Temperature-related risk factors associated with the colonization of broiler-chicken flocks with Campylobacter spp. in Iceland, 2001-2004.

Our objective was to identify temperature-related risk factors associated with the colonization of broiler-chicken flocks with Campylobacter spp. in Iceland, with an underlying assumption that at minimum ambient temperatures, flies (Musca domestica) play a role in the epidemiology and seasonality of Campylobacter. At slaughter, pooled caecal samples were obtained from 792 flocks that hatched between March 15 and September 15 each year from 2001 to 2004, and cultured for Campylobacter. Daily temperature data (average, maximum and minimum temperatures) were obtained from the Icelandic Meteorological Office. We determined the closest weather station to each farm using ArcGIS 9. We then used logistic-regression models (with a random-effects term for farm) to examine associations between flock positivity and temperature-related predictors. The overall prevalence of Campylobacter was 27.4%, and the month with the highest prevalence (54.4%) was August. The final model included cumulative degree-days (CDD) above an average temperature of 4.4 degrees C and the presence of 1 or more days below a maximum temperature of 8.9 degrees C (threshold below which fly activity was expected to be substantially reduced and egg laying was not expected to occur) during the period 2-4 weeks (i.e. days 8-28) before slaughter. Below 79 CDD, the risk of flock Campylobacter colonization was generally low. Between 79 and 139 CDD, the risk increased gradually, but was lower for flocks raised during periods with 1 or more days <8.9 degrees C than flocks raised without days <8.9 degrees C. The risk increased sharply under conditions of high CDD (>139) and when the maximum temperature remained >8.9 degrees C during the period 2-4 weeks before slaughter.

Laryngeal Chondritis in Sheep in Iceland.

Laryngeal chondritis is a chronic disease in sheep with low morbidity, high mortality and unresolved pathogenesis. The disease has been recognized recently in Iceland and affects both ewes and rams. Animals of different ages are affected, but lambs and yearlings predominate. The disease is seen in housed animals and most cases occur during the late winter months. We report the gross and microscopical findings in 45 cases of laryngeal chondritis in Icelandic sheep.

Expression and purification of Clostridium perfringens beta-toxin glutathione S-transferase fusion protein.

The beta-toxin gene from Clostridium perfringens type C was cloned and expressed as a glutathione S-transferase fusion protein in Escherichia coli. The DNA sequence was determined and compared to the type B sequence. Two nucleotide differences were found in the protein coding sequence, resulting in one amino acid difference between the two proteins. The purified beta-toxin fusion protein is not toxic in mice, but rabbit antiserum raised against it neutralises the toxic effect of C. perfringens type C culture filtrate in mice.

Site-directed mutagenesis of Clostridium perfringens beta-toxin: expression of wild-type and mutant toxins in Bacillus subtilis.

Recombinant beta-toxin has been expressed and secreted from Bacillus subtilis. Biological activity was tested in vivo and in vitro. The lethal dose in mice was determined. Hemolysis of rabbit and sheep erythrocytes was tested but no effect was observed. Seven mutant proteins were produced. Targets for mutagenesis were mostly selected on the basis of the similarity between beta-toxin and alpha-toxin from Staphylococcus aureus, a pore-forming toxin. Mutations of two amino acids affected the lethal dose in mice. Both residues have counterparts in the membrane binding region of alpha-toxin. Alteration of the single cysteine residue did not affect protein function, contrary to previous suggestions.

Paratuberculosis in Iceland: epidemiology and control measures, past and present.

Paratuberculosis as well as the slow virus infections maedi/visna and jaagsiekte came to Iceland in 1933 when 20 sheep of the Karakul breed were imported from Halle, Germany. At least five of these sheep were subclinical carriers of paratuberculosis. Within 16 years paratuberculosis together with the other Karakul diseases (maedi/visna and jaagsiekte) almost ruined sheep farming, the main agricultural industry in Iceland. The first clinical case of paratuberculosis in sheep was confirmed in 1938, and in cattle in 1944. The first cattle cases of paratuberculosis appeared on farms where the disease had been prevalent in sheep for years. The virulence in cattle appeared to be considerably lower than in sheep. Extensive measures were used to control the spread of paratuberculosis in sheep. Hundreds of kilometres of fences were put up and used together with natural geographic borders to restrict the movement of sheep from infected areas. Serological and other immunological tests were also used to detect and dispose of infected individuals. These measures proved inadequate and the disease could not be eradicated. Culling and restocking of uninfected sheep in endemic areas eradicated maedi/visna and jaagsiekte but not paratuberculosis. Experiments showed that vaccination against paratuberculosis could reduce mortality in sheep by 94%. Vaccination of sheep in endemic areas has been compulsory in Iceland since 1966 and as a result losses have been reduced considerably. Today, serology is used to detect and control infection in cattle herds. Furthermore, serology is used to control vaccination of sheep and screen for infection in non-endemic areas. The complement fixation (CF) test for paratuberculosis has been used until now, but recently we have started comparing the CF test with the CSL absorbed ELISA test.

