9), and humans (n=10) using pulsed-field gel electrophoresis (PFGE) and to investigate the virulence of the identified strains in Nile tilapia (Oreochromis niloticus). The PFGE types were determined by dendogram analyses and the in vivo virulence was evaluated by experimental infection (using i.p. and immersion routes) of Nile tilapia. Among the fish strains, 5 different PFGE patterns were observed and 21 strains showed the same genetic pattern. In some farms two or three profiles occurred simultaneously. The bovine and human strains exhibited high genetic diversity and few relationships were established among S. agalactiae strains from the three host origins analyzed. Eight S. agalactiae strains from fish caused high mortality of Nile tilapia. Three bovine strains infected Nile tilapia (by i.p. route) and two of those strains caused clinical signs of meningoencephalitis. All human strains (n=5) infected Nile tilapia (by i.p. route) and meningoencephalitis was induced by one strain (by both i.p. and immersion routes). In conclusion, the analyzed strains from the three natural hosts did not show genetic relatedness, yet some of the bovine and human strains were able to infect fish and cause meningoencephalitis. We suggest that genetic linkage is not a prerequisite for S. agalactiae to cross the host-specific barrier.

Experimental exposure of Artemia to Hepatopancreatic parvo-like Virus and Subsequent transmission to post-larvae of Penaeus monodon

The different life stages of Artemia franciscana were experimentally exposed to Hepatopancreatic parvo-like virus (HPV), in order to evaluate the possibility of Artemia acting as reservoir or carrier for HPV. All the five developmental stages of Artemia were challenged with HPV both by immersion and oral infection routes. The viral infectivity to Artemia was studied by PCR but not much difference in mortality between control and challenge groups were observed. To confirm the vector status of Artemia for HPV, the HPV exposed Artemia were fed to postlarval forms of Penaeus monodon. Post-larvae of P. monodon were fed with HPV exposed Artemia and could get infected upon feeding on them. Mortality was observed in the post-larvae, which were fed with HPV exposed Artemia, and whereas no mortality was observed in post-larvae fed with Artemia not exposed to HPV and these post-larvae were PCR negative for HPV, as well. Results of this experiment suggest that Artemia might be a possible horizontal transmission pathway for HPV. Further research however is required with histology, immunohistochemistry and transmission electron microcopy to determine whether the Artemia are actually infected with this virus or whether they are simply mechanical carriers. This will enable us to understand better whether Artemia is a carrier of this virus and if so the mechanism involved.

Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P. fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections.

Experimental study of the susceptibility of Atlantic cod, Gadus morhua (L.), to infection with an IPNV strain pathogenic for Atlantic salmon, Salmo salar L

Intraperitoneal (IP) injection, cohabitation and immersion routes of infection were used to determine if Atlantic cod, Gadus morhua (L.), of 1 and 3 g are susceptible to infectious pancreatic necrosis (IPN). Mortalities of cod injected IP were significantly higher when challenged with infectious pancreatic necrosis virus (IPNV) than with phosphate buffered saline. This is the first report of Atlantic cod mortalities caused by IPNV. Fish challenged by cohabitation had significantly higher mortalities than the controls, but mortalities of Atlantic cod challenged with IPNV by immersion were not significantly different from controls. Titres of IPNV in the tissues of infected fish were sometimes very high (range 10(2)-10(10) infectious units per gram of tissue) suggesting virus replication and titres of fish that died were generally higher than those of fish which survived. However, the relatively low mortality rates when challenged by cohabitation and immersion (20% and 17%, respectively), compared to the IP injection challenge (100%) suggest that 1 and 3 g cod have low susceptibility to IPN when challenged by more natural routes. These data strongly suggest that the cause of death of experimentally challenged cod was IPNV and further histological evidence for this came from 1 g cod challenged IP with IPNV in which the pancreas showed severe necrosis and heavy immunostaining for IPNV coincidentally with the peak of mortalities.

