SOME STUDIES ON LISTERIA SPECIES ISOLATED FROM CHICKEN FLOCKS IN ASSIUT PROVINCE.

Animal Health Research Institute,

Assiut Laboratory.

Some studies on Listeria species isolated from chicken flocks in Assiut Province.

(With 4 Tables and 7 Figures)

By

FATMA A. MOUSTAFA and AZHAR M. Abdel AZIZ

(Received at 30/3/2005)

بعض الدراسات عن ميکروبات الليستيريا المعزولة من قطعان الدواجن

فى محافظة أسيوط

 فاطمه عبد المجيد مصطفى ، أزهار محمد عبد العزيز

تعتبر ميکروبات الليستريا من الميکروبات واسعة الإنتشار فى الطبيعة ومنها ميکروب الليستيريا مونوسيتوجينس الذى يسبب الإصابة بمرض الليستيروزيس، ومن هنا کان الاهتمام بالبحث لمعرفة مدى تواجده فى بعض مزارع الدواجن بمحافظة أسيوط. وقد تم العزل من أعمار تتراوح من 1-24 أسبوع. ومن عينات أرتبطت بتاريخ مرض معين. وقد أسفرت النتائج عن تواجد الليستيريا بنسبة 19.2% من العينات المفحوصة أما بالنسبة لميکروب الليستيريا مونوسيتوجينس فقد تم عزل 14 عترة بنسبة 60.9% أنطبقت عليها صفات الميکروب المورفولوجية والبيوکيميائية وتم تصنيفه إلى نوع سرولوجى رقم O₁ . تم إجراء العدوى الصناعية بحقن الميکروب فى أجنة بيض الدجاج عن طريق الغشاء الکوريوالأنتويس ،هذا وقد تم تسجيل الاعراض والافات التشريحية وتم کذلک مناقشتها. کذلک تم  إجراء العدوى الصناعية فى کتاکيت عمر 6 أسابيع هذا وقد تم ملاحظة الاعراض وتسجيل الصفات التشريحية ومناقشتها، بأجراء اختبار الحساسية لميکروب الليستيريا مونوسيتوجينس تبين ان الميکروب  شديد الحساسية لکل من الأموکساسلين والکلورمفينکول والأمبسيلين وحمض النلدکسک بينما کان الميکروب مقاوم لکل من سلفات الکولستين وحمض الأوکسالنيک.

Summary

Listeria infections caused by microorganisms of the genus Listeria, occur worldwide and in a variety of animals, birds and man. In this study isolation of Listeria was done from different ages (1-24- week old) from freshly dead chickens (baby, chick, broilers and layers). Double sample were taken from each bird, one from intestine and the other was pooled samples include liver, heart and spleen. 23 (19.2%) strains of Listeria spp. were obtained, from which 14 strains were identified as L. monocytogenes at a percentage of 60.9%. The experimental infection of L. monocytogenes in chicken embryo was carried out by chorioallantoic membrane (CAM) route. Intraperitonial infection of 6 weeks old chicken revealed 25% of birds died during observation period. Signs and postmortem changes were observed and discussed. Contact birds showed depression and inappetance. The in-vitro susceptibility of L. monocytogenes isolates to a variety of antibiotics revealed  that isolates were highly sensitive to Amoxycillin, Chloramphenicol, Ampicillin, and Nalidixic acid while they were resistant to Colistin sulphate and oxolinic acid.

Key words: Listeria species, chicken

Introduction

Listeria has attracted the wide attention of microbiologists in recent years. Listeria species are widely spread environmental microorganisms found in soil, water, vegetation and many animal species (Best et al., 1990). Genus Listeria is composed of many species of which Listeria monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. inovanovi and L. denitrificans have been identified (Rocourt and Cossart 1997).

Listeriosis is caused by L. monocytogenes which considered the major pathogenic species that under certain conditions can cause serious or even fatal disease in man, animals and fowls (Thamm, 1962).  Mortality rate vary from a few to as much as 40%, sudden death may occur in adult chickens while young birds had slow wasting before death (Allen Packer, 1975; Dalton et al., 2004).

Listeriosis in fowl is seen as torticollis, drowsiness and septicemic condition with focal necrosis of the liver, necrosis of the myocardium, pericarditis and occasionally encephalitis (Gross, 1984 and Kurazono et al., 2003).

