Farm specific risk factors for Campylobacter colonisation in Danish and Norwegian broilers.

Campylobacteriosis has become the leading bacterial zoonosis in humans in the European Union and other developed countries. There are many sources of human Campylobacter infections, but broilers and broiler meat have been shown to be the most important. In order to implement effective interventions that reduce the probability of Campylobacter colonisation of broiler flocks, it is essential to fully understand the risk factors involved. We present a bi-national risk factor survey comprising Campylobacter data from more than 5200 Danish and Norwegian indoor, conventional broiler flocks and the responses to a standardised questionnaire, with more than 40 explanatory variables from 277 Danish and Norwegian farms. We explored several models by using different combinations of the Danish and Norwegian data, including models with single-country datasets. All models were analysed using a generalized linear model using backwards elimination and forward selection. The results show that Norwegian broiler flocks had a lower risk of being colonised than Danish flocks. Farm specific variables that increased the risk of flocks becoming colonised with Campylobacter in both countries were: broiler houses older than five years; longer downtime (no. of days between flocks), probably a consequence of longer downtimes being associated with less focus on maintaining a high biosecurity level; broiler houses without a separate ante-room or barrier; and the use of the drinker nipples with cups or bells compared with nipples without cups. Additional country specific risk factors were also identified. For Norway, the risk of colonisation increased with increasing numbers of houses on a farm and when the water used for the broilers originated from surface water or bore holes instead of mains. For Denmark, having boot dips or low stocking density increased the risk of a flock becoming Campylobacter positive. The different model approaches allowed us to explore the effect of having a large number of data available to identify the significant variables. To a large extent, the country specific models identified risk factors that were also found in the bi-national model. However, the bi-national model identified more risk factors than the country specific models. This indicated that combining the data sets from the two countries did not disrupt the results but was beneficial due to the greater strength achieved in the statistical analyses and the possibility of examining interactions terms with the variable Country.

Space-time patterns of Campylobacter spp. colonization in broiler flocks, 2002-2006.

This study was performed to investigate space-time patterns of Campylobacter spp. colonization in broiler flocks in Norway. Data on the Campylobacter spp. status at the time of slaughter of 16 054 broiler flocks from 580 farms between 2002 and 2006 was included in the study. Spatial relative risk maps together with maps of space-time clustering were generated, the latter by using spatial scan statistics. These maps identified the same areas almost every year where there was a higher risk for a broiler flock to test positive for Campylobacter spp. during the summer months. A modified K-function analysis showed significant clustering at distances between 2.5 and 4 km within different years. The identification of geographical areas with higher risk for Campylobacter spp. colonization in broilers indicates that there are risk factors associated with Campylobacter spp. colonization in broiler flocks varying with region and time, e.g. climate, landscape or geography. These need to be further explored. The results also showed clustering at shorter distances indicating that there are risk factors for Campylobacter spp. acting in a more narrow scale as well.

Trends in Campylobacter incidence in broilers and humans in six European countries, 1997-2007.

The objective of this study was to examine incidences of Campylobacter in broilers and humans, and to describe seasonal variation and long-term trends by comparing longitudinal surveillance data in six Northern European countries (Denmark, Finland, Iceland, Norway, Sweden and the Netherlands). Due to high degree of seasonality and autocorrelation, seasonally adjusted (de-seasonalized) and trend adjusted data (de-trended) were used for comparing incidences within and between the six countries. De-seasonalized time series were obtained by fitting the incidence time series to mean monthly temperature and then removing this effect from the data. Long-term trends were fitted to the de-seasonalized time series. The incidence of Campylobacter colonization in broiler flocks and incidence of campylobacteriosis in humans showed a concordant seasonality for all the countries. There was a strong association between the incidence in both broilers and humans in a given month and the mean temperature of the northern hemisphere in the same month, as well as the preceding month, as shown by the cross-correlations and the chosen Generalized Additive Model. Denmark and Sweden showed a steadily decreasing trend for Campylobacter in broilers and human campylobacteriosis in the period 2001-2007. In Iceland, there was a decreasing trend for campylobacteriosis in humans from 1999 to 2007, whilst the broiler trend for Campylobacter was stable from 2001 to 2004, then falling thereafter. In Norway, the human campylobacteriosis trend showed a steady increase throughout the period. On the other hand, the Norwegian broiler trend for Campylobacter showed a decrease from 2001 until 2004, but was thereafter stable. There was no significant decrease or increase in incidence for human campylobacteriosis in the Netherlands, and the trend for Campylobacter in broilers was close to stable. The seasonality seen in broiler and human closely follows the temperature, and was probably caused, at least partly, by temperature related factors.

