ccug 11284 is a wild - type strain that was originally isolated from bovine feces . before each experiment , bacteria were grown on conventional blood agar plates ( columbia agar ii containing 8% vol / vol whole horse blood ) at 42c for 20 h in a microaerobic environment , using a campygen gas generating system ( cn0025a ; oxoid ltd . , basingstoke , uk ) and a bbl gaspak system ( bd , franklin lakes , nj ) .
bacterial cells were harvested and diluted in a peptone - yeast extract - glucose ( pyg ) medium and used as stock solution for all treatments .
the stock solution was striven to obtain a concentration of approximately 10 cfu / ml , as were detected by plate counting .
a. polyphaga stock cultures were maintained in pyg medium at 27c in 75 cm culture flasks ( sarstedt , nrnbrecht , germany ) , as described by axelsson - olsson et al .
, a. polyphaga were seeded into 12-well culture plates ( fischer scientific gtf ab , switzerland ) in pyg medium ( 1 ml / well ) and incubated at 27c for 24 h , until the trophozoites formed confluent layers at the bottom of the wells . commercially available milk with a ph of 6.4 ( protein 3.4 g , sugar 5 g , fat 1.5 g , ca 120 mg , vitamin a 25 g , vitamin d 0.38 g ) and orange juice with a ph of 3.9 ( protein 0.7 g , sugar 18 g , fat < 0.5 g , na 0.003 g , vitamin c 30 mg ) were used for all experiments .
basingstoke , uk ) was added to the products to inhibit growth of other bacteria than c. jejuni .
to mimic the conditions of storage in the fridge or at the bench , experiments were incubated at room temperature and 4c . to test
whether the presence of amoeba in two different beverage products , milk and orange juice , influenced the survival of c. jejuni , the following three treatments were used : c. jejuni preincubated with a. polyphaga before the addition of product ( treatment a ) , c. jejuni mixed with a. polyphaga after the addition of product ( treatment b ) , and c. jejuni in product without a. polyphaga ( treatment c ) . for treatment a , 12-well plates with confluent a. polyphaga layers in pyg medium were inoculated with 100 l of the c. jejuni stock solution , generating a concentration of 10 cfu / ml and a multiplicity of infection ( moi ) of one bacteria per amoeba , in each well . before inoculation with c. jejuni , the medium in all wells were gently removed and replaced with 1 ml fresh pyg medium .
the plates were incubated for 3 h at 32c to allow the bacterial cells to attach to and invade amoebae , and thereafter the pyg medium was gently removed and replaced by 2 ml of product .
plates with confluent a. polyphaga were prepared by gently removing the pyg medium and replacing it with 2 ml of product . for the control treatment ( treatment c ) , plates without amoebae
after the addition of product , the plates for treatment b and c were inoculated with 100 l of the c. jejuni stock solution generating a concentration of 510 cfu / ml and an moi of one bacteria per amoeba in treatment b , in each well .
three plates ( treatments a  c ) were incubated at room temperature and at 4c , respectively .
all plates were incubated in an aerobic environment and each treatment was done in triplicate wells , resulting in three similar wells for each temperature , treatment and product .
from each well , a 100-l sample was taken at time zero ( the addition of product ) and at 3 , 6 , 18 , 24 , and 48 h. all samples were 10-fold serially diluted in pyg medium and spread on blood agar plates for colony counting .
three independent experiments were performed on separate occasions . to make sure that the ph level was not affected ,
the ph level of the fluid in each well was measured after 48 h , when experiments were completed .
compared to initial ph ( milk : 6.41 and juice : 3.89 ) only a small increase in ph was observed ( milk : 0.4 and juice : 0.1 ) . for pasteurization experiments ,
the same settings were used , as described above for treatments a , b , and c. directly after the addition of product , samples of 100 l were taken from the different treatments , a , b , and c and added to tubes containing 500 l of milk or juice .
the tubes were gently shaken at 1,400 rpm ( ms2 minishaker ika , germany ) and then incubated in a water bath ( heto dt hetotherm , denmark ) .