L. monocytogenes was isolated from various poultry species at different incidences. Gitter (1976) isolated 10 isolates of L. monocytogenes out of 135 swabs from 68 birds included 62 chickens. Cooper (1989) isolated L. monocytogenes in significant numbers from the brain stem of three out of four birds.  Petersen and Madsen (2000) isolated L. monocytogenes from two of 71 broiler flocks of cloacal swabs from broiler. Njagi et al. (2004a) were able to isolate two L. monocytogenes and seven L. species from the oropharyngeal swab but non from cloacal swabs of chickens.

Antibiotic sensitivity pattern of L. monocytogenes was studied by Antunes et al. (2002) who recorded that L. monocytogenes was resistant to Enrofloxacin and Clindamycin.  Njagi et al. (2004b) showed that Listeria isolates were sensitive to Gentamycin, Kanamycin, Tetracycline, Chloramphenicol and resistant to Ampicillin, Augmentin and Cefuroxime.

Therefore, this work was conducted to cover the following items:

- Isolation and identification of Listeria species from different ages of chickens.

- Pathogenicity of L. monocytogenes for newly hatched chickens exposed to natural infection.

- Pathogenicity of L. monocytogenes to 6-weeks old chickens.

- In vitro sensitivity testing of L. monocytogenes against different  antibiotics.

Materials and Methods

1- Collection of samples:

            Chickens: One hundred and twenty samples of different ages (1-24 week old) were collected from freshly dead chickens (baby chicks, broilers, layers) from different Chicken farms at Assiut governorate.  Tissue samples from intestine, liver, heart and spleen collected from these cases were subjected to bacteriological isolation of Listeria organisms.

2-  Isolation of Listeria spp.

            As described by (Gray et al., 1948 and Curtis et al., 1989) samples were collected from each bird, the first one was as intestinal content, while the second one was pooled of liver, heart and spleen.  Both samples were aseptically added to Listeria enrichment broth (Biolife) and incubated at 35±1°C. After 48h. incubation a loopful of enrichment broth was streaked on the surface of Oxford agar medium and incubated at 35±1°C for 24-48 h.

3-  Identification of Listeria spp.

            Identification and species differentiation was carried out according to Mackie and MacCartney (1962), Finegold and Martin (1982) and Warburton et al. (2003) including Gram^’stain, motility test, catalase reaction, Voges-Proskauer test, carbohydrate fermentation and the Christie-Atkins-Munch-Peterson (CAMP test).

4- Serotyping of L. monocytogenes strains:

            Biochemically identified isolates were serotyped with Listeria 0 antisera type I (Difco) by using slide agglutination test according to Difco, 1984.

5- Antimicrobial susceptibility testing for L. monocytogenes:

            L. monocytogenes were tested for their sensitivity and resistance patterns aganist 11 different antimicrobial agents (Gentamycin, Kanamycin, Amoxycillin, Chloramphenicol, Ampicillin, Enrofloxacin, Colistin Sulphate, Cephradine, Oxolinic acid, Norfloxacin and Nalidixic acid) by disk diffusion method (Harvey and Gilmour, 2001).  All plates were incubated at 35±1°C for 24 hours and examined for inhibition zones and the results were recorded.

6-  Pathogenicity test:

a. Effect of L. monocytogenes on chicken embryos and newly hatched chicks exposed to natural infection:

            Typical morphologically and biochemically suspected colonies were picked up into Tryptose broth and incubated at 35°C for 18 hours.  Amount of 0.2 mL of 10⁵ CFU of broth culture was inoculated in twenty-11-day old embryonating chicken eggs on chorioallantoic membrane (CAM) and incubated at 38°C in humidity with frequent turning and twice daily candling. Died embryos were exposed and examined. The survived and hatching chicks were added to 10 birds of the same age. Five birds were kept as non-infected control. The chicks of both groups were examined daily for 15 days, clinical signs and mortality were recorded (Graham et al., 1940; Basher et al., 1984a and Lattmann et al., 1989).

b. Experimental infection of 6 weeks old chickens with L. monocytogenes:

            Two groups of 6 weeks old chickens proved by coloacal swab to be free from Listeria. The first group of 20 birds was intraperitoneally infected with 0.5 ml of 18 hour tryptose broth culture of isolated L. monocytogenes. Contact group of 10 birds was left in contact with the infected one.  Another group of 5 birds was kept as non-infected group control and received 0.5 mL sterile tryptose broth intraperitoneally. All birds were kept under observation for 6 weeks, clinical signs, postmortem lesions and mortalities were recorded (Gross, 1984; Cooper, 1989 and Kurazono et al.,  2003).

