A role for flies (Diptera) in the transmission of Campylobacter to broilers?

Campylobacter is the leading cause of bacterial diarrhoeal disease worldwide, with raw and undercooked poultry meat and products the primary source of infection. Colonization of broiler chicken flocks with Campylobacter has proved difficult to prevent, even with high levels of biosecurity. Dipteran flies are proven carriers of Campylobacter and their ingress into broiler houses may contribute to its transmission to broiler chickens. However, this has not been investigated in the UK. Campylobacter was cultured from 2195 flies collected from four UK broiler farms. Of flies cultured individually, 0·22% [2/902, 95% confidence interval (CI) 0-0·53] were positive by culture for Campylobacter spp. Additionally, 1293 flies were grouped by family and cultured in 127 batches: 4/127 (3·15%, 95% CI 0·11-6·19) from three broiler farms were positive for Campylobacter. Multilocus sequence typing of isolates demonstrated that the flies were carrying broiler-associated sequence types, responsible for human enteric illness. Malaise traps were used to survey the dipteran species diversity on study farms and also revealed up to 612 flies present around broiler-house ventilation inlets over a 2-h period. Therefore, despite the low prevalence of Campylobacter cultured from flies, the risk of transmission by this route may be high, particularly during summer when fly populations are greatest.

Intestinal colonization of broiler chickens by Campylobacter spp. in an experimental infection study.

Consumption of poultry meat is considered as one of the main sources of human campylobacteriosis, and there is clearly a need for new surveillance and control measures based on quantitative data on Campylobacter spp. colonization dynamics in broiler chickens. We conducted four experimental infection trials, using four isolators during each infection trial to evaluate colonization of individual broiler chickens by Campylobacter jejuni over time. Individual and pooled faecal samples were obtained at days 4, 7 and 12 post-inoculation (p.i.) and caecal samples at day 12 p.i. There were large differences between broiler chickens in the number of C. jejuni in caecal and faecal material. Faecal samples of C. jejuni ranged from 4·0 to 9·4 log c.f.u./g and from 4·8 to 9·3 log c.f.u./g in the caeca. Faecal c.f.u./g decreased with time p.i. Most variation in c.f.u. for faecal and caecal samples was attributed to broiler chickens and a minor part to isolators, whereas infection trials did not affect the total variance. The results showed that pooled samples within isolators had lower c.f.u./g compared to the arithmetic mean of the individual samples. There was a significant correlation between faecal c.f.u./g at days 4 and 7 p.i., days 7 and 12 p.i. and for caecal and faecal c.f.u./g at day 12 p.i.

The effects of temperature and innate immunity on transmission of Campylobacter jejuni (Campylobacterales: Campylobacteraceae) between life stages of Musca domestica (Diptera: Muscidae).

The house fly (Musca domestica L.) is a well-established vector of human pathogens, including Campylobacter spp., which can cause infection of broiler chicken flocks, and through contaminated broiler meat can cause outbreaks of campylobacteriosis in humans. We investigated whether Campylobacter jejuni (Jones) could be transferred between life stages of M. domestica (larvae-pupae-adults) and determined bacterial counts of C. jejuni at different time points after bacterial exposure. C. jejuni was transmitted from infected larvae to pupae, but not to the adult stage. Infected larvae maintained at 25 degrees C had mean bacterial numbers of 6.5 +/- 0.2 SE log10 (colony forming units [CFU]/g) that subsequently dropped to 3.6 +/- 0.3 SE log10 (CFU/g) 8 h after infection. Pupae originating from infected larvae contained mean bacterial numbers of 5.3 +/- 0.1 SE log10 (CFU/g), and these numbers dropped to 4.8 +/- 0.1 SE log10 (CFU/g) 24 h after pupation. The decline in C. jejuni numbers during pupal development coincided with increased expression of antimicrobial peptides, including cecropin, diptericin, attacin, and defensin, in the larva-pupa transition stage and a later second peak in older pupae (4 or 48 h). Conversely, there was a reduced expression of the digestive enzyme, lysozyme, in pupae and adults compared with larvae.