incubation conditions for milk tubes were 7274c for 15 sec ( equivalent to swedish low pasteurization guidelines ) .
incubation conditions for juice tubes were 85c for 15 sec ( equivalent to swedish pasteurization guidelines ) .
after heating , the sample tubes were put on ice and 100-l samples were spread on blood agar for colony counting .
all experiments were done in triplicates , resulting in three similar wells for each treatment and product . for each well and each time point , a measure of c. jejuni cell survival was calculated by dividing the bacterial concentration of the sample ( estimated from colony counts ) by the bacterial concentration of that well at time 0 h ( the addition of product ) .
the c. jejuni strain ccug 11284 and the a. polyphaga strain ( linc ap-1 ) were used in all experiments .
ccug 11284 is a wild - type strain that was originally isolated from bovine feces . before each experiment , bacteria were grown on conventional blood agar plates ( columbia agar ii containing 8% vol / vol whole horse blood ) at 42c for 20 h in a microaerobic environment , using a campygen gas generating system ( cn0025a ; oxoid ltd . , basingstoke , uk ) and a bbl gaspak system ( bd , franklin lakes , nj ) .
bacterial cells were harvested and diluted in a peptone - yeast extract - glucose ( pyg ) medium and used as stock solution for all treatments .
the stock solution was striven to obtain a concentration of approximately 10 cfu / ml , as were detected by plate counting .
a. polyphaga stock cultures were maintained in pyg medium at 27c in 75 cm culture flasks ( sarstedt , nrnbrecht , germany ) , as described by axelsson - olsson et al .
, a. polyphaga were seeded into 12-well culture plates ( fischer scientific gtf ab , switzerland ) in pyg medium ( 1 ml / well ) and incubated at 27c for 24 h , until the trophozoites formed confluent layers at the bottom of the wells . commercially available milk with a ph of 6.4 ( protein 3.4 g , sugar 5 g , fat 1.5 g , ca 120 mg , vitamin a 25 g , vitamin d 0.38 g ) and orange juice with a ph of 3.9 ( protein 0.7 g , sugar 18 g , fat < 0.5 g , na 0.003 g , vitamin c 30 mg ) were used for all experiments .
basingstoke , uk ) was added to the products to inhibit growth of other bacteria than c. jejuni .
to mimic the conditions of storage in the fridge or at the bench , experiments were incubated at room temperature and 4c . to test whether the presence of amoeba in two different beverage products , milk and orange juice , influenced the survival of c. jejuni , the following three treatments were used : c. jejuni preincubated with a. polyphaga before the addition of product ( treatment a ) , c. jejuni mixed with a. polyphaga after the addition of product ( treatment b ) , and c. jejuni in product without a. polyphaga ( treatment c ) . for treatment
a , 12-well plates with confluent a. polyphaga layers in pyg medium were inoculated with 100 l of the c. jejuni stock solution , generating a concentration of 10 cfu / ml and a multiplicity of infection ( moi ) of one bacteria per amoeba , in each well . before inoculation with c. jejuni , the medium in all wells were gently removed and replaced with 1 ml fresh pyg medium .
the plates were incubated for 3 h at 32c to allow the bacterial cells to attach to and invade amoebae , and thereafter the pyg medium was gently removed and replaced by 2 ml of product .
plates with confluent a. polyphaga were prepared by gently removing the pyg medium and replacing it with 2 ml of product . for the control treatment ( treatment c ) , plates without amoebae
after the addition of product , the plates for treatment b and c were inoculated with 100 l of the c. jejuni stock solution generating a concentration of 510 cfu / ml and an moi of one bacteria per amoeba in treatment b , in each well .
three plates ( treatments a  c ) were incubated at room temperature and at 4c , respectively .
all plates were incubated in an aerobic environment and each treatment was done in triplicate wells , resulting in three similar wells for each temperature , treatment and product . from each
well , a 100-l sample was taken at time zero ( the addition of product ) and at 3 , 6 , 18 , 24 , and 48 h. all samples were 10-fold serially diluted in pyg medium and spread on blood agar plates for colony counting .