Molt stage and cuticle damage influence white spot syndrome virus immersion infection in penaeid shrimp

Transmission of white spot syndrome virus (WSSV) in shrimp has been reported to occur by feeding and immersion. In the present study, the impact of the molt process and artificial lesions in the cuticle on shrimp susceptibility to WSSV was examined using intramuscular and immersion routes. For the intramuscular route, Penaeus ( Litopenaeus) vannamei shrimp ( n = 450) were injected with 10 −2.3 up to 10 2.7 shrimp infectious dose 50% end point (SID 50) of WSSV in early and late post-molt, inter-molt, early and late pre-molt; resp. A-, B-, C-, D1- and D2-stage. The resulting infection titers demonstrated that no difference ( p > 0.05) in susceptibility existed between different molt stages when virus was injected. For the waterborne route, shrimp in different molt stages were immersed in seawater containing 10 4 SID 50 ml −1 of WSSV. In a first study, P. vannamei ( n = 125) incubated in cell culture flasks, became infected with WSSV mostly in post-molt stages. In a second study, 2 groups of P. vannamei ( n = 100) and P. monodon ( n = 100) were transferred into plastic bags to prevent damage to the cuticle; and in 1 group a pleopod was cut off prior to incubation. Induction of damage increased infection significantly ( p < 0.05) in A-stage from 0–40% to 60–100%, in B-stage from 0–20% to 40–60%, in C-stage from 0–20 to 20–60%, while infection was 0% in D-stages with both immersion methods. This study proved that shrimp are more susceptible to WSSV infection via immersion after molting than in the period before molting and wounding facilitates infection.

Anesthetic Considerations for Amphibians

The popularity of amphibians in research, zoological exhibits, and as pets is on the rise. With this increased popularity comes a need for veterinarians to develop methods for managing these animals for various diagnostic and surgical procedures. For many of these procedures, the provision of anesthesia is a must. Fortunately, there are a number of different anesthetics available to the veterinary clinician for anesthetizing amphibians, including tricaine methanesulfonate, clove oil, propofol, isoflurane, and ketamine. In addition to the variety of anesthetics at our disposal, there is also a wider range of methods for delivering the anesthetics than are generally available for higher vertebrates, including immersion, topical, and intracoelomic routes of delivery. The purpose of this article is to review the different methods that can be used to successfully manage an amphibian patient through an anesthetic event.

DETECTION AND IDENTIFICATION OF VIRULENT YERSINIA RUCKERI: THE CAUSATIVE AGENT OF ENTERIC REDMOUTH DISEASE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) CULTURED IN FARS PROVINCE, IRAN

Summary From the winter 2002 to spring 2006, 126 moribund rainbow trout with clinical signs of external body haemorrhages around and within the oral cavity were sampled from 10 rainbow trout farms situated in the northwest and west of Fars province, Iran and examined for the detection and identification of Yersinia ruckeri, the causative agent of enteric redmouth disease. Fish kidneys were cultured aseptically on brain heart infusion (BHI) agar plates and incubated at 25oC for 48 h. Using conventional biochemical tests, Y. ruckeri was detected in 7 fish (5.5% of total fish sampled). This was also confirmed using specific polymerase chain reaction (PCR) assay. The 16S rDNA PCR assays produced amplicons of 409 bp when applied to Y. ruckeri isolates as well as a reference strain. Results of antibiogram tests on Y. ruckeri isolates showed a high susceptibility to enrofloxacin, norfloxacin, ofloxacin, trimethoprim and oxytetracycline. In pathogenicity tests, dilution of 4 × 10 8 colony forming unit/ml of Y. ruckeri by immersion route in challenge experiments showed 70 ± 8.2% mortality during 14 days post-infection. Experimentally infected fish showed typical haemorrhages in mouth, blackening of skin, exophthalmia and a wide haemorrhages on the internal organs.

