The persistence of Salmonella following desiccation under feed processing environmental conditions: a subject of relevance.

UNLABELLED: Although Salmonella persistence has been predominantly linked to biofilm formation, the physiological state of Salmonella should also be considered as a possible pathway for persistence and survival in the feed industry. Hence, the purpose of this study was to assess the extent of viability of Salmonella cells through long-term desiccation periods under conditions typically found in feed processing environments, and whether these same cells could resuscitate and cause salmonellosis in vivo. We showed that upon desiccation, Salmonella Agona, a representative feed industry isolate and Salmonella Typhimurium ATCC 14028, a laboratory strain, were induced into a nonculturable state at 35 and 85% relative humidity conditions, at defined temperatures of 30 and 12°C, respectively. Although the reduction in culturable cells was more than 6 log10 , metabolic activity was found in more than 1% of the population. Desiccation-induced nonculturable Salm. Typhimurium could not be revived and were nonvirulent in a mouse model following infection through oral gavage. These results suggest that the specific conditions for reviving nonculturable Salmonella after long periods of desiccation are yet to be fully identified. The need for mapping key factors involved in the persistence of Salmonella would help better detect it and improve feed safety measures. SIGNIFICANCE AND IMPACT OF THE STUDY: While Salmonella has been shown to persist for years in feed processing environments, it is still unknown how temperature and humidity affect the persistence of Salmonella cells over time in terms of their metabolic states and cultivability. Here, we show that long-term exposure to feed processing environmental conditions induces Salmonella into a nonculturable state even though about 1% of the population remains metabolically active. This has significant implications when monitoring Salmonella from the environment which could yield false-negative results using conventional pre-enrichment detection methods.

Synthetic brominated furanone F202 prevents biofilm formation by potentially human pathogenic Escherichia coli O103:H2 and Salmonella ser. Agona on abiotic surfaces.

AIMS: Investigate the use of a synthetic brominated furanone (F202) against the establishment of biofilm by Salmonella ser. Agona and E. coli O103:H2 under temperature conditions relevant for the food and feed industry as well as under temperature conditions optimum for growth. METHODS AND RESULTS: Effect of F202 on biofilm formation by Salmonella ser. Agona and E. coli O103:H2 was evaluated using a microtiter plate assay and confocal microscopy. Effect of F202 on bacterial motility was investigated using swimming and swarming assays. Influence on flagellar synthesis by F202 was examined by flagellar staining. Results showed that F202 inhibited biofilm formation without being bactericidal. F202 was found to affect both swimming and swarming motility without, however, affecting the expression of flagella. CONCLUSIONS: F202 showed its potential as a biofilm inhibitor of Salmonella ser. Agona and E. coli O103:H2 under temperature conditions relevant for the feed and food industry as well as temperatures optimum for growth. One potential mode of action of F202 was found to be by targeting flagellar function. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study gives valuable new knowledge to the potential use of furanones as a tool in biofilm management in the food and feed industry.

Biofilm as an environment for dissemination of stx genes by transduction.

Dissemination of Shiga toxin (Stx)-encoding bacteriophages is the most likely mechanism for the spread of Stx-encoding genes and the emergence of new Stx-producing Escherichia coli (STEC). Biofilm has been reported to be a place where horizontal gene transfer by plasmid conjugation and DNA transformation may occur, and in this study, horizontal gene transfer by transduction has been demonstrated. Transfer of Stx-encoding bacteriophages to potentially pathogenic E. coli in biofilm was observed at both 20°C and 37°C. The infection rates were higher at 37°C than at 20°C. To our knowledge, this study is the first to show lateral gene transfer in biofilm mediated by a temperate bacteriophage. The study shows that the biofilm environment can be suitable for transduction events and can thereby be an environment for the emergence of new pathogenic E. coli.

A synthetic furanone potentiates the effect of disinfectants on Salmonella in biofilm.

