Survival and humoral antibody response of Atlantic salmon, Salmo salar L., vaccinated against Aeromonas salmonicida ssp. achromogenes

Atlantic salmon were vaccinated against Aeromonas salmonicida ssp. achromogenes (Asa) by injection with three vaccines developed in our laboratory and an autogenous bacterin (IcelandBiojec.OO, IBOO) produced by a commercial vaccine producer. The humoral antibody responses to bacterial antigens were monitored by ELISA and Western blotting. The fish were challenged by infection with Asa 6 and 12 weeks post-vaccination. Protection was induced in all groups of vaccinated fish. The protection achieved was time-dependent. The autogenous bacterin, IBOO, induced a protective immune response later than our experimental vaccines. All the vaccines tested induced specific antibody response that increased between 6 and 12 weeks after vaccination. The antibody response was mainly directed against the A-layer protein, but antibodies to other bacterial components were also detected. Significant correlation was obtained between the antibody titre to extracellular Asa antigens, induced by the different vaccine preparations, and survival of vaccinated fish challenged by a virulent Asa strain. Furthermore, the detection of antibodies directed against an extracellular toxic metallo-caseinase, AsaP1, in fish sera correlated with protection.     

Protection against atypical furunculosis in Atlantic halibut,Hippoglossus hippoglossus (L.); comparison of a commercial furunculosis vaccine and an autogenous vaccine

Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine.     

Efficacy of furunculosis vaccines in turbot, Scophthalmus maximus (L.): evaluation of immersion, oral and injection delivery

The commercial furunculosis vaccine Aquavac Furovac 5 and an autogenous vaccine, based on the challenge strain, induced immune protection in turbot, Scophthalmus maximus (L.), as shown in challenge tests 120 days post-immunization by injection (relative percentage of survival, RPS = 72-99%). This protective effect lasted for at least 6 months post-immunization at appreciable levels (RPS = 50-52%). Neither the autogenous vaccine nor the commercial vaccine was able to induce significant levels of protection against Aeromonas salmonicida in turbot when administered by immersion. Antibody levels were high or moderate in fish vaccinated by injection with the different vaccines and very low in fish vaccinated by immersion. The field results show that delivering an oral boost after the primary vaccination by injection did not enhance protection of turbot against furunculosis and that water-based (autogenous vaccine) and oil adjuvanted (Alpha Ject 1200) vaccines administered by injection conferred similar levels of protection (RPS > 80%) in turbot.     

Comparison of different challenge methods to evaluate the efficacy of furunculosis vaccines in Atlantic salmon, Salmo salar L.

Four challenge methods, intraperitoneal (i.p.) and intramuscular (i.m.) inoculation, bath and cohabitation exposure, were evaluated as methods for testing the efficacy of furunculosis vaccines in Atlantic salmon, Salmo salar L. Groups of fish vaccinated with one of two different vaccines containing aluminium phosphate or animal/vegetable oil as an adjuvant were challenged with Aeromonas salmonicida 6 and 12 weeks after vaccination. Relative per cent survival (RPS) was calculated daily during a 3-week observation period post-challenge. A large variation in protection measured by RPS, both between methods and between different time points for each method, was found. Towards the end of the observation period, RPS tended to be similar in the i.p. and cohabitation challenge groups. A high degree of protection was demonstrated for the oil adjuvanted vaccine. The i.m. challenge produced very low RPS numbers for both vaccines, but this was most marked for the aluminium-phosphate-adjuvanted vaccine. The bath exposure resulted in RPS values intermediate to the cohabitation and injection methods. The study also demonstrated that, after i.p. and i.m. challenges, the initial peak mortality caused by the inoculation was followed by a secondary increase in mortality, probably because of shedding of bacteria into the water during the first mortality phase and hence contributing to a superinfection state.     