three independent experiments were performed on separate occasions . to make sure that the ph level was not affected ,
the ph level of the fluid in each well was measured after 48 h , when experiments were completed .
compared to initial ph ( milk : 6.41 and juice : 3.89 ) only a small increase in ph was observed ( milk : 0.4 and juice : 0.1 ) .
for pasteurization experiments , the same settings were used , as described above for treatments a , b , and c. directly after the addition of product , samples of 100 l were taken from the different treatments , a , b , and c and added to tubes containing 500 l of milk or juice .
the tubes were gently shaken at 1,400 rpm ( ms2 minishaker ika , germany ) and then incubated in a water bath ( heto dt hetotherm , denmark ) .
incubation conditions for milk tubes were 7274c for 15 sec ( equivalent to swedish low pasteurization guidelines ) .
incubation conditions for juice tubes were 85c for 15 sec ( equivalent to swedish pasteurization guidelines ) .
after heating , the sample tubes were put on ice and 100-l samples were spread on blood agar for colony counting .
all experiments were done in triplicates , resulting in three similar wells for each treatment and product .
for each well and each time point , a measure of c. jejuni cell survival was calculated by dividing the bacterial concentration of the sample ( estimated from colony counts ) by the bacterial concentration of that well at time 0 h ( the addition of product ) .
the experimental setup included three different treatments ( a , b , and c ) ; see materials and methods section . in milk , the highest c.
jejuni survival was seen in treatment a , where bacteria were pre - incubated with amoebae before addition of milk ( 2.8% , 18 h ; 3.8% , 24 h ; 0.8% , 48 h ; table 1 ; fig .
treatment b showed 0.05% survival at 18 h and reached a fraction of 6.710of the inoculum at 48 h ( equivalent to 14 cfu / ml ; table 1 ) .
after 3 h , the survival of c. jejuni without amoebae ( treatment c ) decreased more rapidly compared to co - cultures ( treatments a and b ) , and the fraction of the inoculum surviving after 18 h was only 3.510 ( equivalent to 6 cfu / ml ; table 1 ) .
no bacteria could be detected after 24 h. at 1848 h , treatment a had significantly higher bacterial survival than treatment c ( kruskal  wallis test with dunn 's multiple comparison test and bonferroni correction for multiple tests ; 18 h , p=0.0003 ; 24 h , p<0.0001 ; 48 h , p=0.0007 ; fig .
. survival of c. jejuni co - incubated with a. polyphaga in milk ( a ) or orange juice ( b ) at room temperature after 0 h , 3 h , 6 h , 24 h , and 48 h. data are based on three independent experiments with c. jejuni treated in three different ways treatment a ( dots ) , c. jejuni preincubated with a. polyphaga before the addition of product ; treatment b ( squares ) , c. jejuni inoculated to a. polyphaga after the addition of product ; and treatment c ( triangles ) , c. jejuni in product without a. polyphaga . to use the log10 scale , a constant
meanssem ( missing data points : one out of nine replicates for : milk treatment b at 0 h , milk treatment a at 3 h and juice treatment b at 3 h ) . mean fraction of the c. jejuni inoculum surviving after 18 h , 24 h , and 48 h for treatments a , b , and c when incubated in milk and juice at room temperature ( rt ) and at 4c 
 c. jejuni incubated in juice showed similar trends as in milk albeit with more pronounced differences between co - cultures and bacteria incubated alone .
the highest c. jejuni survival was seen in treatment a ( 88.4% , 18 h ; 36.9% , 24 h ; 0% , 48 h ; table 1 ; fig .
1b ) and the lowest survival was found in treatment c , c. jejuni without amoebae .
after 3 h , the survival of c. jejuni incubated without amoebae decreased rapidly compared to co - cultures ( treatments a and b ) and no viable bacteria could be detected after 18 h ( table 1 , fig .
