Animal Health Research Institute

Assiut Regional Laboratory

Isolation of Yersinia  enterocolitica

and Pseudomonas aeruginosa from commercial Hens and ducks ’eggs

(With 2 Tables)

By

Amal A. Abdel- Haleem and M.M. Ali

(Received at 24/9/2005)

عزل اليرسينيا انتيروکوليتکا والسيدوموناس ايروجينوزا

من بيض الدجاج والبط التجارى

أمال على عبد الحليم ، محمد مصطفى على

لما کان للبيض من اهمية کبيرة من حيث قيمته الغذائية الا انة قد يصبح مصدرا لا يستهان به لنقل العديد من الامراض التى تهدد صحة الانسان وذلک لأنة عرضة للتلوث بانواع مختلفة من الميکروبات الممرضة والمفسدة والتى تصل اليه من مصادر متعددة منذ انتاجه واثناء توزيعه حتى يصل الى المستهلک. لذلک قمنا بجمع عدد 225 عينة من بيض الفراخ البلدى وبيض فراخ المزارع وکذلک بيض البط ممثلة فى 45 مجموعة (15 لکل نوع) جمعت عشوائيا من اسواق مدينة اسيوط ومحلات البقالة المختلفة. وقد تم  فحص البيض بکتريولوجيا لمعرفة مدى تلوث قشر البيض من الخارج وکذلک محتوياته بميکروبى اليرسينيا انتيروکوليتکا والسيدوموناس ايروجينوزا. ولقد تبين من الفحص ان ميکروب اليرسينيا انتيروکوليتکا تواجد فى عدد 3 عينات من محتويات بيض الفراخ البلدى  بنسبة 20% وامکن عزلة من عينة واحدة من على قشر ذلک البيض بنسبة 7‚6% بينما لم يتم عزل الميکروب من بيض فراح المزارع سواءمن المحتويات او من على القشر. بينما فى بيض البط التجارى امکن عزله من على القشرة بنسبة 7‚6% (عينة واحدة) وکذلک من المحتويات بنسبة 20% (3 عينات) .اما با لنسبة لميکروب السيدوموناس ايروجينوزا فقد تم عزل الميکروب من عينة واحدة من قشر بيض فراخ المزارع بنسبة 7‚6% وکذلک من عينة واحدة من المحتويات بنفس النسبة , بينما لم يتم عزل الميکروب من محتويات أو من على قشر بيض الفراخ البلدى. وبفحص قشر بيض البط تواجد الميکروب فىعينة واحدة(7‚6%) وتواجد فى عدد 2 عينة(3‚13%) من محتوياته. ولقد ناقش البحث اسباب التلوث بهذين الميکروبين وخطورة تلوث منتجات البيض اثتاء التصنيع والخطر الذى قد يشکله هذا على صحة الانسان , کذلک الطرق الواجب اتباعها لمنع تلوث البيض بهما.

Summary

Commercial Hens and ducks’ eggs (225 eggs) representing 45 groups   (5 eggs each as a sample) were collected from Assiut city markets and different groceries. Hens’ eggs represent farm hens and native breeds. The collected samples were examined for incidence of Yersinia enterocolitica and Pseudomonas aeruginosa on egg shells and in contents                                                                                       

Y. enterocolitica could not be isolated from shells ad contents of farm hens’ eggs (shell and content), and the organism recovered from one sample (6.7%) of shells and 3 samples (20%) of the contents of native breeds eggs. Ducks’ eggs were contaminated by the same ratio of native breeds hens’ eggs. Pseudomonas aeruginosa could not recover from any of native breed eggs (shells and contents) but could be isolated from 6.7% of shells and contents of farm eggs (one sample each). Ducks’ eggs proved to contain the organism in the content (2 samples) and on its shell (one sample). The public Health hazard of the isolated organisms and suggestive measures were discussed.                                               

Key words: Y. enterocolitica, P. aeruginosa, eggs

Introduction

It has been recognized that eggs are important and popular food in all countries and almost for all ages. Eggs provide a unique well balanced source of nutrients, including high quality protein, vitamins, useful amount of minerals and easily digested lipids, However, the nutrients that make eggs a high quality food for humans are also a good growth medium for bacteria. In the rare event that egg contains bacteria, as the inside of an egg was once considered almost sterile (Brooks and Taylor, 1955 and Frazier and Westhoff, 1978). But, over the recent years, different types of microorganisms have been found inside eggs. As, if the ovary is infected with bacterial pathogens, the egg may become infected before it is laid. Besides, other types of microorganisms could be deposited along with dirt on the outside of an egg and from fecal matters, from hens, by lining of the nest, by wash if the eggs are to be washed, by handling and perhaps by materials in which eggs are packed(Board and Fuller, 1994 and Cox et al., 2000).

