IS900 restriction fragment length polymorphism (RFLP) analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats and cattle in Norway.

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats.

Description of the infection status in a Norwegian cattle herd naturally infected by Mycobacterium avium subsp. paratuberculosis.

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-gamma (IFN-gamma) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-gamma immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test, 10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-gamma and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-gamma immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.

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.

Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway.

Milk samples from 340 individual goats in 34 dairy herds throughout Norway were examined for Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) by culture and immunomagnetic separation combined with PCR (IMS-PCR). The samples included three categories; (A) vaccinated dairy goats in herds with paratuberculosis; (B) vaccinated dairy goats in herds with no history of paratuberculosis; (C) unvaccinated goats in herds with no history of paratuberculosis. Viable M.a. paratuberculosis were not detected by culture in any sample, but 24 samples (7.1%) tested positive by IMS-PCR when the PCR products were visualised by dot blot hybridisation. PCR products from five milk samples originating from five different herds were sequenced; all showed 99% homology with the IS900 sequence from M.a. paratuberculosis.M.a. paratuberculosis were detected in all sampled categories. The percentage of IMS-PCR positive samples from herds where paratuberculosis had previously been reported was significantly lower than from herds where the infection had never been diagnosed (3.3 and 9.1%, respectively, P=0.048). Similar proportions of milk samples from vaccinated and non-vaccinated goats tested positive for the presence of M.a. paratuberculosis. Vaccinated goats older than 4 years tested positive more often than vaccinated animals less than 2 years old. Samples collected in May tested significantly more often positive than milk sampled during February-March (13.8 and 2.9%, respectively, P=0.001). This study showed that raw goats' milk in Norway might be contaminated with M.a. paratuberculosis.

Prevalence of Salmonella typhimurium infection in Norwegian hedgehog populations associated with two human disease outbreaks.

Faecal carriage of salmonella was investigated in 320 hedgehogs from Moss municipality in south-eastern Norway, Askøy, Bergen and Os municipalities in central-western Norway, and five municipalities in south-western and central Norway. The sampling in Moss was carried out 1 year after a human outbreak of salmonellosis, whereas the sampling in Askøy, Bergen and Os was carried out during a human outbreak. Both outbreaks were caused by Salmonella Typhimurium 4,5,12:i:1,2. No salmonella were detected in the hedgehogs from south-western (0/115) and central (0/24) Norway. Thirty-nine percent (39/99) of the animals sampled on Jeløy, and 41% (34/82) of those from Askøy, Bergen and Os, carried S. Typhimurium 4,5,12:i:1,2. The PFGE profile of isolates from hedgehogs and human beings were identical within each of the two outbreak areas. A significantly higher carrier rate of S. Typhimurium occurred among hedgehogs sampled at feeding places, compared to those caught elsewhere. The salmonella-infected hedgehog populations most likely constituted the primary source of infection during both of the human disease outbreaks, and the Norwegian hedgehog is suggested as a reservoir host of S. Typhimurium 4,5,12:i:1,2.

A cross-sectional study of bovine tuberculosis in dairy farms in Asmara, Eritrea.

Bovine tuberculosis in dairy cattle in Asmara, Eritrea, was studied using a cross-sectional study to describe its prevalence and to identify factors associated with it. A total of 72 randomly selected herds were included in the study. The comparative intradermal tuberculin test was used for the diagnosis. Of 1813 individual animals tested, 14.5% were reactors. Thirty herds (41.7%) had at least one reactor but, by defining a reactor herd as any herd with two or more reactors, only 19 (26.4%) herds were classified as reactor herds. Based upon individual animal specificity of 98.5%, the calculated herd specificity was > 99%. A multiple logistic model showed that the presence of exotic breeds was associated with a high risk (odds ratio = 5.70; 95% confidence interval 1.13-28.8). An increased risk was also linked to large herds. Keeping the animals always indoors reduced the risk, but could not be fitted to the model owing to empty cells.

Subclinical paratuberculosis in goats following experimental infection. An immunological and microbiological study.