1b ) . the relative survival of c. jejuni in treatment b ( 4.1% , 18 h ; 2.0% , 24 h ; 0.02% , 48 h ; table 1 ) was lower than in treatment a but higher than in c. however , due to the higher start concentration in treatment b , compared to treatment a , the bacterial concentration in the juice was still
45 cfu / ml at 48 h. statistically significant differences in survival were seen between : a and b ( kruskal  wallis test with dunn 's multiple comparison test and bonferroni correction for multiple tests : 18 h , p=0.0428 ; 24 h , p=0.0428 ; 48 h , p=0.0029 ; a and c : 18 h , p<0.0001 ; 24 h , p<0.0001 ; b and c : 18 h , p=0.0428 ; 24 h , p=0.0428 ; 48 h , p=0.0029 ; fig . 1b ; table 1 ) . at 4c , c. jejuni generally survived better in both milk and juice and the differences were less pronounced between co - cultures and bacteria incubated alone . in milk , the highest c.
jejuni survival was seen in treatment a for time point up to 24 h ( 90.3% , 18 h ; 34.3% , 24 h ; table 1 ; fig .
at these time points , treatment c ( 24.1% , 18 h ; 20.1% , 24 h ; table 1 ) gave a higher survival than treatment b ( 19.2% , 18 h ; 18.2% , 24 h ; table 1 ) .
at 48 h , the highest c. jejuni survival was seen in treatment c ( 3.3% table 1 ) , and the survival in treatments a and b were 0.4 and 0.6% , respectively ( table 1 ) .
statistically significant differences in survival were seen at 18 h between : a and b as well as a and c ( kruskal  wallis test with dunn 's multiple comparison test and bonferroni correction for multiple tests : a and b:18 h , p=0.001 ; a and c : 18 h , p=0.0045 ; fig .
survival of c. jejuni co - incubated with a. polyphaga in milk ( a ) or orange juice ( b ) at 4c after 0 h , 3 h , 6 h , 24 h , and 48 h. data are based on three independent experiments with c. jejuni treated in three different ways treatment a ( dots ) , c. jejuni preincubated with a. polyphaga before the addition of product ; treatment b ( squares ) , c. jejuni inoculated to a. polyphaga after the addition of product ; and treatment c ( triangles ) , c. jejuni in product without a. polyphaga . to use the log10 scale , a constant 1 had to be added to all cfu values to manage zeros .
meanssem ( missing data points : one out of nine replicates for juice treatment c at 18 h ) .
when c. jejuni were incubated in juice at 4c , the differences seen between co - cultures ( treatments a and b ) and c. jejuni incubated without amoebae ( treatment c ) , were similar to those seen in juice at room temperature , although the bacteria in treatment c survived longer compared to incubation at room temperature .
the highest c. jejuni survival was seen in treatment a : 90.6% , 18 h ; 56.7% , 24 h ; 0.35% , 48 h ; table 1 ) and the lowest survival was found in treatment c : 3.3% , 18 h ; 2.0% , 24 h ; 0.00074% ; 1 cfu / ml ; 48 h ; table 1 ) .
the survival of c. jejuni in treatment b : 7.4% , 18 h ; 3.4% , 24 h ; 0.01% , equivalent to 18 cfu / ml ; 48 h , table 1 ) was lower than a but higher than c. statistically significant differences in survival were seen at 1824 h between a and b , and 1848 h between a and c ( kruskal  wallis test with dunn 's multiple comparison test and bonferroni correction for multiple tests ; a and b : 18 h , p=0.0125 ; 24 h , p=0.005 ; a and c : 18 h , p=0.0001 ; 24 h , p=0.0002 ; 48 h , p=0.0489 ; fig .
a. polyphaga did not show any protective effect on c. jejuni when heated to recommended pasteurization temperatures , neither in milk nor in orange juice ( data not shown ) .
growth was totally inhibited in all samples from treatments a , b , and c. results were based on three independent experiments .
the experimental setup included three different treatments ( a , b , and c ) ; see materials and methods section . in milk , the highest c.
