Potential Zoonotic Pathways of Salmonella Enteritidis in Laying Farms

Introduction

C ontamination with Salmonella Enteritidis is an important public health problem worldwide. No sufficient published data could be obtained about the actual existence of Salmonella Enteritidis in Egypt, and hence the epidemiological picture is not currently clear in this country. The predominant serotype associated with the egg-borne Salmonellosis in human is Salmonella enterica serotype Enteritidis (Gantois et al. 2008). Infected hens can transmit Salmonella Enteritidis by physical contact with other hens through the allocation of infected feed and water as well as aerosols (Lahellec et al. 1986, Baskerville et al. 1992, Nakamura et al. 1994, 1997, Holt et al. 1998). Salmonella Enteritidis is able to colonize the reproductive tissues of birds (Okamura et al. 2001). A wide range of Salmonella serovars could be isolated from eggshells, including Salmonella Enteritidis (Paul and Batchelor 1988, Humphrey et al. 1989, De Louvois 1993); the contamination with Salmonella Enteritidis through shell is much higher than contamination through the egg contents (Bichler et al. 1996, Miyamoto et al. 1997). Moreover, Salmonella on the shell surface may contaminate the interior egg contents by penetrating the shell and its associated membranes (Bradshaw et al. 1990, Gast and Beard 1990, Humphrey et al. 1991b). Egg contents may be infected with Salmonellae after breaking of the contaminated eggshell or through passage from the shell (Murchie et al. 2008).

This study was carried out to assess the distribution of Salmonella Enteritidis in Egyptian laying farms and its potential contamination pathways from birds to eggs and from eggshells to egg packagers.

Materials and Methods

Study design

The current cross-sectional study was carried out in the period between February and December 2008. It involved 10 laying hen farms located nearby other farms that are confirmed to have Salmonella Enteritidis infections. The distance between each investigated farm and the infected one ranged between 500 and 2000 m; the studied farms were located in the Delta of Egypt (Fig. 1). If the farm under investigation contained many flocks, only one flock was selected from the middle of the farm for such study. The number of laying hens in each studied flock was about 4500 to 5000. The laboratory work was carried out in the Department of Hygiene and Preventive Medicine, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt.

OPEN IN VIEWER

FIG. 1.

Locations of examined laying farms in the Delta of Egypt.

Cloacal swabs (30/flock) were collected from hens using a systematic random sampling plan. Before egg handling, the eggshell swabs were collected (40/flock) by passing the sterile swab over the whole shell. Before packing process, hand swabs were collected from all packagers (n = 38) in studied flocks. After the packing process, hand swabs were recollected before hand washing. The obtained swabs were inoculated directly into buffered peptone water (BPW) (1.07228; Merck, Darmstadt, Germany). From each examined flock, 15 laying hens were obtained using systematic random sampling strategy. The birds were subjected to aseptic necropsy in the laboratory and then separate pools (25 g/pool) of the ovary and oviduct from each hen were collected and inoculated into BPW. After the preliminary screening of infection percentage within the examined flocks, the overall infection percentage was calculated to specify the cutoffs. All samples from flocks that exceeded the mean percentages were summed for calculating their relative risks compared with total samples from flocks that had lower infection percentages than the mean values. A brief questionnaire proposed for all egg packagers was done; questions included their hygienic behavior in the farms and if they accustomed to wear protective gloves during egg packing. Packagers were also asked if they usually suffered from repeated Salmonellosis by former documented hospital reports intended for Salmonella Enteritidis (diagnosis of Salmonella infection among the workers was confirmed via fecal culture), if they used to smoke during the egg packing process, and for the smokers, do they accustomed to wash or disinfect their hands before smoking?

Results

The total positive samples for Salmonella Enteritidis prevalence in ovary, oviduct, cloaca, and eggshells within screened laying farm hens were 49/150, 43/150, 165/300, and 261/400 with overall percentages of 32.7%, 28.7%, 55.0%, and 65.3%, respectively.

At the day of laying, eggs were collected and carried in baskets for packing process. All examined packagers did not accustom to wear personal protective gloves and/or white coats during packing of eggs, and the smoker packagers were not accustomed to wash or disinfect their hands before smoking.