Pandemic influenza A(H1N1)v: human to pig transmission in Norway?

In Norway there is an ongoing outbreak in pigs of infections with pandemic influenza A(H1N1)v virus. The first herd was confirmed positive on 10 October 2009. As of 26 October, a total of 23 herds have been diagnosed as positive. The majority of the herds seem to have been infected by humans. Sequence analysis of pig viruses from the index farm shows that they are identical or virtually identical to human viruses from the same geographical region.

Comparison of Campylobacter fla-SVR genotypes isolated from humans and poultry in three European regions.

AIMS: The genetic diversity of Campylobacter isolated from human infection and from poultry was assessed in strains originating in three different European regions in order to compare these two hosts and to investigate European regional differences. METHODS AND RESULTS: Randomly chosen isolates originated from Norway, Iceland and Basque Country in Spain were genotyped by sequencing of the short variable region (SVR) of flaA. A total of 293 strains were investigated, c. 100 per country with half originated from either host. The results indicate extensive diversity in both hosts and identified differences in the nature and distribution of genotypes between the countries. These differences could in part be related to geographical location, in that Campylobacter genotypes from Iceland and Norway were more similar to each other than either was to Basque Country. CONCLUSIONS: Differences between the countries exceeded the observed differences between human and poultry isolates within a country. SIGNIFICANCE AND IMPACT OF THE STUDY: Regional differences are extensive and should not be ignored when comparing genotyping data originating from different international studies.

Risk factors associated with the presence of Campylobacter species in Norwegian broiler flocks.

A case-control study was conducted in 2005 to identify risk factors for the presence of Campylobacter spp. in Norwegian broiler flocks. A total of 131 broiler farms (44 cases and 87 controls) were included in the study, and 1 flock from each farm was included in the statistical analyses. Data on farm and flock level were collected by means of a questionnaire designed for the present study. The relationship and strength of association between the presence of Campylobacter spp. in the broiler flocks and several factors were investigated by unconditional logistic regression. The following factors were found to be associated with an increased risk of testing positive for Campylobacter spp.: water from a private water source, swine holdings closer than 2 km, a specific slaughterhouse, a hired animal caretaker, transport personnel passing through the hygiene barrier when delivering day-old chickens, less than 9 d between depopulation and restocking, and multiple broiler houses on the farm.

Genotyping of thermotolerant Campylobacter from poultry slaughterhouse by amplified fragment length polymorphism.

AIM: To examine the occurrence, diversity and transmission of Campylobacter in a poultry slaughterhouse. METHODS AND RESULTS: During a 4-week period, a slaughterhouse was sampled alternately during slaughtering and the following mornings post-disinfection. Samples were taken from poultry at six stages in the slaughter process and from 25 environmental sites. For positive broiler flocks slaughtered on one occasion, 92% and 48% of the environmental sites were positive during slaughter and post-disinfection, respectively. For positive laying hen flocks slaughtered on three occasions, 8-56% and 12-20% of the environmental sites were positive during slaughter and post-disinfection, respectively. Genetic fingerprinting by amplified fragment length polymorphism (AFLP) of the 109 isolates obtained resulted in 28 different AFLP clones. Five AFLP clones were present for more than 1 week. CONCLUSIONS: Slaughtering of Campylobacter-positive broilers resulted in extensive contamination of the slaughterhouse, including the air. A high proportion of the laying hen flocks were Campylobacter positive, but these caused less environmental contamination than the broilers. This, together with the freezing of all layer carcasses, results in a lower public health risk from laying hens, when compared with broilers. SIGNIFICANCE AND IMPACT OF THE STUDY: When slaughtering Campylobacter-positive broilers, the implementation of preventive measures is important to reduce contamination of negative carcasses and to protect the workers against infection.