Molecular epidemiological confirmation and circumstances of occurrence of sheep (S) strains of Mycobacterium avium subsp. paratuberculosis in cases of paratuberculosis in cattle in Australia and sheep and cattle in Iceland.

Distinct strains of Mycobacterium avium subsp. paratuberculosis with a tendency to segregate in either sheep, or cattle and other ruminants, have been described and are known as S and C strains, respectively. These strains can be distinguished by a polymorphism in the IS1311 element and other DNA-based methods. C strains are relatively easy to culture from tissues and faeces of animals with paratuberculosis but S strains are difficult to culture. A retrospective survey of archival formalin-fixed paraffin-embedded tissue samples from culture negative Australian paratuberculous cattle was undertaken to determine whether infection in these cases was due to S strains. Polymerase chain reaction and restriction endonuclease analysis of the amplified product was used to identify the polymorphism in IS1311. Three cases of bovine paratuberculosis due to S strain were confirmed from three different farms. A serological survey led to the identification of a further two cases on one of these farms. S strains were also identified in archival tissues from paratuberculous sheep and cattle from Iceland, confirming epidemiological and microbiological evidence that paratuberculosis in Iceland was due to S strain following importation of infected sheep from Europe. In each bovine case in both Iceland and Australia there had been direct or indirect contact of calves with paratuberculous sheep. We were unable to determine whether S strains had established endemic infection in cattle or whether repeated infection from sheep had occurred. Limited epidemiological evidence suggests that transmission of S strains to cattle in Australia has been uncommon under extensive grazing conditions. In Iceland, different husbandry practices appear to have favoured transmission of S strains to cattle.

Suspected Lyme borreliosis in sheep.

Two cases of suspected Lyme borreliosis in lambs are described. The clinical signs observed resemble those described for Lyme borreliosis in dogs and horses. The lambs were approximately six months old and came from two different flocks in a district heavily infested with Ixodes ricinus ticks. The lambs had pronounced lameness in one leg, generally bad condition and poor appetite. They had high serum levels of IgG antibodies to Borrelia burgdorferi when tested in an ELISA. The other lambs in the flocks were negative to B burgdorferi antibodies. Attempts were made to isolate spirochaetes from one of the lambs, but were unsuccessful.

A national epidemic of campylobacteriosis in Iceland, lessons learned.

Epidemics often result in organizational, policy and technical changes within a country. In 1999, an epidemic of campylobacteriosis was reported in Iceland. The recent availability of fresh poultry products in the marketplace was suggested as the source of infection. This paper reports on the context of the epidemic, reviews interventions implemented to prevent campylobacteriosis, and discusses lessons learned. A retrospective study of interventions implemented in Iceland from June 1995 to December 2007 was conducted by interviewing key informants and reviewing Iceland's literature. Cumulative incidence rates of domestic campylobacteriosis by year and average incidence rates per epidemic period were calculated. Interventions included on-farm surveillance of Campylobacter, producer education, enhanced biosecurity measures, changes in poultry processing, a leak-proof packaging policy, a freezing policy for products from Campylobacter-positive poultry flocks, consumer education, and the creation of a legislated inter-organizational response committee. These interventions appear to have collectively contributed to a decrease in campylobacteriosis' incidence rate near pre-epidemic baseline levels. Expert consultations revealed that the implementation of a Campylobacter surveillance program in poultry and the freezing policy were critical to controlling the disease in the Icelandic population. It was also recognized that new multidisciplinary collaborations among public health, veterinary, and food safety authorities and a sustained co-operation from the poultry industry were integral factors to the mitigation of the epidemic. Iceland's response to the campylobacteriosis epidemic is a lesson learned of inter-disciplinary and inter-organizational precautionary public health action in the face of a complex public health issue.

Prevalence and genetic relatedness of antimicrobial-resistant Escherichia coli isolated from animals, foods and humans in Iceland.