Results

Obtained results are recorded in Tables 1-4 and Figures 1-7.

Table 1: Incidence of Listeria species recovered from the examined chickens.

Table 2: Biotyping of L. monocytogenes isolated from chickens.

Table 3: Results of pathogenicity of L. monocytogenes in experimentally and naturally infected chicks.

Table 4:   Antibiotic sensitivity pattern of the isolated L. monocytogenes strains.

+++ = highly sensitive                           + = weakly sensitive.

++ = moderately sensitive                       - = resistance

Discussion

Listeria monocytogenes, the most common causative agent of Listeriosis, is a widespread pathogen and has been also isolated from the gastrointestinal tract of a symptomatic animals and persons as well as the environment (Glass et al., 1995). Chickens, geese, ducks, turkeys and canaries are the most susceptible birds to Listeriosis. Despite the low incidence of infection by this pathogen, its association with high mortality rates (25-30%) makes Listeriosis a serious health problem (Datton et al., 2004).

Results presented in Table (1) declared that the isolation was done from intestine and pooled samples (liver, heart and spleen) at a percentage of 20% and 12.5%, respectively in Dairoute. Listeria were isolated from El Kosseia at a percentage of 12.5% and 7.5%, respectively but in Faculty of Agriculture Farm the percentage of isolation was 5%. In this study Isolation was done from different ages (1-24 week old). The relatively  higher incidence of Listeria spp. may be attributed to that some flocks in Dairoute were suffering from enteritis, fatty liver, drop of egg production. These results are in agreement with those of Hoffman and Lenarz (1942) and Peperkamp and Jansen (1947) who isolated L. monocytogenes from chickens suffering from lymphomatosis and enteritis. On the other hand, in El Kosseia, the recorded results of isolation in some flocks were associated with Newcastle infections while, coccidiosis was diagnosed in other flocks. The same findings were previously described by Bolin and Turn (1951) who found L. monocytogenes in cases of Newcastle disease infections, and Vander Schaaf et al. (1956) who isolated the organism from broilers suffered from coccidiosis.

From both intestine and pooled sample from liver, spleen, heart, L. monocytogenes and the other Listeria species were isolated at rates of 60.9% and 39.1% respectively. These results were more or less higher if compared with the rates of isolation that obtained by Gitter (1976), Cooper (1989), Petersen and Madsen (2000) and Njagi (2004a).

The results in table (1) revealed that the intestinal specimens showed a higher rate of isolation of L. monocytogenes compared with the pooled samples of liver, heart and spleen. These results proved the presence of L. monocytogenes in the alimentary tract of healthy birds (Allen Packer, 1975 and Gross, 1984).

The results obtained in the present work showed that L. monocytogenes could be isolated from one week old chicks. This may be attributed to the role played by eggs in transmission of infection via shell contamination. Our explaination is in agreement with the observation of Dedie (1955) who suggested that eggs contaminated by fecal material may constitute a source of infection.  In the same manner, Petran and Swanson (1993) and Nagah and Enas (1995) who isolated L. monocytogenes from the egg shell at a rate of 8.3%, isolated L. monocytogenes from the egg shell at the rate and 17.77%, respectively. Asmaa (1997) recovered L. monocytogenes and other Listeria species at the rate of 11.7% and 5% respectively. 

All tested isolates identified as L. monocytogenes gave small gram positive rode cells occure singly in short chains, motile in semisolid agar giving umbrella like growth. Catalase positive, voges-proskauer positive, carbohydrate fermentation positive produce acid with no gas.  On CAMP test hemolysis of L. monocytogenes was enhanced near Staphylococcus aureus streak (Table 2). These results are similar to those reported by Finegold and Martin (1982) and Warburton et al. (2003).