Temperature and population density effects on locomotor activity of Musca domestica (Diptera: Muscidae).

The behavior of ectotherm organisms is affected by both abiotic and biotic factors. However, a limited number of studies have investigated the synergistic effects on behavioral traits. This study examined the effect of temperature and density on locomotor activity of Musca domestica (L.). Locomotor activity was measured for both sexes and at four densities (with mixed sexes) during a full light and dark (L:D) cycle at temperatures ranging from 10 to 40°C. Locomotor activity during daytime increased with temperature at all densities until reaching 30°C and then decreased. High-density treatments significantly reduced the locomotor activity per fly, except at 15°C. For both sexes, daytime activity also increased with temperature until reaching 30 and 35°C for males and females, respectively, and thereafter decreased. Furthermore, males showed a significantly higher and more predictable locomotor activity than females. During nighttime, locomotor activity was considerably lower for all treatments. Altogether the results of the current study show that there is a significant interaction of temperature and density on daytime locomotor activity of M. domestica and that houseflies are likely to show significant changes in locomotor activity with change in temperature.

The dynamics of Salmonella occurrence in commercial laying hen flocks throughout a laying period.

Contaminated eggs and egg products have been recognized for many years as an important source of Salmonella infections in humans in the European Union and in the United States. Longitudinal studies can help to increase our knowledge about the dynamics of the occurrence of Salmonella in the course of a laying period. The total of 41 laying hen flocks-18 in Belgium, six in Denmark and 17 in Germany-were followed during an entire laying period. Samples taken from the empty cleaned and disinfected poultry houses were all negative for Salmonella. After hens arrived on the farms, five pooled faecal samples, one pooled dust sample and 40 cloacal swabs (Belgium and Germany) or 40 swabs from fresh droppings (Denmark) were taken four times from 18 flocks, three times from 21 flocks and two times from two flocks in the course of the laying period. Ten flocks (two Belgian and eight German flocks) tested up to three times positive for Salmonella. Forty-three out of 50 positive samples contained Salmonella Enteritidis phage type 4 (29 isolates) or phage type 8 (14 isolates). The probability of subsequent Salmonella-positive findings increased significantly in Salmonella-positive flocks (P<0.05, odds ratio = 6.4). However, the probability of finding Salmonella did not depend on the time of sampling in the laying period or the season.

Retention of Campylobacter (Campylobacterales: Campylobacteraceae) in the house fly (Diptera: Muscidae).

The house fly (Musca domestica L.) may transmit Campylobacter to broiler flocks. We assessed the retention time of house flies for Campylobacter jejuni at five temperatures and three doses. Flies were inoculated individually at their proboscis with 1.6 x 10(7) CFU (colony forming units) of C. jejuni and incubated at 15, 20, 25, 30, and 35 degrees C. Furthermore, a dose experiment was conducted at 25 degrees C where individual flies were inoculated in three series: 6.5 x 10(6), 6.0 x 10(4), and 8.2 x 10(2) C.jejuni CFU. Whole flies were tested for C. jejuni carriage at 0, 6, 12, 18, and 24 h by initial preenrichment in Bolton broth, which afterwards was streaked on modified mCCDA agar plates and incubated under micro-aerobic conditions. The results showed that the time C. jejuni remained in flies declined over time with ascending temperatures and when reducing the inoculation dose. All flies stayed Campylobacter positive 24 h postinoculation at 15 degrees C whereas only one-third of the flies were positive at 20 degrees C and few to none at 25, 30, and 35 degrees C. When combinations of temperature and retention time were expressed as accumulated day-degrees, data could be adequately fitted using a generalized linear mixed model that included a linear effect of day-degrees and the difference between the lowest and the two highest doses. Based on model predictions of selected combinations of temperature and dose, the time for 50% and 1% of flies containing Campylobacter was calculated. It is suggested that house flies are mainly short distance carriers of C. jejuni.

Influxed insects as vectors for Campylobacter jejuni and Campylobacter coli in Danish broiler houses.