An experimental oral infection of goats with a caprine isolate of Mycobacterium a. subsp. paratuberculosis was used to investigate immunological and bacteriological events during the subclinical phase of infection. Seven goats at 5-8 weeks of age were given a bacterial suspension in milk-replacement three times weekly for 9 weeks. Six animals were kept as controls. Cellular recall responses against M. a. paratuberculosis were analysed by means of a lymphocyte proliferation test, an IFN-gamma assay and an IL-2 receptor assay. All inoculated animals had detectable CMI responses from 9 weeks post-inoculation and through the 2 years of study, although the responses were highest during the first year. Antibodies against M. a. paratuberculosis could be detected from weeks 15-20 in four of the seven animals, and one additional animal became antibody positive at week 35, while two inoculated animals did not produce significant antibody titres during the experiment. At about 1-year post-inoculation, two animals became faecal shedders, while two others started to excrete bacteria into faeces about 2 years post-inoculation. The appearance of M. a. paratuberculosis in faeces was not associated with a decline in cellular responses as far as could be assessed using the current methods for measuring CMI. Pathological lesions due to M. a. paratuberculosis infection and presence of bacteria were recorded in the intestine and/or mesenteric lymph nodes of five animals while lymph node changes suggestive of paratuberculosis were observed in one animal. Only the two animals with no signs of an active infection at necropsy showed a considerable decline in the cellular parameters during the last year of the study, particularly in the IFN-gamma assay. The two animals with the highest levels of M. a. paratuberculosis responsive CD8+ lymphocytes in the circulation about 1-year post-inoculation had no detectable lesions in the distal ileum and colon at necropsy, while high numbers of gammadelta T-cells responsive to M. a. paratuberculosis in the circulation were associated with disseminated lesions in the distal ileum and colon.

Prevalence of antibodies to Brucella spp. in cattle, sheep, goats, horses and camels in the State of Eritrea; influence of husbandry systems.

Samples from 2427 cattle, 661 goats, 104 sheep, 98 camels and 82 horses were screened for brucella infections by the Rose Bengal Test and positive reactors confirmed by the complement fixation test. In cattle, the highest individual seroprevalence was in dairy herds kept under the intensive husbandry system, with an individual prevalence of 8.2% and unit (herd) seroprevalence of 35.9%. This was followed by the pastoral husbandry system in the Western Lowlands with 5.0% individual but a higher unit (vaccination site) prevalence of 46.1%. The lowest was in the mixed crop-livestock system in the Southern Highlands with individual 0.3% and unit (village) prevalence of 2.4%. In sheep and goats, no positive animals were detected in the mixed crop-livestock areas. In the Eastern Lowlands individual prevalences of 3.8% (goats) and 1.4% (sheep) and unit prevalence of 33.3% (goats) and 16.7% were found, while 14.3% of individual goats and 56.3% of the units in the Western Lowlands were positive. No positive horses were found. The present study documents the first serological evidence of Brucella spp. infection in camels (3.1%) in Eritrea.

Risk factors for Brucella spp. infection In dairy cattle farms in Asmara, State of Eritrea.

A cross-sectional study was conducted to identify risk factors for herd infection by Brucella spp. in dairy cattle in the suburbs of Asmara, Eritrea. Data were collected from 64 herds, randomly selected from a total of 99 herds with a minimum herd size of 9 cows. A questionnaire was used to gather data on management, hygiene and herd structure. Serum samples collected from all pregnant heifers, cows and bulls, were screened for Brucella infection by the Rose Bengal test (RBT), and all RBT-positive sera re-tested with the complement-fixation test (CFT) for confirmation. A seropositive herd was defined as one in which at least one animal tested positive in the CFT. There were 23 (36%) positive herds among the 64 studied. Both multiple logistic and multiple betabinomial regression modeling were used to analyze the data. Mixed-breed herds, compared to single (exotic)-breed herds, were found to be independently associated with increased herd seroprevalence (OR=5.2, 95% confidence interval 1. 4-18.7) in the multiple logistic model with the herd infection status as the dependent variable. The importance of this variable was supported by the multiple betabinomial regression model (OR=3.3, 1.4-7.6) with animal-level prevalence within herd as the outcome variable. Both models also revealed the presence of a negative association between seropositivity and cattle stocking density.