Our results concerning the inoculation of L. monocytogenes in chick embryos via CAM as shown in (Table 3), revealed that the tested strains were highly pathogenic, causing deaths after 48 hours post-inouclation. Died embryos showed severe congestion of internal organs and necrotic foci on the liver and heart, congestion and curling of embryos and severe enlargement and congestion of yolk sac (Fig. 1). The remaining inoculated chicks died on the 9^th day post-inoculation, died embryos showed stunting with signs of depression, dullness, ruffled feathers, incordination of gait and postration death (Fig. 2). The hatched chicks which died after three days during observation period showed tremor, unilateral and bilateral toe paralysis (Fig. 3).  At postmortem examination, there was engorgement of the unabsorbed yolk sac, distention of the gall bladder and congestion of the internal organs.

Contact chicks showed dullness, shivering, muscular tremor and the rate of mortality was 30% after five days.  All chicks showed severe congestion in all body muscles and internal organs, necrotic foci on the liver, distention of the gall bladder (Fig. 4).  There was congestion of the meninges and oedematous fluid on it (Fig. 5). L. monocytogenes was reisolated from dead embryos and contact chicks.

The recovery of L. monocytogenes from intestinal contents and various organs (liver, spleen, heart and kidneys) of experimentally and naturally infected chicks is in harmony with findings of Graham et al. (1940) and Basher et al. (1984a) who explained the pathogenicity of L. monocytogenes for newly hatched chicks exposed to natural infection where organism entered through alimentary tract and dissemination followed bacteraemia and toe paralysis were observed in some birds. Lattmann et al. (1989) studied the pathogenicity of 10-day old fertilized hen’s eggs infected with Listeria species and found that L. monocytogenes caused death 100% of the infected chicken embryos within 96 hours.

The intraperitoneal infection of broilers aged 6-week old resulted in death rate of 25% during observation period. Nervous signs (torticollis and incoordiantion) were observed after 7 days of infection. Depression, ruffled feathers and inappetence were also observed (Fig. 6). These clinical signs were only noticed in 12/20 birds (60%).  Most of examined birds showed congestion of internal organs, brain, peticheal haemorrhages on the proventriculus and thigh muscles, distended gall bladder, pericarditis and airsacculitis (Fig. 7). The same clinical signs and lesions were described by Allen Packer (1975), Gross (1984) and Cooper (1989).

Contact birds showed depression and inappetence which were the only noticed clinical signs. These results are not similar to those described by Dedie (1955) who did not observe any signs of illness in contact birds. L. monocytogenes was reisolated from all diseased and dead birds as well as clinically infected contact birds.

In vitro susceptibility of L. monocytogenes isolates to eleven different antibiotics illustrated in Table (5) revealed that 100% of L. monocytogenes isolates were sensitive to Amoxycillin, Chloramphenicol, Ampicilline and Nalidixic Acid. 71.4% of isolates were sensitive to Enerofloxacin and Norfloxacin, 50% of isolates were sensitive to Gentamycin and Kanamycin. 35.7% of isolates were sensitive to Cephradine, while, all isolates were resistant to Colistin sulphate and Oxalinic acid.  These results are in difference to those of Antuness et al. (2002) who showed that L. monocytogenes was resistant to Enrofloxacin and with those of Njagi et al. (2004b) who reported that Listeria isolates were sensitive to Gentamycin and Kanamycin and resistant to Ampicillin.  On the other hand, our results are in agreement with those of Smaill (2000) who stated that Listeria species were sensitive to Amoxycillin and Ampicillin but were resistant to Gentamycin.      

It is noteworthy, to state that L. monocytogenes is widely distributed among fowl of different ages and breeds.  The organism play a role in many cases especially as secondary invador following viral, parasitic, nutritional disturbances and bacterial diseases. Listeria occure normally in alimentary tract of some healthy birds, soil, litter secreation and excretions of carriers or infected chickens.  So cleaning, disinfection of houses, equipments and utensils is very important to minimize the infection in poultry farms. Egg shell contamination is considered a major way transmission (Board, 1977). So, hygienic treatment of eggs is very important before setting in incubators because the organism is transmitted to human or animals which can carry this microorganism in their gasterointestinal tract even without symptoms or may retain it for several months, thus constituting potential public health hazard (Farber and Peterkin, 1991). In the meantime, control of Listeriosis could be achieved by awareness of the ubiquity of these organisms and especially of those, environments that favor their multiplication.  So collaboration of medical, veterinary and chicken meat hygienists, farmers and technologists is essential.