jejuni survival was seen in treatment a , where bacteria were pre - incubated with amoebae before addition of milk ( 2.8% , 18 h ; 3.8% , 24 h ; 0.8% , 48 h ; table 1 ; fig .
treatment b showed 0.05% survival at 18 h and reached a fraction of 6.710of the inoculum at 48 h ( equivalent to 14 cfu / ml ; table 1 ) .
after 3 h , the survival of c. jejuni without amoebae ( treatment c ) decreased more rapidly compared to co - cultures ( treatments a and b ) , and the fraction of the inoculum surviving after 18 h was only 3.510 ( equivalent to 6 cfu / ml ; table 1 ) .
no bacteria could be detected after 24 h. at 1848 h , treatment a had significantly higher bacterial survival than treatment c ( kruskal  wallis test with dunn 's multiple comparison test and bonferroni correction for multiple tests ; 18 h , p=0.0003 ; 24 h , p<0.0001 ; 48 h , p=0.0007 ; fig .
. survival of c. jejuni co - incubated with a. polyphaga in milk ( a ) or orange juice ( b ) at room temperature after 0 h , 3 h , 6 h , 24 h , and 48 h. data are based on three independent experiments with c. jejuni treated in three different ways treatment a ( dots ) , c. jejuni preincubated with a. polyphaga before the addition of product ; treatment b ( squares ) , c. jejuni inoculated to a. polyphaga after the addition of product ; and treatment c ( triangles ) , c. jejuni in product without a. polyphaga . to use the log10 scale , a constant
meanssem ( missing data points : one out of nine replicates for : milk treatment b at 0 h , milk treatment a at 3 h and juice treatment b at 3 h ) . mean fraction of the c. jejuni inoculum surviving after 18 h , 24 h , and 48 h for treatments a , b , and c when incubated in milk and juice at room temperature ( rt ) and at 4c 
 c. jejuni incubated in juice showed similar trends as in milk albeit with more pronounced differences between co - cultures and bacteria incubated alone .
the highest c. jejuni survival was seen in treatment a ( 88.4% , 18 h ; 36.9% , 24 h ; 0% , 48 h ; table 1 ; fig .
1b ) and the lowest survival was found in treatment c , c. jejuni without amoebae .
after 3 h , the survival of c. jejuni incubated without amoebae decreased rapidly compared to co - cultures ( treatments a and b ) and no viable bacteria could be detected after 18 h ( table 1 , fig .
1b ) . the relative survival of c. jejuni in treatment b ( 4.1% , 18 h ; 2.0% , 24 h ; 0.02% , 48 h ; table 1 ) was lower than in treatment a but higher than in c. however , due to the higher start concentration in treatment b , compared to treatment a , the bacterial concentration in the juice was still
45 cfu / ml at 48 h. statistically significant differences in survival were seen between : a and b ( kruskal  wallis test with dunn 's multiple comparison test and bonferroni correction for multiple tests : 18 h , p=0.0428 ; 24 h , p=0.0428 ; 48 h , p=0.0029 ; a and c : 18 h , p<0.0001 ; 24 h , p<0.0001 ; b and c : 18 h , p=0.0428 ; 24 h , p=0.0428 ; 48 h , p=0.0029 ; fig . 1b ; table 1 ) .
at 4c , c. jejuni generally survived better in both milk and juice and the differences were less pronounced between co - cultures and bacteria incubated alone . in milk , the highest c.
jejuni survival was seen in treatment a for time point up to 24 h ( 90.3% , 18 h ; 34.3% , 24 h ; table 1 ; fig .
at these time points , treatment c ( 24.1% , 18 h ; 20.1% , 24 h ; table 1 ) gave a higher survival than treatment b ( 19.2% , 18 h ; 18.2% , 24 h ; table 1 ) . at 48 h , the highest c.
jejuni survival was seen in treatment c ( 3.3% table 1 ) , and the survival in treatments a and b were 0.4 and 0.6% , respectively ( table 1 ) .
statistically significant differences in survival were seen at 18 h between : a and b as well as a and c ( kruskal  wallis test with dunn 's multiple comparison test and bonferroni correction for multiple tests : a and b:18 h , p=0.001 ; a and c : 18 h , p=0.0045 ; fig .