Antigen retention and enzyme reactivity in the spleen of Atlantic salmon, Salmo salar L., following administration of injectable furunculosis vaccines

Abstract. The retention of vaccine components and phenotypes of leucocyte populations were examined in the spleen of Atlantic salmon, Salmo salar L., 12 weeks after intraperitoneal administration of three different furunculosis vaccines. There were marked differences between the vaccine groups as judged by serum antibody response and survival following challenge with Aeromonas salmonicida. Abundant vaccine components were present in the spleen following administration of the two adjuvanted vaccines but not the non-adjuvanted vaccine. The non-adjuvanted group showed a disrupted pattern of silver staining in the splenic ellipsoids, suggesting possible toxic changes. Altered levels of enzyme reactivity in the spleens of vaccinated fish suggested activation of macrophages. Computer-assisted morphometric analysis was used to demonstrate that a significant ( P < 0.05) increase in acid phosphatase reactivity associated with the melanomacrophage accumulations only occurred in the group that had shown a good response to challenge (14% mortality when control group = 60%), and a high level of anti- A . salmonicida antibodies. The findings of the present study suggest that the retention of antigen and the activation of macrophages in melanomacrophage accumulations of Atlantic salmon are of significance in vaccination against furunculosis.     

Experimental infection of turbot, Scophthalmus maximus (L.), by Aeromonas salmonicida subsp. achromogenes and evaluation of cross protection induced by a furunculosis vaccine

Turbot was shown to be sensitive to injection challenges by Aeromonas salmonicida subsp. achromogenes (Asa). A systemic disease was induced and the bacterium was isolated from various internal organs. Histopathological changes involved haemorrhages, necrosis and degeneration in skin and muscle, haemorrhages and necrosis in kidney, degeneration in the heart muscle, and fusion of the secondary gill lamellae. A polyvalent commercial salmon vaccine, containing A. salmonicida subsp. salmonicida as one of five antigens, did not confer protection in turbot against an experimental Asa infection 13 weeks post-vaccination. Vaccination induced a significant antibody response against Asa cells but not against extracellular products of the bacterium. The results of the study indicate that Asa may be a potential threat to turbot farming and that the development of new turbot vaccines is needed.     

The development of live vaccines for furunculosis lacking the A-layer and O-antigen of Aeromonas salmonicida

Abstract. Mutants of Aeromonas salmonicida strains lacking either the A-protein, O-antigen or both of these major surface antigens were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), for their suitability as live vaccines (LV). All of these mutants were shown to be attenuated, as fish receiving ∼5 × 10⁷ of the respective strains showed no clinical signs of furunculosis. Immersion vaccination of fish in 5 × 10⁷ cfu ml^-1 of these strains with an identical immersion dose 14 days later resulted in significant protection by all strains from challenge with a heterologous virulent strain of A. salmonicida 5 weeks later. The levels of protection conferred were all greater than or equal to that provided by an injected bacterin using the same vaccination schedule. With one exception, all LV strains that still possessed a functional O-antigen provided protective indices (PI) four- to seven-fold greater than the PI for the fish injected with bacterin. When antibody responses of vaccinated fish were compared, it was found that only vaccination by bacterin gave rise to a measurable agglutinating litre. Western immunoblots using the immune fish sera failed to reveal any major differences in antigen recognition in fish that received any of the vaccines tested. These data suggest that the immune response generated by the use of live vaccine strains is different from that generated by a bacterin, and that these useful mutations may be incorporated into existing furunculosis LVs for further attenuation.     

Field testing of adjuvanted furunculosis vaccines in Atlantic salmon, Salmo salar L.

Abstract. Two different commercial vaccines against furunculosis, caused by Aeromonas salmonicida subsp. salmonicida , were tested in Atlantic salmon on seven fish farms. Both vaccines were based on formalin-inaclivated bacterins containing aluminium salts as adjuvants. The fish were vaccinated by intraperitoneal injection in the spring approximately one month prior to transfer to sea water, and they were challenged by natural outbreaks of furunculosis. During the first year, six of the farms experienced disease outbreaks. The overall mortality was 7·14% in vaccinated fish and 21·7% in unvaccinated controls, giving a relative percentage survival (RPS) of 67%. In the seventh farm, outbreaks of furunculosis more than one year after vaccination revealed that there was still a trend towards lower mortality in vaccinated fish, though the mean RPS fell to 22%. The use of adjuvants in the vaccines resulted in local lesions in the abdominal cavity of vaccinated fish. However, the severity of the lesions declined gradually, and they did not influence fish quality at the time of slaughtering. Vaccination also had a moderately adverse impact on fish weight gain in most cases.     