The risk of getting a food borne illness from eggs is very low, in spite of the good growth medium for bacteria. Unfortunately, some food poisoning outbreaks due to consumption of eggs had been reported (Philbrook et al., 1960, Adler, 1965 and Bowmer, 1965). Different types of bacteria including Psudomonas have been isolated from eggs. (Longree, 1980). Also, it has been proved that egg shells are pervious to microorganisms such as Salmonella typhi, E.coli, Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens and Alkaligenes bookeri (Haienes and Moran, 1940; Garibaldi and Stokes, 1958 and lifshitz et al., 1964).

Yersinia enterocolitica is a food borne pathogen that is widely distributed in nature, animal and aquatic reservoir. They are able to grow at low temperature (Stern et al. 1980) and can produce toxins in foods (Boyce et al., 1979; Francis et al., 1980). These enterotoxins may be able to resist the temperature used in food processing and storage (Boyce et al., 1979). Y.enterocolitica could lead to enterocolitis, mesenteric lymphadenitis and terminal ileitis. Also it has been reported that Y.enterocolitica invades the epithelium cells of gastrointestinal tract to produce intestinal diseases in animals and humans. There has been interest in the recovery of Y.enterocolitica from foods since the implication of Yersinia entercolitica in food poisoning occurred in New York due to consumption of chocolate milk (Black et al., 1978). The reports on Y.enterocolitica prevalence in eggs and egg products are sketchy, however, it has been shown that Y.enterocolitica can be found on the surface of egg shells (Favir et al., 2000).

Pseudomonas aesuginosa, has been implicated in various human infections, including enteritis, various respiratory diseases, urinary tract infection, infections of bones, joints, nails, skin and wounds (Clement and Millard, 1953; Winso, 1957; Burlina, 1962 and Chernosky and Duckes, 1963). As reported by Haines and Moran (1940), Pseudomonas aeruginosa can grow and then penetrate through intact shell to the egg contents. This phenomenon was previously assured by Garibaldi and Stokes (1958), that egg shells were pervious to Pseudomonas aeruginosa. Ahmed et al (1985) could isolate the organism from the shells of examined hens' eggs. In a study conducted by Das et al. (1996), Pseudomonas aeruginosa was one of the causative organisms of nosocomial diarrhea occurred in Calcutta, the eggs were among the source of infection. Therefore, this work aims to investigate the incidence of Yersinia enterocolitica and Pseudomonas aeruginosa on shells and in contents of commercial hens and ducks' eggs.

Materials and Methods

Collection of samples:

A total of 225 eggs representing 45 groups of commercial hens and ducks’ eggs, were collected randomly from Assiut city markets and different groceries. Hens’ eggs included farms and native breed hens. Each group (5 eggs) represent one sample, was placed in a sterile plastic bag and dispatched to the laboratory with a minimum of delay. The eggs were prepared and examined for the presence of Yersinia enterocolitica and Pseudomonas aeruginosa                          

Preparation of samples:

(A) Egg shells were tested by surface method as described by Moats(1979)

(B) Egg contents. The egg was prepared for evacuation of its content according to Speck (1976).

Experimental techniques:

The rinse solution of egg shells, as well as, the homogenous egg contents was subjected to the following examination:

1- Isolation and identification of Yersinia entrocolitica

(a) - Enrichment procedure: 1 ml of rinse solution of eggshells, as well as, from the homogenous egg contents was placed aseptically into enrichment broth (Trypticase soy broth) and incubated at 37^oCfor 24 hours (Greenwood and Hooper, 1989).

 (b) Isolation technique: A loopful of the incubated broth was streaked directly onto Cefsuldin Irgasan Novobiocin (CIN) as described by Schiemann (1979), and then incubated at 37c for 24h. The presumptive colonies were identified according to Schiemann and Devenish (1982).