Grouping by plasmid profiles of atypical Aeromonas salmonicida isolated from fish, with special reference to salmonid fish.

Plasmid profile analyses were performed for 113 strains of atypical Aeromonas salmonicida and the reference strain A. salmonicida subsp. salmonicida ATCC 14174. The atypical A. salmonicida strains comprised 98 strains obtained from fish originating from 54 farms and 2 lakes in Norway, 10 strains from Canada (2), Denmark (2), Finland (1), Iceland (1) and Sweden (4), the reference strains NCMB 1109 and ATCC 15711 (Haemophilus piscium) of A. salmonicida subsp. achromogenes, and the type cultures A. salmonicida subsp. achromogenes NCMB 1110, A. salmonicida subsp. masoucida ATCC 27013 and A. salmonicida subsp. smithia CCM 4103. A total of 95 strains of atypical A. salmonicida were separated into 7 groups (I to VII) based on the plasmid profiles. Eighteen strains of atypical A. salmonicida had no common plasmid profile. The type strain NCMB 1110 and the reference strain NCMB 1109 were included in group IV, and the type strain ATCC 27013 in group V, but the other reference and type strains had plasmid profiles different from all the other strains. An epidemiological link was documented between strains collected from different farms/localities in each of groups I, III, V and VII. Physiological and biochemical characterizations were performed for 93 of the strains to investigate phenotypic differences between the plasmid groups. Group VII strains and 3 strains with no common plasmid profile differed from the other groups in being catalase-negative. Differences in phenotypic characteristics were shown between the plasmid groups. However, significant variations in reactions for several phenotypic characteristics also occurred within each of the groups I to VII. The present study indicates that plasmid profiling may give useful epidemiological information during outbreaks of atypical A. salmonicida infections in fish. Additional comprehensive phenotypic characterisation is of limited value since the phenotypic characteristics in each plasmid group are not uniform.

Phenotypic and genotypic characterization of Vibrio viscosus sp. nov. and Vibrio wodanis sp. nov. isolated from Atlantic salmon (Salmo salar) with 'winter ulcer'.

Two groups of Vibrio strains isolated from Atlantic salmon with 'winter ulcer' were characterized phenotypically and genotypically. The data obtained indicated that each of the two groups represented a new species in the genus Vibrio. The names Vibrio viscosus sp. nov. [type strain NVI 88/478T (= NCIMB 13584T)] and Vibrio wodanis sp. nov. [type strain NVI 88/441T (= NCIMB 13582T)] are proposed for the new species. V. viscosus strains exhibited a similar total DNA RFLP pattern and a similar plasmid DNA profile. DNA relatedness (hydroxyapatite method) of the V. viscosus type strain to nine other V. viscosus strains was 81-93% at 60 degrees C. Divergence within related sequences was 0.0-1.5% and relatedness at 75 degrees C was 74-100%. V. wodanis strains exhibited marked heterogeneity on the basis of RFLP analysis and plasmid profiles. DNA relatedness of the V. wodanis type strain to 10 other V. wodanis strains was 66-94% at 60 degrees C. Divergence within related sequences was 0.0-1.5% and relatedness at 75 degrees C was 55-97%. Relatedness between V. viscosus and V. wodanis type strains was approximately 20%. Among other Vibrio species, the closest relative of V. viscosus was Vibrio marinus (ATCC 15381T) (43% relatedness at 60 degrees C) and that of V. wodanis was Vibrio logei (ATCC 15382) (57% relatedness at 60 degrees C). These same pairs were the closest phenotypic relatives. DNA sequence analysis of the 16S rRNA gene of V. viscosus indicated an intimate relationship to V. marinus. A total evaluation of the results, however, supports V. viscosus to be a separate species in the genus Vibrio. The analysis of the sequence of the 16S rRNA gene of V. wodanis supports that V. logei (ATCC 15382) was the most related species. Ability to degrade casein, oxidative production of acid from trehalose and production of lysine decarboxylase are important biochemical tests that will differentiate between V. viscosus, V. wodanis, V. marinus (ATCC 15381T) and V. logei (ATCC 15382).