Exploring the efficacy of vaccine techniques in juvenile sablefish,Anoplopoma fimbria

Wild sablefish, Anoplopoma fimbria, are a valuable commercial species whose populations are declining. Fortunately, sablefish are excellent species for commercial aquaculture. Sablefish raised under high‐density conditions in the marine environment require the use of efficacious vaccines to control disease. Sablefish impacted by disease in net pens may have poor flesh quality and high mortality during grow‐out. As a result, disease can cause financial hardship for sablefish aquaculture operators. The efficacy of a multivalent vaccine preparation for sablefish, administered either by intraperitoneal (i.p.) injection or by immersion, against atypical and typical Aeromonas salmonicida, the causative agents of atypical and typical furunculosis, respectively, was examined. A. salmonicida can affect sablefish at any age and size. Consequently, an efficacious vaccine that can be appropriately and optimally administered to all life stages is desirable. Sablefish vaccinated by immersion at ~1.5 or ~4.5 g with a whole‐cell multivalent vaccine were not protected against either typical or atypical A. salmonicida. Factors that may have contributed to the ineffectiveness of the immersion vaccine are discussed. By contrast, the relative per cent survival (RPS) or potency of the whole‐cell multivalent vaccine injected i.p. in juvenile sablefish at ~50 g against typical and atypical A. salmonicida was 94.3% and 81.7% respectively. The high RPS values indicated that the vaccine successfully initiated an immune response in sablefish upon a second encounter with the pathogen.     

Serological comparison of selected isolates of Aeromonas salmonicida ssp. salmonicida

Abstract. Eight isolates of Acronionus salmonicida ssp. salmonicida were collected during furunculosis epizootics in North American Pacific coast states and provinces. Both virulent and avirulent forms of each isolate, confirmed by challenge and electron microscopy, were examined. Serological comparisons by cross-absorption agglutination tests revealed no serological differences between isolates. Using the double diffusion precipitin test, a single band was observed when antigen from a sonicated virulent strain was reacted with antiserum against a sonicated, virulent strain absorbed with homologous, avirulent strain. The presence of the single band was eliminated by excess sonication.     

Feeding Atlantic salmon Salmo salar L. soybean products: effects on disease resistance (furunculosis), and lysozyme and IgM levels in the intestinal mucosa

Two trials were initiated to investigate the consequences of including various soybean products in diets for Atlantic salmon Salmo salar L. on (1) mortality following infection by Aeromonas salmonicida ssp. salmonicida during a cohabitation challenge, and (2) the lysozyme and IgM content of the intestinal mucosa. Groups of salmon were fed control diets containing fishmeal as the sole protein source (Contr1 and Contr2, respectively), soy concentrate-containing diets (SoyConc1 and SoyConc2, respectively), or diets containing either solvent-extracted soybean meal (SoyMeal, trial 1) or soybean molasses (SoyMol, trial 2), an alcohol extract of soybean meal. Both SoyMeal and SoyMol caused enteritis-like changes in the distal intestine, which were not observed in fish fed the Contr1, Contr2, SoyConc1, or SoyConc2 diets. There were significant differences ( P  < 0.05) in mortality between feeding groups following the A.s. salmonicida challenge: these differences were greatest in fish fed SoyMeal (65.6%), least in fish fed SoyConc1 (60.5%), and intermediate in the fish fed the Contr1 diet (62.9%). The SoyMol diet caused significantly ( P  < 0.0001) increased levels of both lysozyme and IgM in the mid and distal intestinal mucosa. It is concluded that components of soybean meal and soybean molasses cause an inflammatory response in the distal intestine that may lead to increased susceptibility to furunculosis.     