Protection against white spot syndrome virus (WSSV) infection in kuruma shrimp orally vaccinated with WSSV rVP26 and rVP28

White spot syndrome virus (WSSV) is the causative agent of white spot disease (WSD), one of the most serious diseases affecting global shrimp farming. We compared WSSV infection induction in kuruma shrimp Marsupenaeus japonicus by oral, immersion, and intramuscular injection (IM) exposure methods and evaluated the oral vaccine prepared from the recombinant WSSV proteins rVP26 and rVP28. The 50% lethal doses (LD50) of WSSV by oral, immersion, and IM challenges were 10(-0.4), 109-4.4), and 10(-7.7) g shrimp(-1), respectively, indicating that WSSV infection efficiency by oral challenge was significantly less than the other 2 challenge routes. However, in shrimp farms it is believed that WSSV infection is easily and commonly established by the oral route as a result of cannibalization of WSSV-infected shrimp. Kuruma shrimp vaccinated orally with WSSV rVP26 or rVP28 were challenged with WSSV by oral, immersion, and IM routes to compare protection efficacy. The relative percent survival values were 100% for oral challenge, 70 to 71% for immersion, and 34 to 61% for IM. Thus, the protection against WSSV-infection that was induced in kuruma shrimp by oral vaccination with rVP26 or rVP28 seemed equivalent to that obtained through IM vaccination.

Vaccination of larvae of the bony fish gilthead seabream reveals a lack of correlation between lymphocyte development and adaptive immunocompetence

Fish eggs are released and embryos hatch into a pathogenically hostile environment, at a time when their immunological capacity is severely limited. Although the eggs are initially protected by the envelope as well as by several innate and adaptive immune substances, which are transferred to eggs during fish vitellogenesis, it seems that young specimens depend fundamentally on their innate defence mechanisms. Here we show in the gilthead seabream, an immunologically tractable teleost fish model, that the first lymphocyte marker genes, those coding for the two subunits for the recombination activating gene, were detected by RT-PCR around 21–27 days post-hatching (dph). In addition, the transcripts coding for the α and β subunits of the T-cell receptor and the light and heavy chains of immunoglobulin M were detected at 27–48 dph. However, most innate immune genes analyzed were already expressed at hatching, including those coding for the toll-like receptors, pro- and anti-inflammatory molecules, antiviral and antibacterial factors, and phagocyte markers. Using the information from the gene expression study, we also examined the achievement of immunocompetence by analyzing the protection induced by a bacterin against the pathogenic bacterium Photobacterium damselae subsp. piscicida. The results show that vaccination of young larvae of this species by either immersion or oral routes resulted in increased susceptibility to infection of the specimens, and point to the lack of correlation between the achievement of immunocompetence and detection of the adaptive immunity markers.

Innate immunomodulation with recombinant interferon- α enhances resistance of rainbow trout ( Oncorhynchus mykiss) to infectious hematopoietic necrosis virus

We examined the in vivo immunostimulatory effects of a recombinant Atlantic salmon (Salmo salar) interferon-alpha2 (rSasaIFN-alpha2). The mature rSasaIFN-alpha2, expressed and purified from Escherichia coli, was administered to rainbow trout (Oncorhynchus mykiss) via the oral, immersion, or intraperitoneal (IP) injection route. Injection of rSasaIFN-alpha2 at 0.1microg/g fish gave significantly greater protection than a phosphate buffered saline (PBS) injection against a lethal challenge of infectious hematopoietic necrosis virus (IHNV), with a relative percent survival of 39%. Relative percent survival (RPS) increased significantly to 92% when the fish were injected with rSasaIFN-alpha2 at 1microg/g fish. Antiviral protection was evident for up to 7 days post-injection of rSasaIFN-alpha2. Administration of rSasaIFN-alpha2 by the oral or immersion route was not protective, and the fish succumbed to virus infection. The level of systemic IFN-induced expression of the Mx1 gene was significantly greater (p<0.01) in the IFN-injected group than in the PBS-injected group, and this was correlated with the fish survival rates in the challenge study. We used relative quantitative real-time polymerase chain reactions to examine the systemic expression of several other IFN-induced genes (including genes for IFN1, IFN regulatory factors 1 and 2, MHC-I, STAT1, vig-1, and GBP) and found that their expression was significantly increased 1-day post-rSasaIFN-alpha2 injection. Expression of the IFN-gamma and interleukin-1beta genes was not significantly increased. Thus, a salmonid rIFN-alpha can modulate the innate immune response of rainbow trout and mediate early antiviral protection against IHNV.