The vector potential of flies (Diptera: Brachycera) for spread of Campylobacter jejuni and Campylobacter coli on 5 Danish broiler farms was evaluated in a longitudinal field study from April to November 2004. First, the prevalence of C. jejuni- and C. coli-positive flies was determined in 2,816 flies captured from farm surroundings. Each individual fly was macerated, preenriched in Bolton broth for 24 h at 42 degrees C, streaked onto modified Campylobater blood-free selective agar and incubated under microaerobic conditions for 48 h at 42 degrees C. Second, the influx of insects to broiler houses was estimated by trapping of insects (n = 5,936) in ventilation vents. In total, 31 flies (28 of which were of the Muscidae family) caught in farm surroundings were Campylobacter spp.-positive (C. jejuni, n = 7; C. coli, n = 23; other Campylobacter spp., n = 1). Musca domestica (L) (house fly) was more frequently (P < 0.0376) positive than other fly species collected. Other positive fly species were Stomoxys calcitrans (L) (stable fly; n = 4), Muscina stabulans (Fallén) (false stable fly; n = 1), Mesembrina meridiana (L) (noon fly; n = 1) and Hydrotaea sp. (black dump fly; n = 1). One Lucilia caesar (L) (green bottle fly) of the Calliphoridae family and 2 flies of unidentified species were also positive. The prevalence of Campylobacter spp.-positive flies varied from 0.0 in April to a peak of 16.3% in July and decreasing to 2.0% in October on a farm with pig production. On 4 broiler farms without other livestock, the prevalence was constantly below 1.0%. The average influx of insects per broiler rotation was estimated to be 30,728 +/- 2,443 SE (range 2,233 to 180,300), of which 21.4% were flies. The influx of insects correlated with the flow (m(3)/h) of ventilation air (P < 0.0078) and with the outdoor temperature (P < 0.0032). We conclude that the influx of large numbers of flies to broiler houses constitutes a considerable risk for infection of broilers with C. jejuni and C. coli.

The role of litter beetles as potential reservoir for Salmonella enterica and thermophilic Campylobacter spp. between broiler flocks.

We evaluated the role of beetles infesting broiler chicken rearing facilities as potential reservoirs for Salmonella enterica infections between successive broiler flocks. In addition, their role as potential reservoirs for thermophilic Campylobacter spp. was also investigated. Fourteen broiler houses located at 11 different farms were included in the study. The houses were nonrandomly selected on the basis of their salmonella status; nine were persistently contaminated with salmonella whereas five were salmonella negative. For each broiler house, two consecutive broiler flocks (i.e., 28 broiler flocks in all) as well as beetles collected during both rotations of production and in the empty period (after cleaning and disinfection) between these flocks were monitored for the presence of salmonella. Examinations for the presence of campylobacter in the same sample materials were also performed. Beetles sampled during production were positive for salmonella or campylobacter or both. Furthermore, in one house, the occurrence of Salmonella indiana in two consecutive broiler flocks coincided with the presence of S. indiana-contaminated beetles in the empty period between the flocks. The genotype of the identified S. indiana was in all cases identical when analyzed by pulsed-field gel electrophoresis. However, our results also suggest that salmonella from beetles may not always be transmitted to the chickens and that beetles living in contaminated houses can remain free of infection. All cases of campylobacter-positive beetle samples were detected in connection with a positive chicken flock; in no case was campylobacter isolated from beetles taken from the empty period between rotations. Four beetle species were identified during this study. Alphitobius diaperinus was found in all houses and was relatively abundant in most. Typhaea stercorea and Ahasverus advena were found in eight and nine houses, respectively, and were abundant in most of these. Carcinops pumilio was found in small numbers in eight houses. No other insect species was identified. These investigations have shown that beetles in broiler houses infrequently are positive for salmonella. However, transmission of S. indiana between two consecutive broiler flocks can coincide with the presence of salmonella-contaminated beetles in the empty period, indicating that the beetles were the reservoir of S. indiana between the two flocks. Concerning campylobacter, the results suggest that beetles do not play a significant role as a reservoir of campylobacter from one rotation to the next.