Antimicrobial resistance in Campylobacter jejuni from broilers and broiler house environments in Norway.

Antimicrobial susceptibility in Campylobacter jejuni collected from the environment outside four broiler houses (n = 63) and from the environment inside these broiler houses (including broiler droppings) (n = 36) from May to September 2004 was studied and compared with isolates from Norwegian broilers analyzed within the frame of the Norwegian monitoring program of antimicrobial resistance in feed, food, and animals (NORM-VET) in 2004 (n = 75). The MICs of oxytetracycline, ampicillin, erythromycin, gentamicin, enrofloxacin, and nalidixic acid were obtained by the broth microdilution method VetMIC. The present study, which to our knowledge is the first Norwegian study on the occurrence of antimicrobial resistance in Campylobacter spp. from the environment of broiler houses, revealed a very low occurrence of antimicrobial resistance in C. jejuni from the broilers and broiler house environments studied. All isolates originating from the four broiler houses studied were susceptible to all the antimicrobial agents tested, except for one isolate from the outdoor environment (courtyard soil), which was resistant to oxytetracycline (MIC, 8 mg/liter). For the isolates from broilers (NORM-VET), low prevalences of resistance to oxytetracycline (1.3%) and ampicillin (4%) were observed. No quinolone resistance was observed. The results for the broiler isolates are in agreement with the earlier findings of a very low prevalence of resistance in Campylobacter from broilers in Norway, which reflects the low usage of antimicrobials in Norwegian broiler production. Furthermore, the present data are in accordance with antimicrobial susceptibility data for C. jejuni from domestically acquired human cases.

Enumeration of thermotolerant Campylobacter spp. from poultry carcasses at the end of the slaughter-line.

AIM: To enumerate Campylobacter on poultry carcasses at the end of the slaughter-line, and investigate the extent to which Campylobacter from a positive flock were transmitted to other flocks during slaughter. METHODS AND RESULTS: The presence (in caeca) and the level (from carcasses) of Campylobacter were determined. The isolates were fingerprinted by amplified fragment length polymorphism (AFLP). A total of three of 13 broiler flocks and three of four-layer flocks harboured caecal Campylobacter. Carcasses from the caeca-positive broiler flocks were Campylobacter positive with numbers ranging from 2.6 x 10(4) to 2.6 x 10(6) CFU per carcass. Two caeca-negative broiler flocks, slaughtered directly after the positive broiler flocks, had the first carcasses contaminated with Campylobacter, with numbers below 2 x 10(4) CFU per carcass of the same AFLP haplotypes as the preceding flock. Campylobacter was detected on carcasses from only one of the caeca-positive layer flocks in numbers below 2 x 10(4) CFU per carcass. No Campylobacter was detected on carcasses from a flock succeeding the positive-layer flocks. CONCLUSION: Carcasses from Campylobacter-positive broiler flocks were heavily contaminated with Campylobacter, and transmitted low levels of Campylobacter to carcasses from negative flocks, slaughtered directly after. Campylobacter-positive layer flocks had low numbers of Campylobacter on the carcasses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate limited cross-contamination of Campylobacter between flocks at the slaughterhouse, reducing the advantage of logistic slaughter.

Genotyping of Campylobacter jejuni from broiler carcasses and slaughterhouse environment by amplified fragment length polymorphism.

