Distribution of Campylobacter jejuni Penner serotypes in broiler flocks 1998-2000 in a small Danish community with special reference to serotype 4-complex.

During the period January 1998-December 2001, all Danish broiler flocks were monitored bacteriologically for thermophilic campylobacters and isolates were stored at -80 degrees C. Six neighbouring broiler farms in a small community were selected for detailed examination of all Campylobacter jejuni isolated (n = 180) from these farms during 1998-2000 using Penner serotyping and pulsed-field gel electrophoresis (PFGE). The area and the farms were selected according to their prevalence of campylobacter so that both farms with low and high frequencies of campylobacter positive flocks were included in the study. The frequency of campylobacter positive flocks on the six farms ranged from 24.5 to 72.7%. One hundred and eighty of the isolates were C. jejuni (included in this study), 14 isolates were C. coli whereas 7 isolates belonged to other species but were not further identified. By serotyping of all C. jejuni 56 isolates (31.5%) were assigned to the 4-complex, 32 isolates (18.0%) to serotype 2, 12 isolates (6.7%) to serotype 11, and 11 isolates (6.2%) were assigned to serotype 12. In three farms, 4-complex was the most prevalent serotype, in one farm it was the second most frequently isolated serotype, while serotypes 2 and 1,44, respectively, were the most frequently isolated from the two remaining farms. This serotype distribution differed from the overall country-wide distribution where serotypes 2 and 1,44 are the most prevalent. All serotype 4-complex isolates from the six selected farms were compared by PFGE to serotype 4-complex isolates from the rest of the country. The results showed that there was a high level of diversity among isolates from the whole country, whereas isolates from the six farms were very homogeneous and only displayed one or a few different PFGE patterns on each farm. It is suggested that certain campylobacter clones persist in a confined geographical area, probably at the farm, and that the broiler houses may be repeatedly infected with a few C. jejuni clones during succeeding broiler flocks. New clones may be introduced, however, the sources and vehicles are yet unknown.

Evaluation of PCR for detection of Campylobacter in a national broiler surveillance programme in Denmark.

AIMS: To develop and evaluate a rapid and sensitive PCR method for detection of Campylobacter spp. directly from chicken faeces. METHODS AND RESULTS: DNA was isolated from faecal swabs using magnetic beads followed by PCR using a prealiquoted PCR mixture, which had been stored in the freezer. The result could be obtained in <6 h. The method was evaluated on 1282 samples from the Danish surveillance programme for Campylobacter in broilers by comparing with conventional culture. The diagnostic specificity was calculated to be 0.99. The detection limits of the PCR method and of the conventional culture were compared using spiked control material. For both methods the detection limit was 36 CFU ml-1. CONCLUSIONS: It was concluded that the PCR proved useful for detection of Campylobacter in pooled cloacal swabs from broilers. SIGNIFICANCE AND IMPACT OF THE STUDY: By taking cloacal samples in the broiler flocks the technique can be used as an important tool for planning and directing the broiler slaughtering process. This will be a great help in minimizing the risk of contaminating Campylobacter-free flocks at the abattoir.

Resistance to quinolones in Campylobacter jejuni and Campylobacter coli from Danish broilers at farm level.