Study of the infectivity of saline-stored Campylobacter jejuni for day-old chicks.

The culturability of three Campylobacter jejuni strains and their infectivity for day-old chicks were assessed following storage of the strains in saline. The potential for colonization of chicks was weakened during the storage period and terminated 3 to 4 weeks before the strains became nonculturable. The results from this study suggest that the role of starved and aged but still culturable campylobacters may be diminutive, but even more, that the role of viable but nonculturable stages in campylobacter epidemiology may be negligible. Even high levels of maternally derived anti-campylobacter outer membrane protein serum antibodies in day-old chicks did not protect the chicks from campylobacter colonization.

Role of batch depletion of broiler houses on the occurrence of Campylobacter spp. in chicken flocks.

AIMS: The effect of batch depletion of broiler houses for campylobacter occurrence in broiler flocks was estimated in 10 flocks, each comprising a separate female and male batch. METHODS AND RESULTS: The chicks were sampled first by cloacal swabs in the broiler houses before the start of the depopulation and secondly, on arrival at the abattoir. Females were slaughtered at 5 weeks of age, males at 6 weeks. The number of campylobacter-positive batches increased from five to seven female batches, and from five to 10 male batches, between the two sampling rounds. CONCLUSION: It is concluded that batch depletion of broiler houses increased the prevalence of Campylobacter spp.-infected broilers in the flocks, that the introduction occurred when catching the first batch, and that campylobacter spreads through the entire flock within a week. SIGNIFICANCE AND IMPACT OF THE STUDY: The results from this study emphasize the need to manage depopulation of broiler houses as quickly as possible and in one batch only.

Fusaria and fumonisins in maize from Ghana and their co-occurrence with aflatoxins.

Fifteen maize samples from four markets and processing sites in Accra, Ghana were analysed for fumonisins B1, B2, and B3. All samples contained fumonisins. Total fumonisin levels for 14 samples ranged from 70 to 4222 microg kg(-1). One sample of visibly mouldy kernels contained 52 670 microg kg(-1) total fumonisins. Mycological examination of the samples showed Aspergillus spp. as the most dominant fungi (76.4%) followed by Penicillium spp. (19.9%). Fusarium formed 2.6% with Fusarium verticillioides as the predominant Fusarium species. Thirty-two Fusarium strains representing five species isolated from the maize samples were tested for the production of fumonisins in maize substrates. From 95% (21 of 22) of the F. verticillioides strains tested, all three types of fumonisins were produced. Total fumonisin levels ranged from 127 to 11 052 microg g(-1). Additional studies on maize samples from 15 processing sites in Accra revealed a co-occurrence of both fumonisins and aflatoxins in 53% (8 of 15) of the samples.

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

Haematological, biochemical and toxicological investigations in spontaneous cases with different frequency of porcine nephropathy in Bulgaria.

Haematological, biochemical and toxicological investigations of blood and urine of normally slaughtered pigs exhibiting different frequency (1-2%, 10-20% and 50-60%) of changes characterized as "enlarged mottled kidneys", at the slaughtering meat inspection were carried out to elucidate the nature of nephropathies encountered in Bulgaria. A content of ochratoxin A, higher in the spring than the autumn, was found in the serum and urine samples. The mean contamination levels of ochratoxin A in consumed feeds ranged from 114 +/- 36 ppb for 1994 to 207 +/- 65 ppb for 1993. The renal changes were characterized by impairment of proximal tubular function (indicated by an increased urinary excretion of glucose and protein) as well as by decreased specific gravity and increased pH in the urine mainly in pigs with 50-60% frequency of nephropathy. The concentration of urea, creatinine and glucose in the blood was increased, whereas the serum protein and cholesterol were decreased in pigs with 10-20% and 50-60% frequency of nephropathy. The mean enzyme levels of gamma-glutamyl transpeptidase and leucine aminopeptidase were significantly increased in the urine. The presence of granular casts and necrotic renal tubular cells were established in the sediment.

Typhaea stercorea (Coleoptera: Mycetophagidae), a carrier of Salmonella enterica serovar Infantis in a Danish broiler house.