survival of c. jejuni co - incubated with a. polyphaga in milk ( a ) or orange juice ( b ) at 4c after 0 h , 3 h , 6 h , 24 h , and 48 h. data are based on three independent experiments with c. jejuni treated in three different ways treatment a ( dots ) , c. jejuni preincubated with a. polyphaga before the addition of product ; treatment b ( squares ) , c. jejuni inoculated to a. polyphaga after the addition of product ; and treatment c ( triangles ) , c. jejuni in product without a. polyphaga . to use the log10 scale , a constant 1 had to be added to all cfu values to manage zeros .
meanssem ( missing data points : one out of nine replicates for juice treatment c at 18 h ) .
when c. jejuni were incubated in juice at 4c , the differences seen between co - cultures ( treatments a and b ) and c. jejuni incubated without amoebae ( treatment c ) , were similar to those seen in juice at room temperature , although the bacteria in treatment c survived longer compared to incubation at room temperature .
the highest c. jejuni survival was seen in treatment a : 90.6% , 18 h ; 56.7% , 24 h ; 0.35% , 48 h ; table 1 ) and the lowest survival was found in treatment c : 3.3% , 18 h ; 2.0% , 24 h ; 0.00074% ; 1 cfu / ml ; 48 h ; table 1 ) .
the survival of c. jejuni in treatment b : 7.4% , 18 h ; 3.4% , 24 h ; 0.01% , equivalent to 18 cfu / ml ; 48 h , table 1 ) was lower than a but higher than c. statistically significant differences in survival were seen at 1824 h between a and b , and 1848 h between a and c ( kruskal  wallis test with dunn 's multiple comparison test and bonferroni correction for multiple tests ; a and b : 18 h , p=0.0125 ; 24 h , p=0.005 ; a and c : 18 h , p=0.0001 ; 24 h , p=0.0002 ; 48 h , p=0.0489 ; fig .

 a. polyphaga did not show any protective effect on c. jejuni when heated to recommended pasteurization temperatures , neither in milk nor in orange juice ( data not shown ) .
growth was totally inhibited in all samples from treatments a , b , and c. results were based on three independent experiments .

 campylobacter causes approximately 200,000 human infections annually in the european union , with sometimes very severe chronic complications such as guillain  barre syndrome ( gbs ) ( 36 , 37 ) . in this study , we investigated the protective effect of the free - living amoeba a. polyphaga on c. jejuni survival in milk and orange juice .
it is well known that drinking unpasteurized milk is a risk for acquiring c. jejuni infections ( 38 ) ; however , the consumption of unpasteurized milk and milk products ( 39 , 40 ) as well as unpasteurized juices is common in many countries ( 12 ) .
the acidic ph in orange juice has been considered lethal to c. jejuni ( 23 , 41 ) , and therefore orange juices have not been considered a risk factor for acquiring campylobacteriosis . on the other hand ,
c. jejuni seem well adapted to survive the acidic milieu of the human stomach as well as disinfection with acid in poultry stables ( 42 , 43 ) .
we have previously shown an increased acid tolerance of c. jejuni ccug 11284 in co - cultures with a. polyphaga ( 26 .
free - living amoebae are common inhabitants of potable water plumbing systems ( 44 , 45 ) and fruit squeezing machines have surfaces that are difficult to clean , with possible formation of biofilms inhabited by amoebae as a result ( 32 , 3446 ) .
hence , such systems could provide entry ports where the presence of amoeba could increase the c. jejuni survival and cause infections . as differences in bacterial survival
were observed in a previous study depending on weather the bacteria were added to a. polyphaga before ( treatment a ) or after addition of acidified medium ( treatment b ) , we evaluated the effect of these two treatments in juice and milk ( 26 ) .
we found significantly higher bacterial survival in co - cultures compared to when c. jejuni were incubated alone ( treatment c ) . in both products and at both temperatures , the highest survival was found in co - cultures where c. jejuni were added to a. polyphaga before the addition of the product ( treatment a ) .
