SURVIVAL OF CAMPYLOBACTER JEJUNI IN NATURALLY AND ARTIFICIALLY CONTAMINATED LAYING HEN
MATERIALS AND METHODS Sample Origin and Preparation
C. jejuni Typing
FlaA typing by Restriction fragment length polymorphism (RFLP) of flaA gene with modifications according to Nachamkin et al. (1993, 1996) was used to type C. jejuni isolates.
The PCR was performed with a total final volume 50 µL using peqGOLD Taq-DNA-Polymerase 'all inclusive' (PeQlab Biotechnologie, Germany) according to the manufacturer’s instructions and 1 ȝL of each primer (Biometra, Germany). PCR was performed with the same Mastercycler used for the mapA gene according to the following cycling conditions:
One incubation cycle for 1 min at 94 °C and then for 30s at 94 °C, 45s at 45 °C and 2 min at 72 °C for 30 cycles. The samples were terminated by a final extension step of 5 min at 72 °C and were maintained at 4 °C until analysis. Seven microliters of PCR product was first
checked for the presence of an expected 1.7 kb band representing a sequence of C. jejuni flaA gene by electrophoresis on a 1.5 % agarose gel for 1 h. If a clear band was obtained, the remaining PCR products were purified using the MinElute PCR Purification kit (QIAGEN, Germany) following the manufacturer’s instructions, producing about 10 µL pure flaA gene fragments. Five microliters of the purified PCR product were subjected to flaA typing using the restriction enzyme DdeI (Roche, Germany) using the protocol recommended by the CAMPYNET research forum (http://campynet.vetinst.dk/Fla.htm) and incubated at 37 ºC for 1 h. The digested DNA was analyzed by 2.5 % agarose gel electrophoresis stained with ethidium bromide at 85 V for 3 h, proceeding as described above.
RESULTS
Table 1 summarizes the results of the experiments and shows the survival times in days of culturable C. jejuni both isolated from artificially and naturally contaminated feces. It also gives the flaA types. The results indicate that the C. jejuni survive up to 3 days longer in naturally contaminated feces than in artificially contaminated feces. Culturable C. jejuni survived in the artificially contaminated feces about 72 h (minimum) to 96 h (maximum) after inoculation. In the naturally contaminated feces, C. jejuni could be cultivated after 120 h (minimum) and 144 h (maximum), respectively. The repetition four months later achieved the same results. Although the results are based on a limited number of experiments (n1 = 6, n2 = 6), when applying the U-test (two-tailed) there is a significant difference (p < 0.01) among the number of days C. jejuni was culturable from artificially and naturally feces.
The same holds true for flaA typing. All biochemically suspected C. jejuni isolates were confirmed by mapA PCR. FlaA typing of culturable C. jejuni isolated from the artificially contaminated feces confirmed the detection of the originally inoculated strain (T1). In the
naturally contaminated feces up to 5 different flaA types were detected (Table 1). These flaA types showed different survival times between 120 and 144 h.
DISCUSSION
The results of this orientating study show that C. jejuni can survive in laying hen feces up to 6 days when removed from the laying hen house and stored under controlled conditions in the laboratory. This is clearly longer than the 2 days reported by Berrang et al. (2004b) who investigated residues of broiler feces in transport cages stored without cleaning after transport using direct plating on Campy Cefex agar after dilution of the collected feces with PBS.
Distinctly longer survival times for C. jejuni are reported for cattle manure (up to 14 days) (e.g. Sinton et al., 2007; Inglis et al., 2010). These large differences may be explained by different reasons. Sinton et al. (2007) investigated samples taken daily from cow pats deposited naturally on a pasture. They used Ester broth for enrichment. The “natural”
microaerophilic milieu in the cow pads and the composition of the cattle manure and the use of Ester broth may have supported the survival of the bacteria. Berrang et al. (2004b) took samples from the transport containers and streaked out the material directly on plates. We used enrichment according to ISO 1173-1 (2006) which is designed to increase the probability of positive findings. Other factors which have an influence on the survival of Campylobacter are ambient temperature and humidity, oxygen concentration and pH-value (Doyle and Roman, 1982; Hazeleger et al., 1995; De Cesare et al., 2003). In order to standardize the experiments, sampling and transportation procedures as well as the preparation of the samples in the laboratory were kept the same. Bolton broth (Humphrey, 1989; Baylis et al., 2000) was used for dilution and enrichment for all samplings and the samples were stored and incubated under controlled laboratory conditions at temperatures and
relative humidity known as typical for laying hen houses (DIN 18910-1, 2004). The identical results in the two experiments may indicate that repeatable methods and conditions were used.
The survival of C. jejuni is significantly longer in contaminated feces collected in the field from an infected flock (up to 6 days) compared to C. jejuni-free feces (up to 4 days) which were contaminated artificially in the laboratory after collection. The reason for this difference is not fully clear. Although the number of inoculated C. jejuni/g feces was about the same as typically found in shedding chickens (Whyte et al., 2001; Wagenaar et al., 2006), the recovery was significantly less. The survival times may be influenced by the composition of the feces which grossly depends on the composition of the feed stuffs (Udayamputhoor et al., 2003), on pharmaceutical residues (Johny et al., 2008, 2010) or on the intestine microflora (Mead, 2002). Also the mucus membrane in the intestines of the birds is reported to have a protective effect on the survival of inoculated C. jejuni (Hermans et al., 2010). A genetic influence is also discussed as shown with Salmonella (Van Hemert et al., 2006). From a technical point of view, it may also be that the second mixing during the inoculation of the Campylobacter-free pooled feces with the bacteria solution may have led to a higher oxygenation than in the naturally contaminated feces which were not mixed a second time. In this regard the applied artificial contamination method could be further standardized.