In December 1994, Salmonella enterica serovar Infantis (S. Infantis) was accidentally introduced into a Danish broiler house by stocking an S. Infantis-infected broiler flock of 39,900 day-old chicks. At the time of the study, the infection had persisted through 6 broiler cycles. Typhaea stercorea (L.), the hairy fungus beetle, was found in large quantities inside and around the broiler house. Various attempts to control the beetle had failed; T. stercorea had been uncontrollable on the farm since Alphitobius diaperinus (Panzer), the lesser mealworm, was eradicated approximately 10 yr earlier. We investigated the ability of T. stercorea to act as a carrier of S. Infantis in the broiler house between 2 broiler cycles. We examined the empty, cleaned, and disinfected broiler house for S. Infantis 3 d before stocking the 7th broiler flock, and S. Infantis was isolated from the beetles only. Of 20 singly examined T. stercorea, 9 (45%) were S. Infantis positive. A rapid spread of the infection was seen in the 7th broiler flock; 100% of the culled chicks were S. Infantis positive 3 d after stocking. Under experimental conditions chicks were observed eating beetles, and when 5 d-old, specified pathogen-free chicks were fed with S. Infantis-positive T. stercorea collected from the broiler house, all 5 chicks became infected in 4 d. We conclude that T. stercorea may act as a potential carrier of S. Infantis between successive broiler cycles.

Porcine nephropathy in Bulgaria: a progressive syndrome of complex or uncertain (mycotoxin) aetiology.

Macroscopic nephropathy was observed in 506 pigs at slaughter in Bulgaria in 1993/94. Histopathological changes were mainly degenerative and proliferative, and were linked with kidney hypertrophy similar to that of the classical Danish Syndrome. Retention cysts formed by dilated tubules, activation or proliferation of capillary and vascular endothelium, and the development of neoplastic tissue were also observed. The most advanced pathology took the form of extensive interstitial fibrosis. Traces of ochratoxin A were found in the kidneys of the majority of 96 cases examined, and in some feed samples taken retrospectively from farms or commercial sources. The dietary ochratoxin concentration (100 micrograms/kg), calculated from serum analyses, closely matched the average of individually analysed feeds. In other feeds no ochratoxin A was detected and the cosmopolitan mycobiota isolated did not include the ochratoxinogenic Penicillium verrucosum that caused the Danish syndrome. Aspergillus ochraceus was rare and the isolates did not synthesise ochratoxin in laboratory culture. The unconfirmed diagnosis of ochratoxicosis suggests a complex or multi-toxin aetiology for this rather common chronic disease in Bulgaria.

Healthy puppies and kittens as carriers of Campylobacter spp., with special reference to Campylobacter upsaliensis.

Living in a household with a dog or cat has previously been identified as a significant risk factor for acquiring campylobacteriosis, in particular, with reference to Campylobacter upsaliensis infection. In a cross-sectional study carried out in Denmark between August and December 1996, 72 healthy puppies and 42 healthy kittens, aged between 11 and 17 weeks, were sampled for fecal campylobacter shedding by culture of rectal swab specimens on blood-free agar base with cefoperazone at 32 mg/liter and amphotericin at 10 mg/liter and on blood-free agar base with cefoperazone at 8 mg/liter, teicoplanin at 4 mg/liter, and amphotericin at 10 mg/liter. Additionally, with respect to the C. upsaliensis transmission potential of poultry, a chicken cloacal swab sample from each of 100 different broiler flocks was included in the study for comparison. We found 21 (29%) of the puppies positive for Campylobacter spp., with a species distribution of 76% C. jejuni, 5% C. coli, and 19% C. upsaliensis. Of the kittens examined, two (5%) excreted campylobacters; both strains were C. upsaliensis. None of the chicken samples examined were found to be positive for C. upsaliensis. We concluded that young puppies and kittens are potential transmitters of human-pathogenic Campylobacter spp., including C. upsaliensis, while poultry seems negligible in C. upsaliensis epidemiology.

