Atle Lillehaug

Recent developments in fish vaccinology.

During the last 10 to 20 years vaccination has become established as an important method for prevention of infectious diseases in farmed fish, mainly salmonid species. So far, most commercial vaccines have been inactivated vaccines administered by injection or immersion. Bacterial infections caused by Gram-negative bacteria such as Vibrio sp., Aeromonas sp., and Yersinia sp. have been effectively controlled by vaccination. With furunculosis, the success is attributed to the use of injectable vaccines containing adjuvants. Vaccines against virus infections, including infectious pancreatic necrosis, have also been used in commercial fish farming. Vaccines against several other bacterial and viral infections have been studied and found to be technically feasible. Pasteurellosis, streptococcosis (lactococcosis) and infections with iridoviruses are candidate diseases for control by immunoprophylaxis in the near future. The overall positive effect of vaccination in farmed fish is reduced mortality. However, for the future of the fish farming industry it is also important that vaccination contributes to a sustainable biological production with negligible consumption of antibiotics. A potential side-effect associated with injectable vaccines is local reactions in the peritoneal cavity. The paper presents recent developments in immunoprophylaxis of fish and some problems that should be addressed by the research community in the years to come.

Relationship between diet and immune response in Atlantic salmon (Salmo salar L.) after feeding various levels of ascorbic acid and omega-3 fatty acids

Abstract Juvenile Atlantic salmon (Salmo salar L.) were fed experimental diets containing megadoses of vitamin C1 and C2, respectively, and increasing amounts of omega-3 fatty acids for periods of 52 and 72 days. General resistance to infection was assessed after challenge with Vibrio salmonicida. Development of specific immune response after vaccination against Yersinia ruckeri was assessed as likelihood of survival after challenge and antibody level. Cell wall strength was assessed by the erythrocyte fragility test, and occurrence of morphological changes in various body organs was investigated. In the present study, no significant increase in general or specific resistance to infection was seen. Fish fed diets with megadoses of vitamin C2 had increased antibody levels and higher incidence of degenerative changes in skeletal muscle. Omega-3 fatty acids exerted an immunosuppressive action with both significantly lower likelihood of survival and lower antibody levels. An increased incidence of degenerative changes was also observed. On the other hand, increased levels of omega-3 fatty acids increased erythrocyte cell wall strength.

Maternal vaccination against subclinical necrotic enteritis in broilers

The inclusion of antibacterial feed additives has until now been the major strategy for controlling Clostridium perfringens-associated necrotic enteritis in broilers. In the present study, the effect of maternal immunization against the disease was examined. Broiler breeder hens were injected intramuscularly with candidate vaccines based on C. perfringens type A and type C toxoids adjuvanted with aluminium hydroxide. Vaccination resulted in a strong serum immunoglobulin G response to C. perfringens alpha-toxin in parent hens, and specific antibodies were transferred to their progeny. Subclinical necrotic enteritis in broilers was induced under field conditions or in a disease model, and the occurrence of specific enteric and hepatic lesions was evaluated in randomly selected birds. In three experiments, estimates of odds ratio for developing such lesions were 0.23, 0.33 and 0.56 in maternally toxoid C-immunized broilers compared with non-immunized controls. In toxoid A-immunized birds, odds ratios were estimated at 0.41, 0.61 and 0.63. From these results, immunoprophylaxis seems to be an interesting alternative for the control of necrotic enteritis in broilers.

Campylobacter spp., Salmonella spp., Verocytotoxic Escherichia coli, and Antibiotic Resistance in Indicator Organisms in Wild Cervids