Protection, immune response and side-effects in European whitefish (Coregonus lavaretus L.) vaccinated against vibriosis and furunculosis

As indicated by market demand and cultivation prospects, the whitefish (Coregonus lavaretus L.) has the potential to become an important alternative to rainbow trout in fish farming in Finland. The fish processing industry has called for a fish species having non-pigmented flesh, and there is a long tradition of whitefish farming for stocking purposes in Finland. However, cultivation in net cages in the brackish water of the coastal area exposes fish to vibriosis (Listonella (Vibrio) anguillarum) and furunculosis (Aeromonas salmonicida salmonicida). Hence, profitable whitefish farming requires the efficient control of both of these diseases. The efficacy of vaccination (Apoject 1800®) was studied in the laboratory using challenge tests and by monitoring specific antibody production (ELISA) and the blood leucocyte pattern. The fish (22 g) were vaccinated at 15°C and the immunity developed during 8 weeks prior to challenge with vibriosis (1.8×10⁵ cfu) and 12 weeks prior to challenge with furunculosis (4.9×10² cfu). The challenge was performed by i.p. injection. Side-effects around the injection site were studied 16 weeks post-vaccination. The immune system of the whitefish responded well and vaccination gave good protection against vibriosis and furunculosis. The RPS was 100% following the Vibrio challenge and 99% following the Aeromonas challenge. The antibody levels continued increasing in the vaccinated group throughout the study period. Lymphocyte and neutrophil counts were elevated in vaccinated compared to non-vaccinated fish at 8 weeks post-vaccination, but not after 16 weeks. Vaccination produced weak adhesions around the injection site in 42% and minor pigmentation in 9% of the 65 fish examined.     

Vaccine-induced protective immunity against Aeromonas salmonicida tested in experimental carp erythrodermatitis

Abstract The development and applicability of a dose-controlled experimental infection with atypical Aeromonas salmonicida in carp is described. The proliferation and clinical manifestations of experimentally induced carp erythrodermatitis mimicked a natural infection. An in-vivo assay was used to evaluate the lethal properties of cell-free culture supernatants and a simple serum-free growth medium was devised for maintaining the virulence of the challenge strain. Depending on the inoculation dose, a sublethal (chronic) to a lethal (acute) infection could be induced, and a dose-response relation was observed between A. salmonicida inoculum size and carp mortality. The dose-controlled experimental infection was used as a challenge test for laboratory evaluation of the efficacy of potential vaccine candidates. The vaccine candidates tested, a cell envelope preparation, purified lipopolysaccharide and purified A-layer (ACE) protein showed no protection or only a feeble one at the best, while formalinized whole cells showed a consistent but only moderate protection. In contrast, when concentrated, detoxified culture supernatant was used, the carp were protected against a subsequent lethal challenge. These observations indicate that immunity against A. salmonicida extracellular products is of prime importance.     

Efficacy studies of passive immunization against Aeromonas salmonicida infection in brook trout, Salvelinus fontinalis (Mitchill)

Abstract. Aeromonas salmonicida , the aetiologic agent of furunculosis, causes high mortality in cultured salmonids. Experiments were conducted to determine the therapeutic and prophylactic efficacy of passive immunization against furunculosis in brook trout, Salvelinus fontinalis (Mitchill), infected by immersion. Rabbit hyperimmune serum was produced against a virulent strain of A. salmonicida and an aliquot of this serum was absorbed with cells of a non-virulent strain of A. salmonicida. Immunoglobulins from aliquots of the absorbed and non-absorbed serum were purified using affinity chromatography. Each serum or immunoglobulin preparation was tested in passive immunization experiments. Brook trout were infected by immersion in a suspension of virulent A. salmonicida , and passively immunized by intraperitoneal injection at the time of experimental infection, or at various periods after experimental infection. Passive immunization of brook trout against furunculosis was therapeutically efficacious when effected either at zero, 24 or 48h post-infection, but not at 72 or 96h. Purified rabbit immunoglobulins specific to virulent A. salmonicida were as protective as the initial rabbit hyperimmune serum in protecting brook trout against furunculosis. To determine the prophylactic efficacy of this treatment, the groups of fish passively immunized at the time of the experimental infection were challenged a second time at either 14, 35, 41 or 56 days after passive immunization. Brook trout were protected against a second experimental bath challenge with virulent A. salmonicida for a period of 35–41 days.     