AIMS: To study a possible effect of a synthetic brominated furanone on biofilm formation and biofilm resistance to disinfectants in Salmonella enterica. METHODS AND RESULTS: The effect of a synthetic furanone on biofilm formation of Salm. enterica serovar Agona and Salm. enterica serovar Typhimurium (11 strains of different origins) was evaluated in a microtiterplate assay. A significant reduction in biofilm build-up in microtiterplates by the furanone was observed for seven of the strains tested. Biofilms by two Salm. Agona feed factory strains and the effects on survival after exposures to disinfectants (hypochlorite and benzalkonium chloride) were assessed for both strains. Pretreatment with furanone significantly potentiated the effect of the two disinfectants for both strains. CONCLUSIONS: The effect of disinfectants on Salmonella in biofilm was significantly enhanced when the biofilm was grown in the presence of furanone. This was probably because of an effect on biofilm architecture, composition and in some cases also biofilm build-up. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study gives valuable new knowledge in the fight against Salmonella biofilm in the environment because of the potentiated effect of conventional disinfectants.

Evaluation of efficacy of disinfectants against Salmonella from the feed industry.

AIMS: To evaluate disinfectants against Salmonella under conditions relevant for the feed industry. MATERIALS AND RESULTS: A survey on the use of disinfectants in feed industry showed that a range of different types was used. Nine disinfectants, reflecting the most commonly used active ingredients, were tested for bactericidal activity on Salmonella isolated from the feed industry. All disinfectants were efficient against Salmonella in suspension. The bactericidal effect varied considerably between different types of active compounds on bacteria dried on surfaces or grown as biofilm. Tenside-based disinfectants and hypochlorite were found to have low bactericidal activity and the efficiency was significantly reduced when the ratio of amount disinfectant per cell decreased. It was shown that concentrations of 70-80% ethanol were effective against Salmonella. Among the disinfectants tested a product containing 70% ethanol was most efficient followed by Virkon S. CONCLUSIONS: Many disinfectants had low bactericidal activity against Salmonella at surfaces while Virkon S and a product containing 70% ethanol were most effective. Another advantage of ethanol-based disinfectants is evaporation of ethanol, resulting in low residual water after use. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of the disinfectants found to be efficient against surface associated Salmonella, may assist the industry in combating Salmonella.

Persistence of orally administered Salmonella enterica serovars Agona and Montevideo in Atlantic salmon (Salmo salar L.).

The objective of our experiments was to study the persistence and dissemination of orally administered Salmonella in smoltified Atlantic salmon. In experiment 1, salmon kept at 15 degrees C were fed for 1 week with feed contaminated with 96 most-probable-number units of Salmonella Agona per 100 g of feed and then starved for 2 weeks. Samples were taken from the gastrointestinal tract and examined for Salmonella 1, 2, 8, 9, 15, and 16 days after the feeding ended. In experiment 2, Salmonella Agona and Montevideo were separately mixed with feed and administered by gastric intubation. Each fish received 1.0 x 10(8), 1.0 x 10(6), or 1.0 x 10(4) CFU. The different groups were kept in parallel at 5 and 15 degrees C and observed for 4 weeks. Every week, three fish in each group were sacrificed, and samples were taken from the skin, the pooled internal organs, the muscle, and the gastrointestinal tract and examined for the presence of Salmonella. The results from the two experiments showed that the persistence of Salmonella in the fish was highly dependent on the dose administered. Salmonella was not recovered from any of the fish that were fed for 1 week with the lowest concentration of Salmonella. In the fish given the highest dose of Salmonella, bacteria persisted for at least 4 weeks in the gastrointestinal tract as well as, to some extent, the internal organs. The present study shows that under practical conditions in Norway, the risk of Salmonella in fish feed being passed on to the consumer of the fish is negligible.

Molecular epidemiology of Salmonella spp. isolates from gulls, fish-meal factories, feed factories, animals and humans in Norway based on pulsed-field gel electrophoresis.