AbstractFaecal samples were collected, as part of the National Health Surveillance Program for Cervids (HOP) in Norway, from wild red deer, roe deer, moose and reindeer during ordinary hunting seasons from 2001 to 2003. Samples from a total of 618 animals were examined for verocytotoxic E. coli (VTEC); 611 animals for Salmonella and 324 animals for Campylobacter. A total of 50 samples were cultivated from each cervid species in order to isolate the indicator bacterial species E. coli and Enterococcus faecalis/E. faecium for antibiotic resistance pattern studies. Salmonella and the potentially human pathogenic verocytotoxic E. coli were not isolated, while Campylobacter jejuni jejuni was found in one roe deer sample only. Antibiotic resistance was found in 13 (7.3%) of the 179 E. coli isolates tested, eight of these being resistant against one type of antibiotic only. The proportion of resistant E. coli isolates was higher in wild reindeer (24%) than in the other cervids (2.2%). E. faecalis or E. faecium were isolated from 19 of the samples, none of these being reindeer. All the strains isolated were resistant against one (84%) or more (16%) antibiotics. A total of 14 E. faecalis-strains were resistant to virginiamycin only. The results indicate that the cervid species studied do not constitute an important infectious reservoir for either the human pathogens or the antibiotic resistant microorganisms included in the study.SammendragUndersøkelse av fecesprøver fra hjortevilt for Campylobacter spp., Salmonella spp., verocytotoksike Escherichia coli og antibiotikaresistens hos indikatororganismer. Fecesprøver ble samlet inn i regi av Helseovervåkingsprogrammet for hjortevilt (HOP) fra kronhjort, rådyr, elg og villrein i løpet av jaktsesongene fra 2001 til 2003. Prøver fra i alt 618 dyr ble undersøkt for verocytotoksiske E. coli (VTEC), 611 dyr for Salmonella og 324 dyr for Campylobacter. For å studere antibiotikaresistens- mønstre ble indikatorbakteriene E. coli og Enterococcus faecalis / E. faeciumi forsøkt isolert fra til sammen 50 prøver fra hver dyreart. Salmonella og de potensielt humanpatogene verocytotoksiske E. coli ble ikke isolert, mens Campylobacter jejuni jejuni ble funnet i prøven fra ett eneste rådyr. Antibiotikaresistens ble påvist hos 13 (7,3%) av de 179 E. coli isolatene som ble testet. Av disse var åtte resistente mot bare en type antibiotika. Andelen resistente E. coli isolater var høgere hos villrein (24%) enn hos det øvrige hjorteviltet (2,2%). E. faecalis eller E. faeciumi ble isolert fra 19 prøver, men ingen av disse var fra villrein. Alle stammene var resistente mot ett (84%) eller flere (16%) antibiotika. Til sammen 14 E. faecalis-stammer var resistente mot bare virginiamycin. Resultatene indikerer at hjortevilt ikke utgjør et smittereservoar av betydning verken for de humanpatogene bakteriene som inngikk i studien, eller for antibiotikaresistente bakterier.

ANTIBODIES TO RUMINANT ALPHA-HERPESVIRUSES AND PESTIVIRUSES IN NORWEGIAN CERVIDS

A serologic survey revealed that Norwegian populations of free-ranging reindeer (Rangifer tarandus tarandus), roe deer (Capreolus capreolus), red deer (Cervus elaphus), and moose (Alces alces) have been exposed to alpha-herpesviruses and pestiviruses. A total of 3,796 serum samples collected during the period 1993–2000 were tested in a neutralization test for antibodies against bovine herpesvirus 1 (BHV-1) or cervid herpesvirus 2 (CerHV-2), and 3,897 samples were tested by a neutralization test and/or enzyme-linked immunosorbent assay for antibodies against bovine viral diarrhea virus (BVDV). Antibodies against alpha-herpesvirus were found in 28.5% of reindeer, 3.0% of roe deer, and 0.5% of red deer, while all moose samples were negative. In reindeer, the prevalence of seropositive animals increased with age and was higher in males than females. Antibodies against BVDV were detected in 12.3% of roe deer, 4.2% of reindeer, 2.0% of moose and 1.1% of red deer. The results indicate that both alpha-herpesvirus and pestivirus are endemic in reindeer and pestivirus is endemic in roe deer in Norway. The viruses may be specific cervid strains. Seropositive red deer and moose may have become exposed as a result of contact with other ruminant species.