References

Allen Packer, R. (1975): (Listeriosis) chapter 10 in Isolation and Identification of Avian Pathogens. 1^st Ed. (Hitchner S.B., Domeomuth, C.H., Purchase, H.G. and Williams, J.E.) Am. Ass. of Avian Pathologist, College Station Texas. pp. 80-83.

Antunes, P.; Reu, C.; Sousa, J.C.; Pestana, N. and Peixe, L. (2002):  Incidence and susceptibility to antimicrobial agents of Listeria spp. and Listeria monocytogenes isolated from poultry carcasses in porto, Portugal. J. Food Prot., 65 (12): 1888-93.

Asmaa, A.A.H. (1997): Occurrence of Listeria species in laying hens.  Assiut Vet. Med. J. 38 (75): 97-103.

Basher, H.A.; Fowler, D.R.; Rodgers, F.G.; Seasman, A. and Woodbine, M. (1984a): Pathogenicity of natural and experimental Listreriosis in newly hatched chicks.  Research in Vet. Science 36 (1): 76-80.

Best, M.; Kennedy, M.E. and Coates, F. (1990): Efficacy of a variety of disinfectants against Listeria spp. Appl. Environ. Microbiol., 56: 377-380.

Board, R.G. (1977): The microbiology of eggs. p. 49-64.  In W.J. Stadelman and O.J. Cotterill (eds): Egg Science and Technology. AVI Publishing Co., Inc., Westport, Connecticut.

Bolin, F.M. and Turn, J. (1951): Non clinical Listeriosis of the chicken.  N.D. Agric. Exp. Station Bull., 13, 107-108.

Cooper, G.L. (1989): An encephalitic form of Listeriosis in Broiler chickens.  Avian Diseases 33: 182-185.

Curtis, G.D.W.; Mitchell, R.G.; King, A.F. and Griffin, E.J. (1989): A selective differential medium for the isolation of L. monocytogenes.  Lett. Appl. Microbiol., 8: 95-98.

Dalton, C.B.; Gregory, J.; Kirk, M.D.; Stafford, R.J.; Givney, R.; Kraa, E. and Gould, D. (2004):  Foodborne disease outbreaks in Australia, 1995 to 2000. Commun. D.S. Intell., 28 (2): 211-224.

Dedie, K. (1955):  Beitrag zur Epizootologie der Listeriose.  Arch. Exp. Vet. Med., 9: 251-264.

Difco Laboratories (1984): Difco manual. 10^th Ed., Detroit, MI, pp. 520-524.

Farber, J.M. and Peterkin, P.I. (1991): L. monocytogenes, a food-borne pathogen. Microbiol. Rev., 55: 476-511.

Finegold, S.M. and Martin, W.J. (1982):  Bailley and Scott Diagnostic Microbiology, 6^th ed., C.V. Mosby Co. St. Louis. Toronto, London.

Gitter, M. (1976): Listeria monocytogenes in (oven ready) poultry.  Vet. Rec. 99 (17) 336.

Glass, K.A.; Prasad, B.B.; Schyter, J.H.; Uljas, H.E.; Farkye, N.Y. and Luchansky, J.B. (1995): Effects of acid type and alta TM 2341 on L. monocytogenes in a Queso Blanco type of cheese. J. Food Prot., 58 (7): 737-741.

Graham, R.; Dunlop, G.L. and Levine, N.D. (1940): Cornell veterinarian 30, 268-290.

Gray, M.L.; Stafseth, H.J.; Thorp, F.; Sholl, L.B. and Riley, W.F. (1948):  J. of Bacteriology 55, 471-476.

Gross, W.B. (1984): Miscellaneous bacterial disease.  In: Diseases of poultry, 8^th Ed. M.S. Hofstad, H.J. Barnes, B.W. Calnek, W.M. Reid and H.W. Yoder, Jr., eds. Iowa State University Press, Ames, Iowa, pp. 257-282.

Harvey, J. and Gilmour, A. (2001): Characterization of recurrent and sporadic L. monocytogenes isolates from raw milk and non dairy foods by pulsed-field gel electrophoresis, monocin typing, plasmid profiling and cadmium and antibiotic resistance determination. Appl. Environ. Microbiol., 67 (2): 840-847.