2- Isolation and identification of Pseudomonas aeruginosa.

 (a) Enrichment procedure. 1ml of shell rinse solution, as well as, from homogenous egg contents was inoculated into Citrimide broth and incubated at 42 C˚ for 48 hours (Shriniwas, 1975).

 (b) Isolation technique: Loopfuls from incubated broth tubes were streaked onto Citrimide agar plates and incubated at 42 C˚ for 24-48 hours (Shrininwas, 1975). Loopfuls from suspected colonies were picked up into agar slants and incubated at 42 C˚ for 24 hours for further identification according to Finegold and Martin (1982).

Results

The obtained results were recorded in Tables 1 and 2

Table 1: Incidence of Y. entrocolitica in the examined samples of Hens and ducks’ eggs.

Tables 2: Incidence of pseudomonas aeruginosa in the examined samples of Hens and ducks’ eggs.

Discussion

As recorded in Table 1, Y. enterocolitica could not be detected on egg shells or in contents samples of the examined hens' eggs collected from poultry farms. The data proved that 1/15 (6.7^oC) of eggshells and 3/15 (20%) of egg contents samples of native breed hens were positive for the organism. Duck's eggs samples were found to be contaminated by Y. enterocolitica on 1/15(6.7%) of their shells and in 3/15(20%) of their contents. No data available to compare with except the research newsletter published by Favir et al. (2000)that Y. enterocolitica could not be detected among the natural flora on any of the egg tested.

Presence of Y. enterocolitica on eggshells and in egg contents samples could be attributed to the fecal matter soiled the eggshells of native breed hens and ducks' eggs. Furthermore, previous isolation of Coilforms, E.coli, Salmonella, Enterococci, Alkaligenes and pseudomonas organisms from eggshells and contents by Alford et al. (1950); Ahmed et al. (1985 &1987); El-prince (1988) and Bastawrows et al. (1997), assures the possibility of contamination by Y. enterocolitica. The study of Henning. (1939) stated that eggshells are frequently contaminated by infected fecal matter and under favorable conditions, the organisms penetrate the shell into the egg contents. Also,Matthes (1984) proved that storage temperature and types of packing materials for transportation influence the penetration through eggshells into the contents.

The summarized data in Table 2 revealed that the hens' eggs of poultry farms were contaminated by Pseudomonas aeruginosa on 1/15(6.7%) of their shells samples and by the same ratio in their contents. In case of egg samples of native bread hens, P.aeruginosa failed to recover from any of their shells or contents. While, 2/5 (13.3%) of ducks' eggs content were positive for P.aeruginosa, and 1/15(6.7%) of their shells carried the organism (Table 2). P. aeruginosa was previously isolated from egg shells of commercial hens' eggs by Garibaldi and Stokes (1958); Lifshitz et al. (1964 &1965) and Ahmed et al (1985) in different percentages. The investigation carried by Moursy et al (1982) pointed out that P. aeruginosa could be detected in 18.18% of contents of unsold aged hens' eggs. However, in a previous study conducted by Miller and Crawford (1953), P. aeruginosa could be isolated from 9% of examined egg content samples of commercial hens' eggs. It was noticed that P. aeruginosa can grow and then penetrate through the intact shell to the egg content and the rate of penetration depends on the temperature of storage and Humidity (Haines and Moran, 1940). Furthermore, Garibaldi and stokes (1958) had proved that eggshells are pervious to P. aeruginosa. The public health hazard of P. aeruginosa has been stated, since its implication   in nosocomial diarrhea occurred in Calcutta, where the eggs were among the source of infection (Das et al., 1996).

From the aforementioned results, it is clearly evident that Y.enterocolitica and P. aeruginosa recovered from the examined egg samples should be considered a public health hazard concern. These pathogens may thrive from shells or contents into other egg products, grow and multiply sufficiently to the risk level of food poisoning.

Recommendation for prevention of their risk include, proper hygienic measure should be adopted in poultry farms side by side with educational program for the employee, and for the public to know how to handle and store egg or egg products. Likewise, the practice of cleaning eggs by removal of dirt and faecal matter, washing by sanitizing solution is recommended and is common in egg industry. Moreover, pasteurization of egg products must be adopted as a statutory requirement.

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