After egg packing, Salmonella Enteritidis was isolated from hands of 25 packagers whom were negative for the hand isolates before packing process while it was isolated from hands of two packagers before and after egg packing process, so that these two packagers were excluded from the assessment. Conversely, 11 packagers were found to be negative for Salmonella Enteritidis before and after work.

The role of ovary, oviduct, and cloaca as potential contamination sources with Salmonella Enteritidis to eggshells is presented in Table 1.

In comparison between Salmonella Enteritidis contamination on eggshell obtained from flocks having >30% positive cloacal isolates (178/240) and from flocks with <30% positive isolates (83/160), the relative risk was 1.42 (95% confidence interval 1.21 to 1.68). There was a significant difference between the two groups (p < 0.0001).

Data shown in Table 2 indicated that there was a significant difference between positive hand swabs for Salmonella Enteritidis isolates obtained from flocks having >60% positive eggshell isolates for Salmonella Enteritidis and flocks with <60% positive eggshell isolates (p = 0.04). The relative risk was 1.83 (95% confidence interval 1.04 to 3.24).

Regarding the appearance of repeated Salmonella infection among egg packagers (n = 25), seven packagers out of the eight, who were accustomed to smoke during their work, had recurrent Salmonellosis. There was a 30-fold increased risk of Salmonella infection in smokers compared with the nonsmoker groups and such increase is statistically significant (p = 0.02).

Discussion

Regarding the lack of documented or published studies concerning Salmonella Enteritidis occurrence in Egypt, this study could be considered as the first globally presented epidemiological assessment about Salmonella Enteritidis inhabitance in Egypt.

Spreading of Salmonella Enteritidis in laying farms may be initiated from the infected birds. Earlier studies reported that Salmonella Enteritidis has been isolated from ovaries, oviducts, and droppings of laying hens (Baskerville et al. 1992, Humphrey et al. 1992, Bichler et al. 1996). Regarding bird specimens, the overall percentages of Salmonella Enteritidis occurrence in ovary and oviduct within screened hens were 32.7% and 28.7%, respectively, so that the 30% infection was chosen as a cutoff. Whereas the general percentage of eggshell contamination with Salmonella Enteritidis was 65.3%, so in this case 60% was chosen as cutoff.

Obtained data indicated that, in infected hens, Salmonella Enteritidis resided the ovary with a percentage that exceeded its occurrence percentage in oviduct. This study indicated high relative risk of eggshell contamination in flocks having > 30% ovary, oviduct, and cloacal isolates of Salmonella Enteritidis than flocks with <30% infections. Gantois et al. (2008) reported that colonized Salmonella Enteritidis in cloaca may contaminate the eggshell during oviposition and the contamination may be originated from infected reproductive organs during egg formation. Also it was confirmed that eggshell contamination could be initiated from the oviduct (Humphrey et al. 1991a).

Isolation of Salmonella Enteritidis from egg packagers' hands in laying farms points out the probability of cross contamination during packing process from the eggshell to the hand (Murchie et al. 2008). It was reported that Salmonella Enteritidis was isolated from fingers subsequent to breaking of intact eggshells (Humphrey et al. 1994). Our results presented higher relative risk of packagers' hand contamination in flocks having >60% eggshell isolates of Salmonella Enteritidis than flocks with <60% contamination percentage.

Isolation of Salmonella Enteritidis from two packagers before and after the egg packing process may give an indication about the risk of eggshell contamination during handling via packagers' hand, so the hands should be regularly disinfected before and during egg handling. Moreover, eggs should be treated with a safe disinfectant before packing process because infected hen may frequently produce contaminated eggs before notification of the disease in the birds.

In addition, shell to shell transmission of Salmonella may occur during packing process (Murchie et al. 2008), and thus, Salmonella Enteritidis that was recovered from eggshell in the present investigation may be distributed from contaminated egg to uncontaminated one through that route especially during collection of eggs in the baskets.

Contamination of eggshells by Salmonella Enteritidis may be considered as a risk factor for packagers and consumers; the infection could occur via hand to mouth route.

Smoking during packing process could be regarded as an exposure factor to contract the infection via hand–cigarette filters to mouth.

Finally, the obtained findings could be considered as a prologue study for detecting the potential pathways of Salmonella Enteritidis in laying farms; further studies are needed to assess the surveillance of Salmonella Enteritidis, especially in the Egyptian laying farms.