The molecular epidemiology of 98 isolates of Salmonella serovar Agona (n = 27), S. Montevideo (n = 42) and S. Senftenberg (n = 29) from wild-living gulls, fish-meal factories, feed factories, humans and domestic animals was investigated using pulsed-field gel electrophoresis (PFGE) and computerized numerical analysis. Two of the S. Agona profiles were identified both in gulls and in two of the factories. In addition, one of these profiles was detected in two infected poultry farms. Two of the S. Montevideo profiles were also identified both in gulls and in two of the factories, and one of these profiles was observed in a human isolate. Four factories shared an identical S. Senftenberg profile. The S. Senftenberg profile found in gulls was not identified in any other source investigated. The presence of isolates with identical PFGE profiles indicates potential epidemiological links between different factories, as well as between gulls and factories.

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.

A field trial with an experimental vaccine against Staphylococcus aureus mastitis in cattle. 1. Clinical parameters.

A total of 108 heifers were included in a placebo-controlled multicenter study on the use of an experimental Staphylococcus aureus mastitis vaccine containing whole, inactivated bacteria with pseudocapsule, alpha and beta toxoids, and a mineral oil as adjuvant. The heifers were injected in the area of the supramammary lymph nodes twice before calving and were observed and sampled throughout the first lactation. None of the vaccinated cows suffered from clinical Staph. aureus mastitis, and only 8.6% suffered from subclinical Staph. aureus mastitis, but a total of 16.0% of the control cows suffered from clinical or subclinical Staph. aureus mastitis. Mean SCC in vaccinated and control cows were the same throughout the lactation. Local swellings at the injection site were palpable in a substantial proportion of the vaccinated cows. In the statistical analyses, when cow was used as the unit of concern, no significant differences occurred between groups. However, when all parameters on udder health were considered together, the results indicated a potential protective effect of this vaccine during the entire lactation.

A field trial with an experimental vaccine against Staphylococcus aureus mastitis in cattle. 2. Antibody response.

A Staphylococcus aureus vaccine containing whole, inactivated bacteria with pseudocapsule and alpha and beta toxoids with a mineral oil as adjuvant, was used in a field trial. Heifers were injected in the area of the supramammary lymph nodes with vaccine or placebo twice before calving and observed and sampled throughout their first lactation. Antibody response toward the pseudocapsule and the alpha toxin was significant in serum from the vaccinated cows. These antibody concentrations were significantly higher in serum and milk during the entire lactation compared with that of the controls. The antibody response to the beta toxin was moderate in serum from vaccinated cows; no differences in antibody concentrations in milk were significant between groups. The antibody response to the pseudocapsule consisted of the IgG1 and IgG2 isotypes, but, in milk, only the concentration of IgG1 was significantly increased in the vaccinated cows during the lactation compared with the control cows.

Relations between the goat MHC (GLA) and antibody response to diphtheria toxoid, human serum albumin and tetanus toxoid, using a twin model.

SUMMARY: Norwegian dairy goats were tested for possible associations between the goat lymphocyte antigen system (GLA) and antibody response to the antigens diphtheria toxoid, human-serum albumin and tetanus toxoid. The serum titres to these antigens following immunization, and their GLA-specificities, were determined in 96 twin pairs. The antibody response and GLA specificity were compared between twins: twins sharing one or more GLA specificities showed a more similar primary-antibody response to diphtheria toxoid than twins with no common specificity. The main point in comparing twins is that the MHC specificity then serves as a marker for the complete haplotype, including MHC class-II genes. However, a gene substitution model that only tests the effect of the observed class-I alleles revealed that animals with GLA specificity Eu2 had a significantly higher anti-diphtheria response than Eu2-negative animals. ZUSAMMENFASSUNG: Untersuchungen über Beziehungen zwischen Ziegen MHC (GLA) und Antikörperreaktion auf Diptherietoxin, humanes Serum Albumin und Tetanustoxin mit Zwillingsmodell Norwegische Milchziegen wurden in Hinblick auf mögliche Assoziationen zwischen Ziegen Lymphozytenantigensystem (GLA) und Antikörperreaktion auf Antigene Diptherie Toxin, humanes Serum Albumin una Tetanustoxin untersucht. Die Serumtiter nach Immunisation und die GLA-Spezifität wurden an 96 Zwillingspaaren erhoben und zwischen Zwillingen verglichen; Zwillinge mit ein oder mehr GLA Spezifitäten zeigten ähnlichere Primärantikörperreaktion auf Diptherie Toxin als solche ohne gemeinsame Spezifität. Der wesentliche Vorteil der Zwillinge ist, daß MHC-Spezifität als Marker für den gesamten Haplotyp dienen kann, einschließlich MHC Klasse-II gene. Allerdings, ein Gen-Substitutionsmodell, das nur die Wirkungen der beobachteten Klasse-I Allele prüft, zeigte, daß Tiere mit GLA-Spezifität EU2 signifikant höhere Antidiptheriespiegel als EU2 negative Tiere hatten.