Virulence of the aetiological agent of goldfish ulcer disease in Atlantic salmon, Salmo salar L.

Abstract. An atypical strain of Aeromonas salmonicida was isolated from goldfish. Carassitis auraius L., with cutaneous ulcerativc lesions. Atlantic salmon, Salmo salar L., weighing 30–40 g were challenged parentcrally and by hath immersion with this strain. It was found to be highly virulent in both forms of challenge and was capable of achieving an LD₅₀ of three colony-forming units in parenterally challenged fish. The gross lesions and histopathology of infected fish bore a close resemblance to classical furunculosis in salmonids. Goldfish ulcer disease is endemic to some regions of Australia and represents a potential threat to the emerging salmonid farming industry.     

Immunogenicity of vaccines from a virulent and an a virulent strain of Aeromonas salmonicida

Abstract. Injected vaccines consisting of formalin-killed cells and extracellular antigens prepared from a virulent and an avirulent strain of Aeromonas salmonicida were tested for their efficacy in protecting juvenile coho salmon. Oncorhychus kisutch (Walbaum). Against experimental furunculosis following active and passive immunization. Sera used in the passive immunization experiments were raised in subadult coho salmon and in rabbits. Results indicated that the avirulent strain was inferior to the virulent strain in its immunogenicity for coho salmon. Thus, even though avirulent cells possessed at least one immunogen–an immunogen that was show in passive immunization experiments to be well recognized by rabbits–the immunogen was only inefficiently protection when vaccinated with avirulent cells. Furher, extracellular antigens of the avirulent strain were not protective for coho salmon even though they elicited the production of anti- A. salmonicida agglutinins. In contrast, the killed cells and extracellular antigens of the virulent strain were both immunogenic in coho salmon. By passively immunizing coho salmon with rabbit sera raised against the virulent and the virulent strain produced at least two immunogens, only one of which was shared in common with the avirulent strain. The extra immunogen possessed by the virulent strain is presumably responsible for its superior immunogenicity in coho salmon; the immunogen had the properties of a protein and is believed to be the A-protein.     

Antibiotic protection against recrudescence of latent Aeromonas salmonicida during furunculosis vaccination

Problems of temporary immunosuppression following vaccination against Aeromonas salmonicida infection had to be overcome in the development of a furunculosis vaccine. Empirical observations have indicated that immunosuppression persists for some time after vaccination, rendering fish, especially subclinical carriers of A. salmonicida, highly vulnerable to bacterial invasion. The efficacy of simultaneous application of furunculosis vaccine and a long-lasting amoxycillin preparation to a population of Atlantic salmon smolts was evaluated. Control groups were treated with either vaccine alone, amoxycillin alone or were untreated. Moderate stress, simulating smolt transfer with a 5°C temperature rise, resulted in a rapid outbreak in mortalities reaching 100% in the vaccinates. Losses among the untreated controls were more gradual and rose to about 50%. Both amoxycillin-treated groups survived well. Further severe stress resulted in total mortalities among the untreated fish but no further losses in the amoxycillin groups. Four months after vaccination, evidence of a specific immune response was confirmed by ELISA, demonstrating circulating antibodies in the blood of vaccinates. In a severe and in a moderate challenge with A. salmonicida., the relative specific protection was 63 and 86%, respectively. Thus, effective protection against furunculosis was achieved without jeopardizing the stock during the vaccination process and with elimination of the carrier state.     

Comparative evaluation of experimental challenge by intraperitoneal injection and cohabitation of Atlantic salmon (Salmo salar L) after vaccination against Piscirickettsia salmonis (EM90‐like)

The Chilean aquaculture has been challenged for years by piscirickettsiosis. A common prophylactic measurement to try to reduce the impact from this disease is vaccination, but the development of vaccines that induce satisfactory protection of the fish in the field has so far not been successful. Experimental challenge models are used to test vaccine efficacy. The aim of this study was to evaluate the performance of experimental vaccines after challenge by the two most widely used challenge routes, intraperitoneal injection and cohabitation. A total of 1,120 Atlantic salmon were vaccinated with non‐commercial experimental vaccines with increasing amounts of an inactivated Piscirickettsia salmonis EM90‐like isolate. Differences in mortality, macroscopic and microscopic pathological changes, bacterial load and immune gene expression were compared after challenge by different routes. The results revealed a similar progression of the diseases after challenge by both routes and no gross differences reflecting the efficacy of the vaccines could be identified. The analysis of the immune genes suggests a possible suppression of the cellular immunity by CD8 T cell and with this stimulation of bacterial survival and replication. Comparative studies of experimental challenge models are valuable with regard to identifying the best model to mimic real‐life conditions and vaccines’ performance.     