the effect of co - culture was most pronounced in juice stored at room temperature , as no c. jejuni survival was detected after 18 h in cultures with c. jejuni alone . also milk
stored at room temperature and juice stored at 4c showed significantly higher bacterial survival in co - cultures , compared to when c. jejuni were incubated alone .
however , in milk stored at 4c the bacterial survival at 24 h was not significantly affected by co - culture with amoebae and at 48 h , c. jejuni incubated alone ( treatment c ) actually survived better than in co - culture .
this is consistent with previous studies reporting good survival of campylobacter in refrigerated milk ( 41 , 47 ) . in our previous study
assessing c. jejuni survival in an acidified medium , we found the highest bacterial survival in co - cultures where c. jejuni were added after the addition of acidic media . in that study we found that the acid milieu triggered c. jejuni motility and uptake into the amoebae .
however , the products tested here are more complex than a defined bacterial growth medium and other constituent of milk and juice may likely have affected the results . in the majority of cases ,
this was not studied by us , and hence possible c. jejuni protection from amoebae present in low concentrations needs to be evaluated in the future .
high concentrations of amoebae could be present in contaminated water or by growth of amoebae in beverages stored at room temperature for a longer period of time . in all experiments
the survival curves for c. jejuni in treatment a were characterized by an increase in bacterial concentration at the beginning of the experiments ( 3 and 6 h ) .
this increase might be explained by extracellular c. jejuni residing in the adherent trophozoite layer . when the medium was changed from pyg to milk or juice these bacteria
together , our results suggest that a. polyphaga can prolong c. jejuni survival both in milk and juice .
it has been shown that legionellae increase their thermal resistance when co - cultured with acanthamoebae , and that intracellular legionellae pneumophila can survive temperatures up to 80c ( 48 ) .
acanthamoebae cysts alone have been shown to survive at temperatures up to 80c and even up to 95c for at least 10 min ( 4850 ) .
we studied if c. jejuni in co - culture with a. polyphaga could survive heating to recommended pasteurization temperatures for milk ( 7274c ) and juice ( 85c ) .
however , a. polyphaga did not have any effect on c. jejuni survival during pasteurization of milk or orange juice , confirming that this is a good method for eliminating c. jejuni in these products . in conclusion ,
amoebae associated c. jejuni in milk and juice survived better than free bacteria both at room temperature and at 4c , but a. polyphaga could not protect the bacteria from pasteurization .
 background 
 campylobacter jejuni is a common cause of human bacterial diarrhea in most parts of the world . 
 most c. jejuni infections are acquired from contaminated poultry , milk , and water . due to health care costs and human suffering , 
 it is important to identify all possible sources of infection . 
 unpasteurized milk has been associated with several outbreaks of c. jejuni infection . 
 campylobacter has been identified on fresh fruit , and other gastrointestinal pathogens such as salmonella , e. coli o157:h7 and cryptosporidium have been involved in fruit juice outbreaks . c. jejuni is sensitive to the acidic environment of fruit juice , but co - cultures with the amoeba , acanthamoeba polyphaga , have previously been shown to protect c. jejuni at low ph.methodsto study the influence of a. polyphaga on the survival of c. jejuni in milk and juice , the bacteria were incubated in the two products at room temperature and at 4c with the following treatments : a ) c. jejuni preincubated with a. polyphaga before the addition of product , b ) c. jejuni mixed with a. polyphaga after the addition of product , and c ) c. jejuni in product without a. polyphaga . 
 bacterial survival was assessed by colony counts on blood agar plates.resultsco-culture with a. polyphaga prolonged the c. jejuni survival both in milk and juice . 
 the effect of co - culture was most pronounced in juice stored at room temperature . on the other hand 
 , a. polyphaga did not have any effect on c. jejuni survival during pasteurization of milk or orange juice , indicating that this is a good method for eliminating c. jejuni in these products.conclusionamoebae-associated c. jejuni in milk and juice might cause c. jejuni infections . 