No evidence of vertical transmission of naturally acquired feline immunodeficiency virus infection.

A naturally occurring feline immunodeficiency virus (FIV) infection in a closed breeding colony of cats, was studied for a period of 9 months. The colony consisted of 25 adult cats, of which six proved to be infected with FIV as judged by serological examination and virus isolation. In all, 48 kittens were monitored for levels of antibodies against FIV during their first 6 months of life. All the kittens (n = 30) born of FIV-infected queens showed maternal antibodies against FIV, although these declined to undetectable levels by the age of 5 months. Antibodies against FIV were not shown in any of 18 kittens born of FIV-negative queens. An attempt to isolate the virus from 12 kittens between 2 and 6 weeks of age did not succeed. None of the cats in the colony seroconverted during the observation period. In conclusion, neither vertical nor horizontal transmission of FIV infection were demonstrated in the colony during the 9-month investigation period.

Seroepidemiological studies of Borrelia burgdorferi infection in sheep in Norway.

An enzyme-linked immunosorbent assay for antibodies to Borrelia burgdorferi in sheep was established. The assay was used in a seroepidemiological survey in which serum samples from 327 Norwegian rams were screened for antibodies to B. burgdorferi. These rams were randomly chosen, clinically healthy animals from all parts of Norway. They came both from areas where Ixodes ricinus is common and from areas where the tick has not been found. The age of the animals varied from 0.5 to 7.5 years. Sera from 10% of the animals tested were seropositive by the enzyme-linked immunosorbent assay, with the percentage of positive animals varying from 0 to 20% between counties. The distribution of seropositive animals was generally in good agreement with the known distribution of I. ricinus, with the highest proportion of seropositive animals being in southern coastal areas of Norway. There were some exceptions, however, because seropositive animals were also found in areas where the tick has not been recorded. The majority of animals appeared to become infected during the first 2 years of life, with 12% of animals that were 1.5 years old being seropositive. The animals were all healthy at the time of serum sampling, and the clinical significance of B. burgdorferi in sheep is still uncertain.

Selection for immune response in goats: the antibody response to diphtheria toxoid after 12 years of selection.

A herd of Norwegian dairy goats was subject to selection for high and low antibody response to diphtheria toxoid for 12 yr, or approximately 5.5 generations because sires were used for one mating season, whereas dams were used several years. The herd comprised approximately 100 milking goats. Only sires were tested and selected, five to seven sires were used in each line each year, and the percentage of male kids used for breeding varied between 15 and 50%. The mean phenotypic values of the lines diverged until the 4th yr, but the lines did not diverge any further. The means of both lines decreased during the experimental period. The restricted maximum likelihood (REML) estimate of the heritability of the trait in the base population was .19, whereas the realized heritability approached zero in the last six cycles of selection. Using the REML-estimate of heritability in an individual animal model, the mean breeding values (BLUP) of the lines were significantly different. The difference in mean BLUP values between the lines increased throughout the study. Yet, this increase was only 40% from the 2nd to the 12th yr. No effect of a major gene was observed in a test based on individual BLUP.