Screening of Feral Pigeon (Colomba livia), Mallard (Anas platyrhynchos) and Graylag Goose (Anser anser) Populations for Campylobacter spp., Salmonella spp., Avian Influenza Virus and Avian Paramyxovirus

A total of 119 fresh faecal samples were collected from graylag geese migrating northwards in April. Also, cloacal swabs were taken from 100 carcasses of graylag geese shot during the hunting season in August. In addition, samples were taken from 200 feral pigeons and five mallards. The cultivation of bacteria detected Campylobacter jejuni jejuni in six of the pigeons, and in one of the mallards. Salmonella diarizona 14:k:z53 was detected in one graylag goose, while all pigeons and mallards were negative for salmonellae. No avian paramyxovirus was found in any of the samples tested. One mallard, from an Oslo river, was influenza A virus positive, confirmed by RT-PCR and by inoculation of embryonated eggs. The isolate termed A/Duck/Norway/1/03 was found to be of H3N8 type based on sequence analyses of the hemagglutinin and neuraminidase segments, and serological tests. This is the first time an avian influenza virus has been isolated in Norway. The study demonstrates that the wild bird species examined may constitute a reservoir for important bird pathogens and zoonotic agents in Norway.SammendragScreening av byduer (Colomba livia), stokkender (Anas platyrhynchos) og grågås (Anser anser) for Campylobacter spp., Salmonella spp., aviært influenza virus og aviært paramyxovirus.Til sammen 119 ferske avføringsprøver ble samlet fra grågås på trekk nordover i april, og kloakksvabere ble tatt fra 100 skrotter av samme fugleart som ble skutt under jakta i august. I tillegg ble det tatt prøver av 200 byduer og fem stokkender. Ved bakteriedyrking ble Campylobacter jejuni jejuni funnet hos seks av duene og hos en av stokkendene. Salmonella diarizona 14 : k : z53 ble funnet hos ei grågås, mens alle duene og stokkendene var negative for salmonella. Aviært paramyxovirus ble ikke påvist i noen av de undersøkte prøvene. Ei stokkand fra ei elv i Oslo ble funnet positiv for influensa A virus med RT-PCR og ved innokkulering i embryonerte egg. Isolatet som benevnes A/Duck/Norway/1/03, ble funnet å være av H3N8 type, basert på sekvensanalyser av hemagglutinin og neuraminidase genene, og ved serologiske undersøkelser. Dette er første gang aviært influensavirus har blitt isolert i Norge. Studien viser at de ville fugleartene som ble undersøkt kan utgjøre et reservoar for viktige fuglepatogene og zoonotiske agens i Norge.

MALIGNANT CATARRHAL FEVER IN FREE-RANGING CERVIDS ASSOCIATED WITH OVHV-2 AND CPHV-2 DNA

Pathologic lesions were summarized in 18 free-ranging cervids (15 moose [Alces alces], two roe deer [Capreolus capreolus], and one red deer [Cervus elaphus[) diagnosed with malignant catarrhal fever (MCF) after examination at the National Veterinary Institute, Oslo 1982–2005. Eye lesions (conjunctivitis, corneal opacity, fibrin clots in the anterior eye chamber) were the most frequent gross finding. Erosive-ulcerative mucosal lesions in the nose and mouth were also commonly found. Histopathology revealed a nonpurulent vasculitis and perivasculitis in the central nervous system (CNS) typical of MCF in 16 of the cases. The diagnosis in the remaining two animals was based upon histologic eye lesions consistent with MCF (CNS not available for examination). Polymerase chain reaction was run on samples from 15 individuals for evidence of MCF-virus DNA, and ovine herpesvirus-2 (OvHV-2) DNA was detected in five moose, one roe deer, and one red deer, and caprine herpesvirus-2 (CpHV-2) DNA was detected in two moose and one roe deer. Sera from 1,000 free-ranging cervids were tested for specific antibodies to MCF-associated viruses (MCFV) by competitive inhibition enzyme-linked immunosorbent assay. The seroprevalences were: red deer 5%, reindeer (Rangifer tarandus) 4%, roe deer 2%, and moose 0.4% (n = 250 for all four species). The results indicate that sheep and goat MCFV may cause serious disease in wild moose, roe deer, and red deer. The seropositive cervids most likely represent individuals infected with either OvHV-2 or CpHV-2, but may also reflect infections with other related MCFV.