Nevertheless, the results underline that both artificially and naturally contaminated feces are a potential source of C. jejuni to infect or re-infect hens coming in contact with such excreta (Willis et al., 2000) and the importance of the horizontal infection pathway (Sahin et al., 2002). Control measures with regard to Campylobacter in laying hen houses have to take in account that C. jejuni may survive in feces for at least 6 days. Too short service periods and insufficient cleaning and disinfection can leave residues of Campylobacter in the animal house which can infect the flock of the next production cycle. Survival times of C. jejuni in
feces may also be considered when laying hen manure is applied to land as fertilizer. It is known that manure and excreta applied to land are attractive for wild birds and insects that can become carriers of Campylobacter (Jones, 2001; Stanley and Jones, 2003). Already the stored contaminated laying hen manure on the farm should be considered as a potential source of infection when insects, including flies and beetles and their larvae which come into contact with contaminated feces, could act as carriers of Campylobacter and may enter new or neighboring flocks where they can be picked up by the birds (Hald et al., 2008). Entrainment or transmission by living or non-living vectors seems to be a general problem which has not yet been sufficiently considered. It is known for instance that the shorter the distance between the litter pile and the poultry house the significantly higher is the risk of flock infection (Cardinale et al., 2004; Arsenault et al., 2007).
By flaA typing, it was shown that no other than the inoculated isolate was recovered from the artificially contaminated feces. Detecting always the same type in each individual fecal sample indicates that there were no cross-contaminations that potentially could have led to false positive PCR reactions. In the naturally contaminated feces 5 different flaA types were found which displayed different survival times. On 2 farms 2 different C. jejuni types were detected simultaneously. It is known that different C. jejuni types can occur in one poultry flock (Hook et al., 2005; Lindmark et al., 2006). This may be a consequence of different infection sources. The most frequent sources and vectors for Campylobacter transmission from feces are the clothes and boots of farm workers (Ramabu et al., 2004), transportation cages (Berrang et al., 2004b), poultry farm waste disposal and insects (Hald et al., 2008) being associated with flock colonization (Riedly et al., 2008, 2011). These multiple sources may be the reason for finding different flaA types in the same flock. There are some indications that the survival of Campylobacter is also dependent on flaA types. This was at
least reported by Newell et al. (2001) investigations in slaughter houses showing that the persistence and survival of Campylobacter on carcasses varied between strains depending on fla types. The risk of transmission increases when flocks of different ages are present on one farm (Allen et al., 2008). Therefore, cleaning and biosecurity are of crucial importance even if such measures can only reduce but cannot fully eliminate the risk of introduction of an infective agent in a flock (Van de Giessen et al., 1998). The here reported time frame of 6 days may help to better assess the risks arising from contaminated feces and improve prevention schemes.
The present study gives a limited insight into the survival of culturable C. jejuni in laying hen feces. More detailed investigations under different environmental conditions are necessary to gain a deeper understanding of the survival of C. jejuni in excreta. This should also include the role of the so-called non-culturable C. jejuni which are frequently addressed (Rollins and Colwell 1986; Tholozan et al., 1999) as the unknown source of infections.
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TABLE 1. Survival in days and flaA typing results of C. jejuni in artificially and naturally contaminated feces from 6 laying hen flocks on 6 different farms
Sampling days Sample
no.
Flock and Farm
Type of
contamination 1 2 3 4 5 6 7 8 9 10 flaA types
1 A artificially + + + - - - - - T1
1 B artificially + + + + - - - - T1
1 C artificially + + + + - - - - T1
1 D naturally + + + + + - - - - - T2
1 E naturally + + + + + + - - - - T3,T4
1 F naturally + + + + + + - - - - T5,T6
2 A artificially + + + - - - - - T1
2 B artificially + + + + - - - - T1
2 C artificially + + + + - - - - T1
2 D naturally + + + + + - - - - - T2
2 E naturally + + + + + + - - - - T3,T4
2 F naturally + + + + + + - - - - T5,T6
FIGURE 1. FlaA typing of C. jejuni isolated from three artificially contaminated feces of flocks C1. Lane 2, 3, 4 and 5: C. jejuni isolated for 4 days from flock C1. Lane 7: control positive, lane 8: control negative. Lane 1 and 6:100 bp DNA marker.
FIGURE 2. Two different C. jejuni types (T3 and T4) isolates from pooled feces of flock E1 were culturable for 6 days. Lane 2, 4, 6, 8, 10 and 12: C. jejuni type 3 (T3) isolated at 1st, 2nd, 3rd, 4th, 5th and 6th day, respectively. Lane 3, 5, 7, 9, 11 and 13: C. jejuni type 4 (T4) isolated at 1st, 2nd, 3rd, 4th, 5th and 6th day, respectively. Lane 15: control negative. Lane1 and 14:100 bp DNA marke