Retention of furunculosis vaccine components in Atlantic salmon, Salmo salar L., following different routes of vaccine administration

The retention of vaccine components was studied in Atlantic salmon, Salmo salar L., following different routes of vaccination against Aeromonas salmonicida. Frozen tissue was collected from the spleen, head kidney, hind gut and liver of fish that had been vaccinated by intraperitoneal injection (monovalent and trivalent vaccines), immersion and oral administration 2,6,8 and 16 weeks previously. The trivalent injection group showed the highest levels of specific antibodies and was the only group to show protection following challenge with virulent A. salmonicida. Following intraperitoneal injection, there was an initial widespread distribution of Aeromonas lipo-polysaccharide, but by 16 weeks lipopolysaccharide was predominantly found in macrophage populations in the spleen, head kidney and abdominal granulomas. Only small amounts of lipopolysaccharide were retained in the head kidney of the immersion group and no lipopolysaccharide was retained in the oral group. Small and inconsistent amounts of A-layer protein were present in the meianomacrophages of the head kidney of all groups. The relative prominence of A-layer protein in the spleen of the trivalent injection group 8 weeks after vaccination was linked to the high levels of specific antibodies, and possible immune-complex trapping and the enhancement of immunological memory.     

Vaccine development for winter ulcer disease, Vibrio viscosus, in Atlantic salmon, Salmo salar L.

Coldwater Vibrio species isolated from Atlantic salmon, Salmo salar L., during winter ulcer disease outbreaks at saltwater sites in Norway and Iceland were characterized phenotypically, tested for virulence, and used to evaluate the efficacy of multivalent, oil-adjuvanted vaccines. The intraperitoneal (i.p.) injection of rainbow trout, Oncorhynchus mykiss (Walbaum), in fresh water with one bacteria species isolated during winter ulcer outbreaks, V. ‘viscosus’, produced rapid mortality and disease signs which resembled those observed during natural outbreaks [10⁵ colony-forming units (cfu) fish^??1]. Another species, V. ‘wodanis’, was not virulent to rainbow trout (10³–10⁶ cfu fish^??1). Although vaccination of rainbow trout with a mineral-oil-adjuvanted, injectable vaccine containing V. anguillarum (serotypes 01 and 02), V. salmonicida and Aeromonas salmonicida did not provide protection against injection challenge with V. viscosus, vaccines which included V. viscosus produced significant protection in Atlantic salmon and rainbow trout. Atlantic salmon vaccinated with an oil-adjuvanted vaccine containing V. viscosus, V. wodanis and atypical A. salmonicida produced a relative percentage survival (RPS) of 97% when challenged i.p. with V. viscosus, demonstrating cross-protection between strains from Iceland and Norway. Short-term efficacy was demonstrated in rainbow trout by injection challenge at 21 and 43 days post-vaccination with an oil-adjuvanted vaccine containing V. viscosus, V. anguillarum (01/02), V. salmonicida and A. salmonicida, which produced an RPS of 96–99%. Rainbow trout challenged with V. viscosus at 52 and 362 days post-vaccination produced an RPS of 93% and 79%, indicating that vaccination provided long-term protection. In a similar manner, rainbow trout injected i.p. with 0.2 mL of a vaccine containing the five bacteria species and infectious pancreatic necrosis virus produced a 90% RPS when challenged with V. viscosus 66 days later. The high RPS under a severe challenge burden, along with disease signs in experimental freshwater challenges which resembled the saltwater disease condition, indicated that V. viscosus is a contributing factor to winter ulcer and that vaccination will protect against the disease.