AIMS: To investigate the prevalence of quinolone resistance among Campylobacter jejuni and Camp. coli isolates from Danish poultry at the farm level, as well as for the whole country. METHODS AND RESULTS: Data and isolates were collected from a national surveillance of Campylobacter in poultry. Quinolone resistance was investigated by determination of minimum inhibitory concentration (MIC) to nalidixic acid and enrofloxacin. Among Camp. jejuni and Camp. coli combined, 7.5% were resistant to nalidixic acid. Quinolone resistance varied considerably from farm to farm, with 0% on some farms and almost 100% on others, but the resistance was evenly distributed geographically. With respect to isolates from farms where resistance was detected, quinolone resistance was higher among Camp. coli (28.7%) than among Camp. jejuni (11.3%). PFGE typing of quinolone-resistant and quinolone-susceptible isolates from four farms indicated that certain resistant isolates belonged to specific clones that were able to persist on the farms during several rotations, even in the absence of selective pressure. Some clones were present and repeatedly isolated in both a quinolone-susceptible and quinolone-resistant variant. CONCLUSIONS: Overall, quinolone resistance among Campylobacter isolates from Danish broilers was 7.5% in 1998 and 1999; it was higher among Camp. coli than Camp. jejuni. Genetic diversity among resistant isolates was lower than among susceptible isolates, and certain clones existed in both a resistant and a susceptible variant. Some resistant clones appeared to persist on the farms and were repeatedly isolated from poultry flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is important for the understanding of persistence and dynamics of Campylobacter in broiler houses. It also highlights the extent, farm-to-farm variation and persistence of quinolone-resistant Campylobacter in broiler houses.

Rapid PCR using nested primers of the 16S rRNA and the hippuricase (hip O) genes to detect Campylobacter jejuni and Campylobacter coli in environmental samples.

Identification of sources Campylobacter infection in the poultry houses is in general problematic due to the lack of reliable methods to detect campylobacteria in environmental samples. Detection of campylobacteria in environmental samples by conventional culture methods is difficult and of limited sensitivity due to the use of selective media, the low number of bacteria in the samples and possibly also due to the presence of non-culturable or sub-lethally injured stages of the bacteria. The present paper describes a rapid PCR assay using nested primers of the 16S rRNA or the hippuricase (hip O) genes to detect Campylobacter jejuni and Campylobacter coli in environmental samples. The sensitivity of the nested PCR was determined to be 0.01 pg/PCR, corresponding to 2-3 colony forming units (cfu) per ml. The nested PCR assays were applied to detect C. jejuni and C. coli in 269 environmental samples collected from ten broiler farms. The sensitivity, specificity and the usefulness of the PCR assay for detection of C. jejuni and C. coli in environmental samples are presented and discussed.

Pre-harvest surveillance of Campylobacter and Salmonella in Danish broiler flocks: a 2-year study.

In national surveillance programmes of broiler flocks carried out in Denmark during 1998 and 1999, 89,110 samples for Campylobacter representing 8911 broiler flocks were taken at 10 different abattoirs, and 44,550 samples for Salmonella were taken from the same flocks in the broiler houses at the farms. Of the swabs, 42.5% were Campylobacter positive. Most positive samples were found during July, August and September, while the lowest number of positive samples were found during January, February, March and April. Of the flocks, 5.5% were Salmonella positive, but no seasonal variation was observed. For each flock, the presence of Campylobacter and Salmonella was recorded in order to estimate the possible correlation between colonisation with the two pathogens. In conclusion, no significant effects on intensive cleaning and disinfection procedures on Campylobacter occurrence could be demonstrated, and no significant correlation between occurrence of Campylobacter and Salmonella infections in Danish broilers could be demonstrated which is in contrast to previous observations on concurrent colonisation of broilers with these two zoonotic pathogens.

Salmonella dublin in Danish dairy herds: frequency of change to positive serological status in bulk tank milk ELISA in relation to serostatus of neighbouring farms.

Bulk tank milk from 1,429 herds were collected in 3 rounds from 19 different geographic areas. The milk samples were tested by use of indirect LPS-ELISA procedure to detect Salmonella dublin antibodies. From the obtained OD-values herd seroprevalence in the given area was determined and GR-scores calculated for each herd by addition of the number of positive sampling rounds by the 5 geographically closest neighbour herds. In the 19 different areas the calculated prevalence ranged from 0.01 to 0.41. Totally 3,697 GR-scores were given. The mean GR-scores in the areas ranged from 0.0 to 6.5. Higher GR-scores were found in herds changing to seropositive status compared with herds seronegative throughout the study period. The results indicate that the risk for a dairy herd to receive S. dublin infection increases with the disease status among the nearest neighbours and with the prevalence of seropositive herds in the geographic area.