Vaccination of Atlantic salmon (Salmo salar L.) against cold-water vibriosis — duration of protection and effect on growth rate

Abstract Atlantic salmon which had been vaccinated against cold-water vibriosis were still protected up to 2 years after vaccination. Previous observations had revealed a mean relative percentage survival (RPS) 1 year after vaccination of 92. In outbreaks of the disease in two farms 1.5 to 2 years after vaccination, the RPS was 50 and 60, respectively, showing that the protection provided by the vaccine declined with time. When the experimental fish at six farms were slaughtered, it was found that vaccinated fish had a lower weight than unvaccinated. This might have been a side-effect of vaccination, though may also have been due to higher mortality among the smaller fish in the unvaccinated group. Results of the field trial showed that only half of the smolt which were transferred to sea-water reached market size 1.5 to 2 years later. The rest was lost due to diseases, accidents, or from unspecified of undetected causes.

SEROSURVEY FOR CANINE DISTEMPER VIRUS, CANINE ADENOVIRUS, LEPTOSPIRA INTERROGANS, AND TOXOPLASMA GONDII IN FREE-RANGING CANIDS IN SCANDINAVIA AND SVALBARD

Prevalence of antibodies reactive to canine distemper virus (CDV), canine adenovirus type 1 (CAV-1), Leptospira interrogans serovars Canicola and Icterohaemorrhagiae, and Toxoplasma gondii were examined in free-ranging Scandinavian canids. Sampling included 275 red foxes (Vulpes vulpes) from mainland Norway, 60 arctic foxes (Vulpes lagopus) from the high-arctic islands of Svalbard, and 98 wolves (Canis lupus) from the joint Swedish-Norwegian population. Methods used included virus neutralization tests for CDV and CAV-1, a microscopic agglutination test for L. interrogans, and a direct agglutination test for T. gondii. High prevalence of antibody to CAV-1 was identified in red foxes (59.6%), wolves (67.7%), and arctic foxes (37.8%). The prevalence of antibody to CDV varied between 9.6% and 12.3% in the three species. Antibodies to L. interrogans serovar Icterohaemorrhagiae were found in 9.9% of the red foxes and 8.4% of the wolves sampled, whereas no antibody-positive arctic foxes were found. All animals were antibody-negative for L. interrogans serovar Canicola. Antibodies to T. gondii were found in 66.9, 51.7, and 18.6% of red foxes, arctic foxes and wolves, respectively. Significantly more adults than juveniles were antibody-positive for CDV in red foxes and arctic foxes, for CAV-1 in wolves, and for T. gondii in red foxes and wolves. There was a general tendency for adult female red foxes to have a higher prevalence of antibodies for CDV than adult males; this difference was statistically significant. The results indicate that CDV and CAV-1 are endemic in red foxes and wolves on the Scandinavian mainland and in arctic foxes on Svalbard. Although infection with L. interrogans serovar Icterohaemorrhagiae was relatively common in wild canids on mainland Norway, it was not found on Svalbard, where the maintenance host (Rattus norvegicus) is absent. All three species are commonly exposed to T. gondii through predation on infected intermediate hosts.

A field trial of vaccination against cold-water vibriosis in Atlantic salmon (Salmo salar L.)

Abstract A vaccine against cold-water vibriosis caused by Vibrio salmonicida in Atlantic salmon was tested in 25 farms using natural challenge. In the experiment, each farm kept about 4000 vaccinated and 1000 unvaccinated fish together in the same net-pen. The vaccination procedure employed was direct immersion, twice, at least 5 weeks apart, at a water temperature above 8°C. Fifteen of the farms reported outbreaks of cold-water vibriosis in the experimental fish during the first year in sea-water. Mean mortality was 1.87% in vaccinated fish and 24.9% in unvaccinated fish, giving a relative percentage survival of 92, the vaccine therefore providing a high level of protection.