We examined the occurrence and diversity of Campylobacter jejuni on broiler carcasses during slaughter of an infected flock and in the slaughterhouse environment during slaughter and postdisinfection before a new production run. During the slaughter of a known C. jejuni infected broiler flock, samples were taken from broiler carcasses at 7 different stages during the process. Thirty-seven sites in the slaughterhouse environment were sampled both during process and postdisinfection. The samples were analyzed for C. jejuni, and genetic fingerprinting was performed using amplified fragment length polymorphism. All carcass samples were positive. Of the environmental samples collected during slaughter, 89% were positive; 100% of those from the arrival, stunning, scalding, defeathering, and evisceration facilities and 67% of those from the cooling and sorting facilities. Postdisinfection, 41% of the samples were positive; 71% of those from the arrival and stunning area, 60% of those from the scalding and defeathering area, and 20% of those from the evisceration, cooling, and sorting area. The C. jejuni isolates (n = 60) recovered were grouped into 4 different amplified fragment length polymorphism clones with a similarity index of 95% or greater. All isolates obtained from the flock and 94% of the isolates obtained from the environment during slaughtering belonged to clone A, whereas 1 environmental isolate belonged to each of the clones B and C. Isolates from clones A, B, and D were present postdisinfection. Only clone B was detected on flocks slaughtered during the previous week. The high level and continuous presence of Campylobacter in the environment constitutes a risk for transmission to negative carcasses. In Norway, where above 96% of the broiler flocks are Campylobacter-negative, this aspect is of special importance. The ability of Campylobacter to remain in the slaughterhouse environment through washing and disinfection is associated with constructional conditions of equipment and buildings, complicating cleaning and providing sufficient moisture. To reduce the probability of the workers acquiring campylobacteriosis, precautions should be taken when slaughtering Campylobacter-positive flocks.

Genetic variability of Campylobacter jejuni isolated from fresh and frozen broiler carcasses.

AIMS: The aim of this study was to determine the genetic variability of Campylobacter jejuni isolates from poultry before and after freezing treatment in order to identify genotypes that would survive the treatment. METHODS AND RESULTS: C. jejuni was isolated from both fresh and frozen halves of the same carcass after freezing for 2 or more than 20 days at -20 degrees C. From 36 carcasses, representing five unrelated flocks in Norway, a total of 209 isolates were included in the study. Thirty-two of the isolates were recovered with a qualitative method while the remaining 177 were isolated using a quantitative method. Isolates were genotyped with fluorescent amplified fragment length polymorphism using MfeI and BglII restriction enzymes. Nine different genotypes were identified, however, one genotype was shown to be dominant in three different flocks. This genotype and the dominant genotype of another flock were found among isolates from fresh and frozen broiler halves. They were also shown to be identical to genotypes frequently identified among strains isolated from humans, cattle and poultry flocks in previous years. CONCLUSIONS: Freezing treatment or isolation method appeared not to select for a particular genotype. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study indicate that the freezing tolerance of strains is not genotype dependent.

Genetic diversity and description of transmission routes for Campylobacter on broiler farms by amplified-fragment length polymorphism.

AIMS: To investigate the genetic diversity of Campylobacter in broilers and in the environment of broiler farms, to compare the genetic profiles and describe critical factors for transmission to broilers. METHODS AND RESULTS: Flocks at three of four investigated farms became colonized with Campylobacter. The total proportion of Campylobacter-positive samples at different farms varied from 20% to 42%. The farm with the poorest biosecurity routines had broilers that became infected earliest, the highest proportion of positive samples and the highest genetic diversity among the broiler Campylobacter isolates. Campylobacter isolates within common amplified-fragment length polymorphism (AFLP) clusters (95-100%) were found to be present in outdoor environment and in broilers at adjacent farms before they were found in the broilers. A large presence of Campylobacter in the farm environment was demonstrated after the broilers were infected. A high genetic diversity was found among Campylobacter present in the outdoor environment, where certain Campylobacter clusters were found for periods of up to 6 weeks. CONCLUSION: Confirmation by AFLP indicates adjacent poultry farms and outdoor environment as major sources of Campylobacter infection of broilers, this being the novel achievements. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide more exact knowledge on transmission of Campylobacter at farm level, helpful for developing optimal preventive strategies.

Antimicrobial resistance in Campylobacter jejuni from humans and broilers in Norway.