Evidence that certain clones of Campylobacter jejuni persist during successive broiler flock rotations.

Through the national surveillance program for Campylobacter spp., nine broiler chicken farms that were infected with Campylobacter jejuni in at least five rotations in 1998 were identified. One additional farm, located at the island of Bornholm where divided slaughter is used extensively, was also selected. Twelve broiler houses located on 10 farms were included in the study. The C. jejuni isolates collected from the selected houses during the surveillance were typed using fla typing and macrorestriction profiling (MRP), and a subset of the isolates, representing each of the identified clones, was serotyped according to the Penner scheme. Pulsed-field gel electrophoresis typing using SmaI and KpnI revealed that the majority of houses (11 of 12) carried identical isolates in two or more broiler flocks. Such persistent clones were found in 63% of all flocks (47 of 75). The majority of persistent clones (7 of 13) had fla type 1/1, but MRPs distinguished between isolates from different houses, and fla type 1/1 clones belonged to different serotypes. Seven houses carried persistent clones that covered an interval of at least four broiler flock rotations, or at least one half year. The dominant fla type (1/1) was represented by 44% of isolates, or by at least one isolate from 31 of 62 broiler flocks. This significantly exceeded the prevalence of fla type 1/1 C. jejuni isolates that we have estimated from other studies and suggests that isolates carrying this fla type are overrepresented in flocks with recurrent Campylobacter problems. The MRPs of clones belonging to fla type 1/1 serotype O:2 isolated from persistently infected flocks shared a high percentage of bands compared to the remaining isolates, indicating that some clones that have the ability to cause persistent infections in broiler farms are highly related to each other.

Testing of bulk tank milk for Salmonella Dublin infection in Danish dairy herds.

The usefulness of enzyme-linked immunosorbent assay (ELISA) was investigated as a simple method to screen for Salmonella Dublin infection in dairy herds, examining bulk tank milk samples for lipopolysaccharide (O:1,9,12) antibodies. The cut-off value for the ELISA on bulk tank milk was established based on individual milk samples (n = 2887) and bulk tank milk from 52 herds. Bulk tank milk samples (n = 5108) were collected from 1464 dairy herds located in 19 different areas. About 10% of the dairy herds in Denmark participated in the study. The percentage of herds changing from test-negative to test-positive in each area was correlated with the incidence of S. Dublin outbreaks in the corresponding county (r = 0.48, n = 19; P < 0.025). The mean level of the OD values obtained in the first and third test rounds was not constant (Pr /t/ = 0.0001). The study demonstrated that the probability of being test-negative in the third test round was 0.926 for a herd with 2 previous test-negative results. It was concluded that the investigated ELISA method was in general accordance with the cases of clinical S. Dublin infection recorded, and that the method has a potential for national screening purposes.

National surveillance of Campylobacter in broilers at slaughter in Denmark in 1998.

A surveillance study for thermophilic Campylobacter spp. in broiler flocks was carried out for the year 1998 in Denmark. The study included examinations of 4286 broiler flocks comprising samples from 57,000 birds. Overall, a flock prevalence of 46.0% was recorded. The species distribution was Campylobacter jejuni 86%, Campylobacter coli 11%, Campylobacter lari 1%, other not further diagnosed species 2%. The prevalence was significantly higher in the period from June to October (3.2 < odds ratio [OR] <1.8, P < 0.0002) and was significantly associated with abattoir (OR < 2.8, P < 0.0001) and the length of the period the broiler houses were left empty between flocks (download period; 6 days or more) (OR = 1.6, P < 0.0198). No association between Campylobacter colonization and the age at slaughter was found. Separating the flocks into batches for slaughter elevated the flock prevalence from 0.41 after the first batch had been slaughtered to 0.46 after all batches had been slaughtered.