Alkyl hydroperoxide reductases C and D are major antigens constitutively expressed by Mycobacterium avium subsp. paratuberculosis.

Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp. paratuberculosis had strong gamma interferon (IFN-gamma) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-gamma production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences.

Control of caprine arthritis-encephalitis virus and Corynebacterium pseudotuberculosis infection in a Norwegian goat herd.

A control programme for caprine arthritis-encephalitis virus (CAEV) and Corynebacterium pseudotuberculosis (C. pseudotuberculosis) infection was established in a Norwegian goat herd comprising approximately 100 milking goats. The herd seroprevalences of antibodies against CAEV and C. pseudotuberculosis were 97% and 94%, respectively. Kids were removed from the infected flock at birth, avoiding any contact between dam and kid. The kids were kept completely segregated from the seropositive flock and fed cow's colostrum and milk. A seronegative flock was established, based on the removed kids and their offspring. Goasts belonging to the seronegative flock were allowed to kid naturally and to mother their kids. The seropositive flock was slaughtered during the second year of the control programme. After washing and disinfection, housing systems and nearby outdoor premises were left empty for 3 months. Of 230 goats examined for antibodies against CAEV with ELISA regularly during 3 years of the control program, altogether 6 were found to be seropositive, while for 10 the result was indeterminate. All 16 animals were immediately culled. During the third year of the control programme, all goats were examined and proved negative for antibodies against C. pseudotuberculosis by a haemolysis inhibition test. Clinical examination revealed no signs of CAE or caseous lymphadenitis.

Corynebacterium pseudotuberculosis infection in goats. VIII. The effect of vaccination against experimental infection.

The effect of an inactivated vaccine against C. pseudotuberculosis infection was tested on castrated male kids from a herd free from caseous lymphadenitis. The animals were divided into 3 groups with 8 animals in each. Group 1 was immunized with crude filtrated C. pseudotuberculosis toxoid and whole killed organisms, while Group 2 in addition was given levamisole. The kids were vaccinated twice at an interval of 4 weeks. Group 3 consisted of unvaccinated animals. All groups were challenged subcutaneously with live bacteria 4 weeks after the last vaccination. Unvaccinated animals showed the most severe course of illness after challenge. Development of abscesses in the regional lymph nodes (Inn. subiliaci) was significantly more common in unvaccinated than in vaccinated kids at necropsy 2 months after challenge. There was, however, no such difference between the vaccinated groups, and there was no difference between any of the groups as regards abscess formation at the inoculation site. In each of the 2 vaccinated groups, there was a titre rise following vaccination in the hemolysis inhibition test, whereas no such rise was seen in the bacterial agglutination test. The titre values in both tests increased significantly after challenge in all the groups, the increase being most rapid in the vaccinated animals. The present investigation indicates that development of caseous lesions in lymph nodes in goats, following subcutaneous inoculation with C. pseudotuberculosis, can be reduced by an inactivated vaccine containing whole organisms and crude toxin.

Corynebacterium pseudotuberculosis infection in goats. IX. The effect of vaccination against natural infection.

A vaccination trial was carried out in 10 infected herds. The trial included 247 female kids, the number of animals in each herd varying from 15 to 38. About half of the animals in each herd were vaccinated twice at 3 to 4 week intervals, the first vaccination being carried out before the age of 4 months. A combination of a crude filtrate of C. pseudotuberculosis toxoid with whole organisms, was used. Overall, the prevalence of animals with superficial swellings was higher in the unvaccinated than in the vaccinated group during the first 1-2 years following immunization. However, in some herds superficial swellings were as common in vaccinated as in unvaccinated animals. An antibody response following vaccination was demonstrated in the hemolysis inhibition test, but not in the bacterial agglutination test. Superficial swellings were more common in vaccinated animals which were negative than in animals which were positive in the hemolysis inhibition test at 1 1/2 months after vaccination. The vaccine used in the present study, was not sufficiently efficacious to be recommended as the only protective measure against caseous lymphadenitis in Norwegian goat herds.