The prevalence of resistant bacteria in food products in Iceland is unknown, and little is known of the prevalence in production animals. The aim of this study was to investigate the prevalence and genetic relatedness of antimicrobial-resistant Escherichia coli from healthy pigs and broiler chicken, pork, broiler meat, slaughterhouse personnel and outpatients in Iceland. A total of 419 E. coli isolates were tested for antimicrobial susceptibility using a microbroth dilution method (VetMIC), and resistant strains were compared using pulsed-field gel electrophoresis (PFGE). All samples were screened for enrofloxacin-resistant strains with selective agar plates. The resistance rates among E. coli isolates were moderate to high from caecal and meat samples of pigs (54.1% and 28%), broilers (33.6% and 52%) and slaughterhouse personnel (39.1%), whereas isolates from outpatients showed moderate resistance rates (23.1%). Of notice was resistance to quinolones (minimum inhibitory concentrations: nalidixic acid > or = 32, ciprofloxacin > or = 0.12 and enrofloxacin > or = 0.5), particularly among broiler and broiler meat isolates (18.2% and 36%), as there is no known antimicrobial selection pressure in the broiler production in Iceland. The majority (78.6%) of the resistant E. coli isolates was genotypically different, based on PFGE fingerprint analyses and clustering was limited. However, the same resistance pattern and pulsotype were found among isolates from broiler meat and a slaughterhouse worker, indicating spread of antimicrobial-resistant E. coli from animals to humans. Diverse resistance patterns and pulsotypes suggest the presence of a large population of resistant E. coli in production animals in Iceland. This study gives baseline information on the prevalence of antimicrobial-resistant E. coli from production animals, and their food products in Iceland and the moderate to high resistance rates emphasize the need for continuing surveillance. Further studies on the origin of the resistant strains and the genetic relatedness of strains of different origin are needed.

Occurrence of Campylobacter spp. in cecal contents among commercial broilers in iceland.

Campylobacter jejuni remains the most frequently reported bacterial cause of human gastroenteritis in Nordic countries. The primary source of transmission to humans is suggested as mishandled raw poultry or consuming improperly prepared chicken. The focus of this report was to characterize the prevalence and cell numbers of the organism within the commercial Icelandic poultry industry. Commercial broiler flocks were sampled from May 2001 through 2003 in a total population study. At the slaughter plant, 40 randomly selected ceca were obtained from each flock, pooled into four samples containing 10 ceca each, and analyzed. Cell numbers and prevalence of Campylobacter spp. were estimated by direct plating of dilutions onto Campy-Cefex agar and incubating the plates at 42 degrees C under microaerobic atmosphere; colonies were confirmed as Campylobacter spp. by microscopy and latex agglutination to provide quantification of cell numbers per gm of cecal material. A total of 15.4% of the flocks carried the organism at at a maximum cell number of 8.1 x 10(7) cfu/g, having a mean raw count of colonized birds at 1.3 x 10(7) cfu/g (geometric mean of 1.5 x 10(6)). During the 3 years of sampling, the prevalence ranged from 17.6% to 17.3% to 12.7% for slaughter years 2001, 2002, and 2003, respectively. Isolation rates varied with numbers of catch lots (groups of birds taken for slaughter)/flock; with one catch lot/flock, the prevalence was 13.7%, with two 17.5%, and with three 33.3%. With increased flock size, isolation rates also increased; flocks of greater than 5,000 birds had a prevalence of 12.0% positive, 14.0% of flocks with 5,000-10,000 birds were positive, and 25.5% of flocks with more than 10,000 birds were positive for Campylobacter spp. Isolation rates varied with the processing lines: M was positive at 17.3%, B was positive at 10.1%, and G at 17.2%. Flocks were more frequently colonized in the warmer months, and younger birds were less frequently colonized than were older slaughtered birds. This study provides descriptive microbiology pertaining to Iceland broilers in a total population study.

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.

A longitudinal study of leucocyte blood counts and lymphocyte responses in pregnancy: a marked early increase of monocyte-lymphocyte ratio.

Seventy-seven pregnant women were studied repeatedly from the first trimester until 4 weeks after delivery. Neutrophil count was increased but constant in all stages of pregnancy. In contrast, there was a marked increase in monocyte numbers in the first and early second trimester, followed by a gradual decrease towards normal before term. A progressive decrease in total lymphocyte count was observed up to 25-28 weeks, when it increased again, although not reaching the non-pregnant level before term. These fluctuations in monocyte and lymphocyte numbers have not been described before and could explain contradictory findings of previous studies. They may also be important in relation to the mechanisms which prevent rejection of the fetal allograft. Absolute T cell counts were not significantly decreased, but a striking reduction was observed in B cell numbers in early pregnancy. Lymphocyte response to T cell mitogens was decreased throughout pregnancy. It is concluded that immunological studies of pregnancy must take account of the dynamic nature of this condition.