Thermophilic Campylobacter spp. in Danish broiler production: a cross-sectional survey and a retrospective analysis of risk factors for occurrence in broiler flocks.

In order to elucidate the rate of thermophilic Campylobacter spp. carriage in Danish broiler production and to identify risk factors for occurrence of campylobacter in broiler flocks, a total of 88 randomly selected broiler flocks were tested for campylobacter infection, and a subsequent study of risk factors based on a questionnaire was conducted. The sample material comprised cloacal swabs from live birds before slaughter, and neck skin samples from carcasses at the end of the processing line. A total of 52% of the flocks were found Campylobacter spp.-positive before slaughter. At the end of processing, 24% of the flocks were positive. The species distribution was 87% Campylobacter jejuni, 8% Campylobacter coli and 5% Campylobacter lari. The following parameters were identified as significant risk factors: lack of a hygiene barrier (odds ratio (OR) = 3.1, 1.1 < OR < 9.3), presence of animals in the vicinity of the broiler house on farms with a missing hygiene barrier (OR = 7.0, 1.6 < OR < 33.9), livestock other than chickens on farms with a missing hygiene barrier (OR = 7.6, 1.4 < OR < 44.9), dividing the flock into batches for staggered slaughter (OR = 6.8, 1.2 < OR < 49.3), a down period of less than 14 days (OR = 5.0, 1.2 < OR < 22.6), and feeding purchased wheat rather than home-grown wheat (OR = 3.1, 1.0

Associations of Neospora caninum seropositivity with gestation number and pregnancy outcome in Danish dairy herds.

The prevalence and distribution of seropositivity towards the protozoan parasite Neospora caninum were studied in single blood samples from 1561 cows from 31 Danish dairy herds. Blood samples were analysed by an indirect enzyme-linked immunoassay and an indirect fluorescent-antibody test. Seroprevalence in 15 herds with previous abortions assigned to neosporosis ranged from 1% to 58%, with a mean frequency of 22%. In eight out of 16 herds without a history of N.caninum related abortions, no seroreactors were found. In the remaining eight herds, the seroprevalence ranged from 6% to 59%. The prevalence and distribution of seropositivity, gestation number prior to sampling, and breed were related to abortions and perinatal deaths using a random-effects logistic-regression model. Abortion risk was significantly increased in seropositive animals (OR = 3) and in > or = 2nd-gestation cows (OR = 3). Perinatal death was significantly influenced by gestation number and breed, but not by serostatus. Reproductive performance and culling risk of cows were not affected by serostatus. Seropositivity increased with "age" (i.e. gestation number) (P = 0.02). In open cows, seropositivity tended to decrease with distance from calving (P = 0.05). The proportion of seropositive pregnant cows increased with trimester (P = 0.02).

Detection of antibodies to Salmonella lipopolysaccharide in muscle fluid from cattle.

OBJECTIVE: To compare muscle fluid with serum samples for detection of antibodies to Salmonella lipopolysaccharide. SAMPLE POPULATION: Muscle fluid and serum samples from 2 cattle populations: 1 from the island of Bornholm with no history of salmonellosis (n = 39), and the other from the S dublin-enzootic areas of Jutland (n = 144). PROCEDURE: Salmonella dublin (O:1,9,12), S typhimurium (O:1,4,5,12), and Salmonella O:9-blocking ELISA were used for testing the samples. RESULTS: In the S dublin ELISA, all serum and muscle fluid samples from cattle on the island of Bornholm had OD450 values well below the cutoff value (0.5). For samples obtained from cattle in the enzootic areas of Jutland, high correlation was found between serum and muscle fluid samples (rs = 0.89, P < 0.001). In addition, 19% (28/144) of the cattle had ELISA-positive muscle fluid and serum samples; 2% (3/144) had positive results for muscle fluid only, whereas 1 animal had positive results for serum only (kappa = 0.91, P < 0.0001; sensitivity and specificity of 97%). The same samples had similar significant correlation in the S typhimurium ELISA (rs = 0.88, P < 0.001, kappa = 0.7, P < 0.001; sensitivity of 73% and specificity of 98%) and the O:9-blocking ELISA (rs = 0.49, P < 0.001). CONCLUSION AND CLINICAL RELEVANCE: Muscle fluid samples taken at slaughter can be used as a practical alternative to serum samples for surveillance of Salmonella infections in cattle.

