the number of flies transported by means of ventilation air into a broiler house in denmark was counted from july 22 , 2003 , to july 28 , 2003 , and the campylobacter carriage rate of flies captured in the environment of the broiler house was estimated .
the study period was chosen because flies generally peak in activity and abundance in july to august in denmark .
in addition , chickens in the broiler house and the animals in the area around the broiler house ( 5 sheep , 4 horses , and 1 dog with 10 puppies ) were tested for carriage of campylobacter .
dna from all c. jejuni isolates was analyzed by pulsed - field gel electrophoresis ( pfge ) to determine if strains from different animals were similar .
the broiler house ( 80 m x 15 m ) was located at universal transverse mercator grid zone 32 , east 564,137 m , north 6,294,759 m ( http://mac.usgs.gov/mac/isb/pubs/factsheets/fs07701.html ) .
the facility was negative - pressure ventilated through a total of 84 wall valves for air intake ( 16.5 cm x 52.5 cm ) and 12 round chimneys ( diameter 62 cm ) for active air outlet through the roof .
the house was emptied , and the chickens ( n = 28,235 ) were slaughtered on july 29 , 2003 .
reports of local weather data from the danish meteorological institute ( www.dmi.dk ) for that week were a maximum day temperature of 25.4c , a minimum night temperature of 11.9c , and days with bright sunshine with no wind or rain .
flies were collected in polyester nets equipped at two wall inlets ( one net at each end of the house ) in the dynamic air flow measured ( 7 ) at a pressure of 21 pa of influx ventilation air ( speed 3.6 m / s , volume 1,213 m / h per inlet valve ) .
after the nets were harvested , flies were visually sorted from other insects and counted .
the flies , identified primarily to the order diptera and the families muscidae and calliphoridae , were counted ; the count showed that 917  standard deviation ( sd ) ( 843.5990.5 ) flies ( [ a + b]/2 x 84 , with a and b representing the number of flies in the two nets ) had entered the broiler house per day through the ventilation system , or approximately 1 fly per 2,700 m of ventilation air ( 1,213 m / h x 84 x 24 h/917 ) .
for this specific broiler house , this amount equals approximately 30,000 flies per broiler cycle in the summer season . to estimate the possibility of roof inlets as entrance route for flies into houses with an air inlet in the roof ,
the result showed 167 flies per day entered through this one chimney , or an average of 4.5 flies per 2,700 m air .
the flies captured in the ventilation system were used for counting only , since they were dead and dried out at the time of harvest . to determine the prevalence of campylobacter in flies around the broiler house ,
a total of 96 flies were captured singly within a distance of 50 m from the house by rackets equipped with small disposable plastic bags .
after species or genus was determined , each fly was transferred with a pair of tweezers to live storage in a sterile plastic tube with 1 ml of saline .
the tubes were kept in an insulated container , which was transported to the laboratory within 24 hours after capture . for campylobacter detection , each fly
the pellet was resuspended in 2 ml of bolton broth ( cm0983 with sr0183 and sr0048 ) ( oxoid , basingstoke , uk ) and vortexed before incubation for enrichment at 37c for 24 h. after enrichment , the tube was again centrifuged , and 100 l of the sample from each of 49 tubes was streaked onto modified cefoperazone charcoal deoxycholate agar ( mccda ) ( blood - free agar base supplemented with cm739 + sr155 ) ( oxoid ) .
these plates were incubated at 42c for 48 h in a microaerobic atmosphere ( 6% o2 , 6% co2 , 4% h2 in n2 ) .
the tubes from the remaining 47 samples were subjected to dna analysis by a nested polymerase chain reaction ( pcr ) ( 8) .
the results of conventional culture showed that , of 49 flies tested , c. jejuni was isolated from 4 ( 8.2% ) ( figure 1a ) ; when a nested pcr was used , 33 ( 70.2% ) of the 47 flies were campylobacter - positive ( figure 1b ) .
the species distribution according to the nested pcr when species - specific primers were used showed that , of 47 samples , 56.4% were positive with c. jejuni primers , 18.0% were positive with c. coli primers , and 25.6% were positive with campylobacter species primers .
coinfection with c. jejuni and c. coli or campylobacter spp . was found in six flies
the reason for dividing the flies into two equal portions , one for conventional culture and the other for pcr , was to avoid reducing the assumed low number of campylobacter on each fly .
campylobacter - positive and -negative by a ) campylobacter bacteriologic culture , and b ) nested polymerase chain reaction .
cloacal or rectal swab samples from 20 broilers , 5 sheep , and 4 horses were cultured on mccda agar , as described above . rectal swabs from 11 dogs were streaked onto cefoperazone amphoricin teicoplanin ( cat ) agar plates ( blood - free agar base with cm739 and sr174 ) ( oxoid ) , and incubated at 42c for 96 h. subsequently , the swabs were tested for campylobacter by our laboratory 's routine pcr for feces ( 9 ) . c. jejuni was isolated from 20 broilers and 4 sheep and c. upsaliensis from 11 dogs .
a total of 28 c. jejuni isolates from 20 broilers , 4 flies , and 4 sheep were fingerprinted with pfge with two different restriction enzymes , smai and kpni . with both enzymes , 27 of the isolates
had an identical pfge pattern ( s6 ) , whereas a single broiler isolate had a slightly different , but closely related pattern ( s7 ) , which probably was derived from the more prevalent pattern .
twenty - seven of 28 isolates from three animal sources , broilers , sheep , and flies , and from both inside and outside the broiler house , belonged to the same clone .
dendrogram of pulsed - field gel electrophoresis types with smai of campylobacter jejuni isolates from chickens in a danish broiler house and from animals outside ( flies and sheep ) . under experimental conditions ( 10 ) , flies are able to transmit campylobacter among chickens .
moreover , a high prevalence of campylobacter - infected flies captured in a broiler house has been found ( 11 ) .
however , no study has yet been able to demonstrate a significant role of flies captured in the houses for transmitting infection from flock to flock ( 5 ) .
our results suggest that the potential of flies to transmit infection depends upon a current supply to the broiler house of campylobacter - infected flies from the outside .
furthermore , the number of flies entering the broiler house must increase as the need for ventilation air increases as a consequence of the growth of chickens .
thus , the risk of introducing campylobacter to the house increases with the age of the chickens .
this study has demonstrated that flies pose a threat of campylobacter infection , from which chickens currently are unprotected from april to october , when insects are in season in the northern hemisphere .
we found that in july hundreds of flies per day passed through the ventilation system into a broiler house and that 8.2% of flies captured in the environment had the potential to transmit c. jejuni from outside animals to chickens in the broiler house .
these results warrant further research on how to combat the summer peak of campylobacter in broilers to improve the safety of the human food supply .
 a total of 8.2% of flies caught outside a broiler house in denmark had the potential to transmit campylobacter jejuni to chickens , and hundreds of flies per day passed through the ventilation system into the broiler house . 
 our study suggests that flies may be an important source of campylobacter infection of broiler flocks in summer . 
The Study
Conclusions