Economic analysis of vaccination applied to ovine listeriosis.

An economic model for determining whether vaccination against a disease would be beneficial financially on individual farms is proposed; it is based on four pieces of information: the costs of a disease case including its treatment, the cost of vaccinating each animal including veterinary fees, the expected incidence of the disease, and the efficacy of the vaccine. The model was applied to ovine listeriosis, which is a serious disease problem in Norway. Vaccination appeared to be beneficial for the average sheep flock of 100 ewes which might expect two or more cases of listeriosis per year. Furthermore, the model suggests the ratio of the price of a single vaccination to the cost of a disease case can be used to plan more efficient vaccine field trials.

Immune response after immunization with an experimental Toxoplasma gondii ISCOM vaccine.

Mice were immunized with ISCOM preparations of tachyzoites from two different strains of Toxoplasma gondii. The antibody response and the cellular response, as measured in vitro, were comparable with those found in chronically infected mice. When challenged with virulent T. gondii tachyzoites, all the immunized mice died, whereas all the chronically infected mice survived. However, the immunized mice generally survived longer than non-immunized animals.

Differential expression of I alpha- and II alpha-globin genes in Norwegian dairy goats.

Fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis were used to determine the ratio between I alpha- and II alpha-globin chains in Norwegian dairy goats. Three different phenotypes, designated normal (N) with I alpha- to II alpha-globin ratio 3:1, reversed (R) with ratio 1:2 and double-reversed (RR) with no I alpha-globin, were described. Family studies indicated that the R animals were heterozygous, and the RR animal homozygous, for a haplotype without a functional I alpha-globin product. Southern blot analysis of goat DNAs digested with six different restriction enzymes showed that the different ratios of alpha chain expression could not be due to a deletion of the I alpha-gene and/or duplication or triplication of the II alpha-globin genes. The homozygous reversed animal with no detectable I alpha-globin had a mild anaemia.

Linkage studies on erythrocyte antigen loci, the major histocompatibility complex and the haemoglobin beta gene cluster in goats.

Linkage studies on seven erythrocyte antigen loci, the major histocompatibility complex (MHC) and the haemoglobin beta gene cluster (HBB) were performed in 48 Norwegian goat families. Close linkage was excluded between the erythrocyte antigen B-system and the MHC, between the erythrocyte antigen N7 locus and HBB, and between the MHC and the HBB.

Erythrocyte antigens in Norwegian goats: serological and genetic studies.

Goat alloantisera and bovine blood typing reagents were used to characterize eight erythrocyte antigen specificities in Norwegian goats by cluster analysis, absorption and family studies. Most of the goat sera were produced by injecting dams once or twice with blood cells or blood from their own kids. The characterized specificities were designated N1-N8. The two specificities N5 and N8 were recognized both by goat alloantisera and by reagents against the bovine factors E'1 and E'2 (N5) and I (N8), which are allelic factors in the bovine B-system. In goat families, the two specificities also behaved as alleles. Consequently, the locus or gene system coding for these specificities was called the B-system of goats. The six other erythrocyte antigens were provisionally assigned to six separate loci. In addition, a bovine anti-sheep R factor reagent reacted with cells from 3.3% of the goats tested, whereas a monoclonal antibody against the Forssman antigen reacted with all the goats tested.

Immunisation against infections caused by Listeria monocytogenes in sheep.

Immunisation against listeriosis in sheep using a live, attenuated vaccine was introduced in Norway in 1984. Since then 65,000 to 80,000 animals have been vaccinated annually. Information obtained by a questionnaire showed that the incidence of listeriosis decreased from approximately 4.0 per cent before the introduction of the vaccine to 1.5 per cent after vaccination started. The incidence of abortions was 0.7 per cent in vaccinated flocks compared to 1.1 per cent in unvaccinated flocks. There were a few adverse reactions in the vaccinated sheep.