In this study comprising isolates from 2001 to 2003, resistance was considerably more widespread among Campylobacter jejuni from humans infected abroad than infected within Norway. The discrepancy was particularly notable for fluoroquinolone resistance (67.4% vs. 6.5%). This is probably a reflection of a low resistance prevalence in Norwegian broiler isolates (1.2% fluoroquinolone resistant).

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.

Bacteriological investigation of infectious keratoconjunctivitis in Norwegian sheep.

Contagious keratoconjunctivitis is a rather common disease in Norwegian sheep. Since the knowledge of its aetiology is limited, the present study was performed to determine the microorganisms involved. Local veterinarians throughout the country collected conjunctival swabs from both sick (n = 43) and healthy (n = 42) sheep on 15 farms with outbreaks of ovine keratoconjunctivitis, and further from healthy sheep (n = 50) on 17 farms not showing any signs of conjunctival disease. All samples were cultivated for bacteria and mycoplasma. Listeria monocytogenes was isolated from 3 cases (1%) in one single herd. Staphylococcus aureus (5%), Corynebacterium spp. (2%) and Escherichia coli (4%) were isolated only in herds with keratoconjunctivitis, but from both sick and healthy animals. Moraxella (Branhamella) ovis was isolated from 28% of sampled animals in affected herds and from 10% of sampled animals in healthy herds. The corresponding numbers for Moraxella spp. were 9%/12%, for Pseudomonas spp. 7%/8%, for Staphylococcus spp. 22//22%, for Bacillus spp. 12%/14%, for Micrococcus spp. 6%/2% and for Streptococcus/Enterococcus spp. 2%/2%. Mycoplasma conjunctivae was isolated from 16 animals with keratoconjunctivitis (37%) and from 3 animals without clinical signs (7%) in farms with keratoconjunctivitis. In farms without clinical signs of keratoconjunctivitis, M. conjunctivae was isolated in 4 animals (8%). To our knowledge, this is the first time M. conjunctivae has been isolated in Norway. Other predisposing agents found were Moraxella (Branhamella) ovis and Listeria monocytogenes. The etiological importance of different microorganisms in ovine keratoconjunctivitis seems to vary; some are probably only present as secondary invaders. Other possible causes of ovine keratoconjunctivitis in Norway, such as Chlamydia psittaci, remain to be investigated.

Risk factors for Campylobacter infection in Norwegian cats and dogs.

Rectal swabs from healthy cats and dogs, and from dogs and cats with clinical diarrhoea were collected approximately every third month from May 2000 to June 2001 from six small-animal practices throughout Norway. A questionnaire was filled in for each animal. Of the 301 healthy cats sampled, 54 (18%) were positive for Campylobacter, compared to 5 out of 31 (16%) cats with diarrhoea. Campylobacter jejuni was isolated from 11 (3%), C. upsaliensis from 42 (13%) and C. coli from 2 (0.6%) of the cats sampled. Isolates from four cats (1%) could not be specified. Of the 529 healthy dogs, 124 (23%) were positive for Campylobacter, compared to 18 of 66 (27%) dogs with diarrhoea. C. jejuni was isolated from 20 (3%) and C. upsaliensis from 117 (20%) of the dogs sampled. Isolates from five dogs (0.8%) could not be specified. Eighteen out of the 20 investigated C. upsaliensis samples were resistant to streptomycin. The clinically healthy animals were included in the analysis to identify factors associated with Campylobacter prevalence. The cat model had low classification ability. The dog-data model indicated increased odds of infection with Campylobacter for dogs

Reduced incidence of Clostridium perfringens-associated lesions and improved performance in broiler chickens treated with normal intestinal bacteria from adult fowl.