Vaccination of rainbow trout against Streptococcus iniae infection: comparison of different routes of administration and different vaccines

Antibody production and clinical efficacy (relative percent survival RPS) were measured in 40±5g rainbow trout after immunization with two types of Streptococcus iniae vaccines consisting of formalin killed cells (FKC) and FKC enriched with the bacterial extracellular products (ECP) administered by intraperitoneal (i.p), immersion and oral routes at 16±1°C for 18 weeks. No significant difference was found in antibody levels among the fish i.p immunized with FKC enriched with ECP plus Freunds' adjuvant (FA), FKC plus FA and FKC vaccines (P>0.05), whilst the antibody production was significantly higher in these three groups than fish immunized by immersion and oral routes of FKC and FKC enriched ECP (P 0.05). The RPSs ranging 82.6-100, 73.9-95 and 73.9-91.7% were obtained in the fish intraperitoneally immunized with FKC enriched ECP plus FA, FKC plus FA and FKC vaccines, respectively, compared to 0% survival for the control fish. Also, RPS in fish vaccinated by the immersion route was in the range 45.8-30.4% after 18 weeks post-vaccination. Efficacy of oral vaccination of fish with FKC plus ECP was in range of 8.7-29% and that of fish orally vaccinated with FKC resulted in 8.7-20.8% protection.

Artemia as a possible vector for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus transmission (XSV) to Macrobrachium rosenbergii post-larvae

Five developmental stages of Artemia were exposed to Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) by immersion and oral routes in order to investigate the possibility of Artemia acting as a reservoir or carrier of these viruses. The second objective was to determine if virus-exposed Artemia were capable of transmitting the disease to post-larvae (PL) of M. rosenbergii. There was no significant difference in percent mortality between Artemia control groups and groups challenged with these viruses. On the other hand, all the developmental stages of Artemia were positive for both viruses by nested RT-PCR, regardless of the challenge route. In horizontal transmission experiments, 100% mortality was observed in M. rosenbergii PL fed with Artemia nauplii exposed to MrNV and XSV by either challenge route. However, no mortality was observed in PL fed with virus-free Artemia. RT-PCR analysis of the M. rosenbergii PL confirmed the presence of MrNV and XSV in the challenge group and absence in the control group.

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout ( Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.

Routes of immunostimulation vis-à-vis survival and growth of Penaeus monodon postlarvae

The present study was carried out to obtain the performance of booster doses of immunostimulants given through different routes. Postlarvae of tiger shrimp Penaeus monodon (PL30) were immunostimulated via immersion and oral routes using heat-killed Vibrio anguillarum. Bath (10 6 CFU/ml for 1 h), bath and in-feed (initially immersed in 10 6 CFU/ml for 1 h followed by two feedings with 10 5 CFU/feeding), and in-feed alone were used as the routes of immunostimulation. A booster dose was given in each of the treatment groups at 20 days postpriming (dpp). Growth and survival were studied following priming and booster until 60 dpp. The survival of shrimps in bath + feed treatment was significantly ( P < 0.05) higher than the bath treatment. However, initial bath priming followed by booster through feed or bath significantly ( P < 0.05) enhanced the growth and survival of the postlarvae compared to the primed or the control. Growth was the best with booster (bath + in-feed)-treated larvae, followed by other booster-treated and primed groups, while it was the least in control groups. The booster dose of immunostimulation, in general, is advantageous in inducing growth and protective response in shrimp, and among the modes, the in-feed route of administration may be more practical as well as productive.

Effectiveness of a divalent vaccine for sole, Solea senegalensis (Kaup), against Vibrio harveyi and Photobacterium damselae subsp. piscicida

The protection of cultured sole, Solea senegalensis, against Vibrio harveyi and Photobacterium damselae subsp. piscicida was evaluated following the use of a divalent vaccine prepared with formalized whole cells and extracellular products of virulent strains of both pathogenic microorganisms and administered by the immersion route. Two prolonged immersions of 5-10 g fish in the divalent bacterin at a 1-month interval gave high levels of protection similar to those obtained when the respective monovalent vaccines were administered by the intraperitoneal route [relative percentage of survival (RPS) values >70%], which indicates that the former procedure can be a useful strategy with small fish. The high protection afforded by the divalent vaccine in sole lasted for 4 months after which the RPS values against both pathogens decreased significantly.