Hoffman, H.A. and Lenarz, C. (1942): A case of Listeriosis in chickens and an additional case in sheep.  J. Am. Vet. Med. Assoc. 100: 340-342.

Kurazono, M.; Nakamura, K.; Yamada, M.; Yonemaru, T. and Sakoda, T. (2003): Pathology of Listerial encephalitis in chickens in Japan. Avian Dis., 47 (4): 1496-1502.

Lattmann, C.; Schwarzkopf, A. and Seeliger, H.P. (1989): Pathogenicity testing of Listeria strains isolated from food in fertilized hen’s eggs. Zentralbl Bakteriol Mikrobiol Hyg., 270 (3): 400-5.

Mackie, T.J. and Maccartney, J.E. (1962): Handbook of practical Bacteriology, 10^th Ed., E. and S. Living Stone Ltd., London.

Nagah, M.S. and Enas El-Preince (1995): Prevalence of Listeria species in Hen’s eggs sold in Assiut city. Assiut, Vet. Med. J. 65: 127-131.

Njagi, L.W.; Mbuthia, P.G.; Bebora, L.C.; Nyaga; P.N.; Minga, U. and Olsen, J.E. (2004a): Carrier status for Listeria monocytogenes and other Listeria species in free range farm and market healthy indigenous chickens and duck. East Afr. Med. J., 81 (10): 529-533.

Njagi, L.W.; Mbuthia, P.G.; Bebora, L.C.; Nyaga, P.N.; Minga, U. and Olsen, J.E. (2004b): Sensitivity of Listeria species, recovered from indigenous chickens to antibiotics and disinfectants. East. Afr. Med. J., 81 (10): 534-7.

Peperkamp, C.W. and Jansen, J. (1977): Listerellosis bij de Kip.  Tijdscher Diergeneesk., 72: 389-390.

Petersen, L. and Madsen, M. (2000): Listeria spp. in broiler flocks: recovery rates and species distribution investigated by conventional culture and the Eia foss method.  Int. J. Food Microbiol. 30; 58 (1-2): 113-6.

Petran, R.L. and Swanson, K.M.J. (1993): Simultaneous growth of Listeria monocytogenes and Listeria innocua. J. Food Prot. 56 (7): 616-618.

Rocourt, J.A. and Cossart, P. (1997): L.monocytogenes.In:Food Microbiology: Fundamentals and frontiers.pp.337-352, M. P. Doyle; L.R. Beuchat and T.J. Montville (eds.). ASM press, Washington, D.C.

Smaill, F. (2000): Antibiotic susceptibility and resistance testing. Can. J. Gastroenterol., 14 (10): 871-875.

Thamm, H. (1962): Zur Epizootiologie der Listeriose. Moonatsh Veterinaemed. 17: 224-370.

Van Der Schaaf, A.; Vandorssen, C.A.; Donker, Voet, J. and Jaartsvels, F.H.J. (1956): Overzicht der onderzoekingen van het uit de practijk ingezonden ziektematerial over het Jaar (1955).  Tijdscher. Diergeneesk 81, 669-679.

Warburton, D.; Boville, Ann, Pagotto, F.; Daley Elaine and Chow. Cindy (2003): The detection of Listeria spp. in foods and environmental samples using placom broth. Health Products and Food Branch (HPFB), Ottawa, Ontario.

     Fig. (1): Listeria infected chicken embryo at 13/14 days showing severe congestion of internal organs, distention of gall bladder, enlargement and congestion of yolk sac.

     Fig. (2): Inoculated chick  that died at day 9 post-inoculation showing stunting, depression, ruffled feathers, incoordination of gait and prostration.

    Fig. (3):  Hatched chicks expressing bilaterial toe paralysis and Tremor ( Right, Middle)- uninfected chick (Left).

    Fig. (4):  Experimental and contact chicks showing sever congestion in all body muscles and internal organs.

     Fig. (5): Experimental and contact chicks showing congested brain and oedematous fluid on the meninges.

     Fig. (6): Intraperitoneally inoculated ckickens showing torticollis, dullness, bilateral toe paralysis and ruffled feathers.

      Fig.(7): Intraperitoneally inoculated ckickens showing congestion of internal                                                           organs, pericarditis, air sacculitis and brain.