The dosing of young chicks with cultures of normal gut flora has been termed "competitive exclusion" (CE). This study was undertaken to examine, under field conditions, the effect of CE treatment on counts of intestinal Clostridium perfringens (CP) and on the occurrence of CP-associated disease in broiler chickens. A farm having recurrent CP-associated health problems was selected as study site. The study comprised four broiler houses, with one treated and one untreated flock per house. Treated birds were sprayed with the CE product Broilact upon arrival at the farm. All flocks were offered feed containing the ionophorous anticoccidial agent narasin. The feed did not contain growth promoters. Treatment was associated with positive but statistically nonsignificant effects on gut health. Delayed intestinal proliferation of CP and delayed appearance of CP-associated gut lesions were found in CE-treated flocks. This delay was associated with improved production performance at slaughter.

Sarcina -like bacteria, Clostridium fallax and Clostridium sordellii in lambs with abomasal bloat, haemorrhage and ulcers.

A study of abomasal disease in lambs aged 2-5 weeks, made during the period 1993-1998, included 67 cases and 45 non-affected controls. Gross pathological findings included various combinations of bloat, haemorrhage and ulcers in the abomasum. Sarcina -like bacteria were found in sections and smears from the abomasum of 79% (53/67) of the cases. From one case, a lamb with abomasal bloat, the anaerobic "packet"-forming Sarcina ventriculi was cultivated from the abomasal contents and identified by biochemical reactions and sequencing of the 16S rRNA gene. Sarcina -like bacteria were observed microscopically in specimens from 94% (44/47) of the lambs with abomasal gas and in 45% (9/20) of those with ulcers or haemorrhage or both but little gas. On culture, abomasal contents from 41 cases yielded Clostridium fallax from 16 (39%) and Clostridium sordellii from eight (20%); abomasal cultures from 30 control lambs were negative for the three bacterial species. Quantitative cultivation, carried out on abomasal contents from live lambs and lambs dead

Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables.

The effect of Clostridium perfringens challenge, number of challenge days, and pre-challenge antibiotic treatment on the induction of necrotic enteritis in broiler chickens raised on litter was studied, and the relationship between bacterial counts and frequency of gut lesions was evaluated. Specific intestinal lesions in randomly selected birds were present despite a lack of disease-specific mortality. Challenge, number of challenge days and frequency of lesions were associated with median counts of C. perfringens. The effect of pre-challenge C. perfringens counts and antibiotics cannot be evaluated unless procedures for the control of pre-challenge infection and methods for the differentiation between wild-type and challenge strains are established.

The antimicrobial susceptibility of Staphylococcus species isolated from canine dermatitis.

In a retrospective study, 1538 strains of beta-haemolysin-producing Staphylococcus species isolated from dermatitis in dogs at three veterinary clinical microbiology laboratories in Norway during 1986-87 and 1993-94 were investigated for their antimicrobial susceptibility. None of the strains was resistant to cloxacillin, cephalexin or the quinolones enrofloxacin and ciprofloxacin. More than 96% of the strains were susceptible to trimethoprim-sulphonamide, bacitracin and fucidic acid. Between 67% and 89% of the strains were susceptible to erythromycin, lincosamides, tetracycline, neomycin and chloramphenicol. Only 37.9% of the strains were susceptible to penicillin. The frequency of penicillin resistance increased significantly between the first and second periods, from 46.0% to 58.6%. The frequency of resistance to lincomycin, clindamycin and erythromycin also increased significantly between the first and second periods, from 3.0%, 2.1% and 3.3% to 25.5%, 19.5% and 24.8%, respectively. A moderate increase in resistance to tetracycline was also noted, from 20.4% in the first to 27.6% in the second period. On the other hand, the frequency of resistance to trimethoprim-sulphonamide decreased significantly from 4.1% in the first to 0.9% in the second period. Many different resistance patterns were observed in each period. However, the proportion of multiresistant strains increased from 2.1% in the first to 10.2% in the second period. There was a decrease in resistance to the combination of trimethoprim-sulphonamide and penicillin from the first to the second period. Resistance to the combination of lincosamides and penicillin increased. For the combinations penicillin-tetracycline-lincosamides, penicillin-lincosamides-erythromycin, and penicillin-tetracycline-lincosamides-erythromycin, there was a striking increase in resistance between the first and the second periods.