Comparison between persisting anti-lipopolysaccharide antibodies and culture at postmortem in salmonella-infected cattle herds.

Herds with recent clinical outbreaks of Salmonella dublin (7 herds) and S. typhimurium (4 herds) infections were followed serologically in O-antigen ELISAs over about one year, divided in four equal sampling phases. Animals found to be persistent high-reactors or seronegative at the end of the study were slaughtered and subsequently cultured for salmonella in a selected number of organ samples. Approximately 3% of all animals had high seroreactions up to 17 months after the outbreaks, and less than half of the seropositive animals in the S. dublin-infected herds were salmonella culture positive at slaughter (14/31). However, one persistently seronegative animal was also culture positive. Furthermore, as much as 70% of the male calves investigated at postmortem in the S. dublin-infected herds were high-reactors, among which approx. 56% were culture positive. Surprisingly, 2 of the 14 animals found culture positive turned out to be culture positive for S. typhimurium only. In the S. typhimurium study, none of the 17 animals investigated at postmortem were salmonella culture positive. All sera from these animals were negative in the O:9 blocking ELISA, and no serum sample was positive in the S. dublin ELISA, alone. In conclusion, although serology based on the O-antigens appears to be useful to identify salmonella-infected herds, it seems to be insufficient for identification of persistently infected animals.

Enzyme-linked immunosorbent assay for screening of milk samples for Salmonella typhimurium in dairy herds.

We investigated the ability of an antibody-specific, O antigen-based ELISA to document Salmonella typhimurium herd infections by screening of milk samples. Three cattle populations, 20 herds with no history of salmonellosis, 8 herds with history of S typhimurium episodes within the previous 7 months, and 220 herds of unknown disease status, were tested. A herd was considered ELISA positive if at least 5% of the cows had OD values > 0.3. Among the 20 herds without history of salmonellosis, only 2 herds were ELISA positive, whereas all 8 herds with a known history of salmonellosis were ELISA positive herd specificity, 0.9 and herd sensitivity, 1.0). A significant correlation (P < 0.001) was found between the OD values of serum and milk samples from cows in the herds with a history of salmonellosis. It was concluded that ELISA testing of individual milk samples can be used for surveillance of herds for S typhimurium infections, but further modifications are needed to test bulk tank milk samples.

Differences in protein expression of Haemophilus somnus grown under conditions of iron-restriction.

Outer membrane protein profiles were compared in 14 H. somnus strains isolated from brain and lung lesions as well as from the genital tract of asymptomatic carriers during in vitro growth under iron-restricted conditions. Ethylenediamine-di-O-hydroxyphenyl acetic acid (EDDA) was used to obtain iron-restricted conditions in media used for this study. The outer membrane protein profiles were studied by the discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoretic system (SDS-PAGE), and the proteins were stained with silver or transferred to nitrocellulose sheets and western blots conducted. Growth under iron-restricted conditions resulted in the induction of outer membrane proteins in most H. somnus strains examined. Studies also indicated differences among H. somnus strains in the number of induced proteins and their molecular weights but the results did not indicate a specific relationship between these strain-dependent differences and tissue trophism. Western blot analysis revealed a high degree of immunological relatedness among strains of H. somnus in their iron-regulated proteins. However, hyperimmune serum used in these assays failed to recognize certain iron-regulated proteins expressed by some H. somnus strains, a finding which may have important implications for the induction of protective immunity in cattle against this bovine pathogen.