Ochratoxin A in cereals and the BCR-M&T-projects.

Reliable analytical procedures and certified reference materials are essential for the establishment and enforcement of tolerance levels for ochratoxin A in foods. The inadequacy of analytical procedures, together with the need for certified reference materials, led the Commission of the European Communities Community Bureau of Reference (BCR) to undertake a project to prepare suitable reference materials for ochratoxin A in wheat, in order to improve methodology and to harmonise agreement of results between member states. The first intercomparison study indicated problems in the analysis due to the influence of co-extractives in the matrix, and demonstrated that further work was necessary to improve recovery, clean-up and reproducibility. The second intercomparison study, in the EC Measurements and Testing Programme, correlated the performance of the different methods for ochratoxin A measurement in a separate batch of contaminated wheat, and compared novel immunoaffinity column methods with the standard laboratory methods. Results were obtained from 26 laboratories within 11 European countries, which therefore gives a good representation of the scope of methods currently used in Europe. Considerable improvements in the determination of ochratoxin A were noted compared with the first intercomparison study.

Ochratoxin A in wheat: an intercomparison of procedures.

The Commission of the European Communities' Community Bureau of Reference (BCR) has undertaken a project to improve methodology and to prepare suitable certified reference materials in order to provide a basis for analytical quality control for the determination of ochratoxin A. The first phase of the project, an intercomparison of procedures for the determination of ochratoxin A in wheat at a level of approximately 13 micrograms/kg, is described. The study involved 24 European laboratories which analysed a naturally contaminated wheat and a 'blank' wheat sample (ochratoxin A content < 1 microgram/kg). The participants used a variety of procedures, including chloroform, methanol, toluene and ethyl acetate for extraction, and silica-, reversed phase- and immunoaffinity columns for clean-up. HPLC (one laboratory used TLC) was applied as the determinative step. Several performance characteristics were checked and the ochratoxin A content was determined. Recoveries were found to range from 25 to 100%. The coefficient of variation from all the results calculated on the basis of peak height was 23%. The study showed that the variation of results was influenced more by the clean-up step than by the extraction solvent. Some laboratories suffered significant day-to-day effects while others found difficulties with interfering peaks in the 'blank' material. It is planned for the next study to improve the recovery range, the clean-up step and the reproducibility (within-laboratory, between-days) and to check the influence of co-extractives from the matrix.

Ochratoxin A in human blood in European countries.

Ochratoxin A is a mycotoxin which contaminates cereals in particular and occurs all over the world. Humans are undoubtedly exposed to this toxin through foods of vegetable and animal origin and through airborne dust. The presence of ochratoxin A in human blood has been suggested as an indicator for indirect assessment of exposure to this nephrotoxic agent. In several countries, therefore, human blood has been collected with the purpose of obtaining more information on the intake of ochratoxin A. Analyses of serum samples in European countries revealed that blood from healthy humans was contaminated with ochratoxin A at concentrations of 0.1-14.4 micrograms/l. The frequency of contamination of human sera seems to indicate continuous, widespread exposure of humans to ochratoxin A.

Porcine nephropathy in Europe.

Numerous surveys conducted in North America, Asia and Europe have revealed that ochratoxin A is a natural contaminant of plant products. Contamination frequencies of up to 40% have been encountered, at levels in the range of 5-500 micrograms/kg. Ochratoxin A is a major causal determinant of the disease porcine nephropathy; but other nephrotoxic mycotoxins, such as citrinin and the fungal quinones, may be involved. The disease is characterized clinically by polyuria and growth depression. Renal lesions in pigs include degeneration of the proximal tubules, interstitial fibrosis and hyalinization of the glomeruli. The disease is endemic, outbreaks being associated with bad weather conditions. A positive correlation has been observed between the prevalence rates of porcine nephropathy and the frequency of ochratoxin A in corresponding feed samples. Surveys for residues of ochratoxin A in kidneys from cases of porcine nephropathy in a number of European countries other than Denmark have demonstrated that 21-42% of samples contain ochratoxin A in the range of 1-100 micrograms/kg.