The invasion of salmonella typhimurium into the cecal wall of chickens infected with Eimeria tenella

Zbl. Bakt. Hyg., I. Abt. Orig. A 253, 344-3 54 (1982) Dep artm ent of Veterinar y Medicine, College of Agriculture, Universiry of Osaka Prefecture , Sakai, Osaka 591, Ja pa n

The Invasion of Salmonella typhimuriurn into the Cecal Wall of Chickens Infected with Eimeria tenella Der Befall der Zokumwand durch Salmonella typhimurium bei mit Eimeria tenella infizierten Hiihnern YOSHIAKI HIKASA, EIICHIRO BABA, TSUNEO FUKATA, and AKIRA ARAKAWA

With 6 Figures ' Received Jun e 14, 1982

Summary The effect of concurre nt Eimeria tenella and Salmonella ty phimu rium infectio ns on the invasion of S. typhimurium into the cecal wall of chickens were stu died. In the experiment 1) groups were composed of bi rds infected with a daily dose of 1 x 103 to 1 x 10" S. typhim urium for 5 days and birds infected with S. ty phimurium as th e same manner one day after having been infected with 20,000 E. tenella oocysts. Chickens were killed 7, 10 and 14 days after coccidial infection. Th e ceca were examined bacteriologically. The number of S. ty phimurium in the cecal washings and cecal wall and the number of chickens po sitive for S. ty phimurium in the ceca were significantly greate r in the concurre nt infections th an in the S. ty phimuri um alone. In experi ment 2) groups were composed of birds infected with E. tenella and S. ty phimurium in the same mann er as experiment 1) and birds infected with 2 x 10· to 2 X 10' S. typhimuriu m for 5 consecutive days. Chickens were killed 7, 10 and 14 (or 15) days after coccidial infection. Th e distribution of S. typhimurium in the ceca was exa mined by fluorescent anti bo dy technique. In coccidia-infected chickens, intense, specific fluorescence was noted in th e epithelia destructed by coccidia, around the coccidia l oocysts parasit ized in th e epithelial cells, and in the lam ina propria and submucosa. In S. typh imurium alone-infected chickens, specific fluorescence was also found in th e epithelia, lamin a pr opria and submucosa, but was only limited in th e lumen and on the surface of epithelial linings. The results indicated that th e invasion of S. typhimurium into the cecal wall was enhanced by E. tenella infection in chickens.

Zusammenfassung Es wurde die Auswirkung einer konkurrierend en Infekt ion mit Eimeria tenella und Salmon ella ty phimurium auf den Befall der Zokurnwand bei Hiihnern untersucht. Im Versuch 1 setzte n sich die Versuchsgruppen aus Tieren zusamm en, die 5 Tage lang raglich mit 1 x 103 - 1 x 10" S. ty phimurium infiziert wor den waren und aus anderen, die in gleicher Weise S. typhim urium 1 T ag nach Infekrion mit 20000 E. tenella-Ooz ysten erhalten hatten.

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Die Hiihner wurden 7 bzw. 10 und 14 Tage nach der Kokzidieninfektion getotet. Die Blinddarrne wurden bakteriologisch untersucht. Bei konkurrierender Infektion waren die Bakterienzahlen in der Spiilfltissigkeit und den Blinddarrnwanden sowie die Zahl der Tiere mit S. typhimurium-positivem Blinddarm signifikant hoher als bei schleimiger S. typhimurium-Infektion. Versuch 2 umfafsre Gruppen von Hiihnern, die wie in Versuch 1 mit E. tenella und S. typhimurium, sowie solche, die 5 Tage hintereinander mit 2 x 106- 2 X 107 S. typhimurium infiziert worden waren. Die Tiere wurden 7, 10 und 14 (oder 15) Tage nach der Kokzidieninfektion getotet. Die Verteilung von S. typhimurium in den Blinddarmen wurde mit Hilfe fluoreszierender Antikorper untersucht. Bei den mit den Kokzidien infizierten Tieren wurde eine intensive spezifische Fluoreszenz in dem von den Kokzidien zerstorten Epithel, urn die in den Epithelzellen parasitierenden Oozysten herum, sowie in der Lamina propria und Submucosa festgestellt. Bei den nur mit S. typhimurium infizierten Hiihnern fand sich ebenfalls eine spezifische Fluoreszenz in Epithel, Lamina propria und Submucosa, diese beschrankte sich aber auf das Darmlumen und die Oberflache der Epithelauskleidung. Die Ergebnissedeuteten an, daB das Eindringen von S. typhimurium in die Zokumwand von Hiihnern durch eine E. tenella-Infektion verstarkt wurde. Introduction Coccidial infection has been shown to enhance salmonella infection in chickens and increase the passage of salmonella from the intestinal tract to the internal organs. Stephens et al. (1964) found that S. typhimurium recoveries from the liver and spleen of E. necatrix-infected chicks were greater than from the organs of chicks infected with salmonella alone. Further studies demonstrated that coccidiosis interferes with the rapid elimination of S. typhimurium from the intestine and internal organs of chicks infected with both E. necatrix and S. typhimurium and indicated that salmonella persist in and permeate the damaged intestine of coccidia-infected chicks (Stephens and Vestal, 1966). Arakawa et al. (1981) reported that E. ten ella infection enhances S. typhimurium infection in the ceca and the liver of chickens exposed to approximately 104 S. typhimurium per bird for 5 consecutive days and killed 7, 10 and 14 days after E. ten ella infection. These doses used were substantially lower than those of reports where 108 to 10 10 salmonella were exposed (Stephens et al., 1964; Stephens and Vestal, 1966; Olesiuk et al., 1969; Sadler et al., 1969; Brownell et al., 1969, 1970; Fanelli et al., 1971; Brown et al., 1975). Furthermore, even at these low exposure levels Baba et al. (1982) found that salmonella remained longer in the ceca and liver of chickens infected with E. tenella than those in birds infected with salmonella alone. It seemed unlikely that under field conditions birds are readily exposed to billion salmonella. The purpose of the present experiment was to study the distribution of S. typhimurium in the cecal washings and cecal wall of chickens infected with E. ten ella and S. typhimurium and to compare with those of chickens infected with S. typhimurium alone. Bacteriological method and fluorescent antibody technique were employed to study the distribution of S. typhimurium. Materials and Methods Birds

White Leghorn, Hy-line", cockerels were purchased from a local commercial hatchery when less than one day old and were kept in electrically heated battery brooders. At 4 days

346

Y.Hikasa, E.Baba, T.Fukata, and A.Arakawa

of age, the birds were transferred to wired-floor batteries in an air conditioned room with continuous artificial illumination. Diets Feed and water were available ad libitum during the study. The compos ition of the basal ration has been described (Arakawa and Obe, 1975). Coccidia Th e E. tene/la strain used in this stud y was originally supplied by Dr. K. Tsunoda of the N ation al Institute of Animal He alth in T sukuba. Sporulated oocysts were prepared from the feces of donor chickens 7 to 8 days after oral inoculation. A dose of 20,000 sporulated oocys ts of the same culture was used in each experiment. Salmonella The S. typhimurium strai n, L.55, was supplied by Dr. S.Satoh of the National Institute of Anim al Health in Tsukuba . It was originally isolated from a young broiler chick in a field outbreak of paratyphoid infection . The organism was grown in Trypticase soy broth (Nissui) at 37 DC for 18 h, and washed, and resuspended into sterile saline solution. Bacteriological examination Mannitol Lysine Crystal Violet Brilliant Green (MLCB) agar (Nissui) was the medium to count S. typbimurium in the inoculum and ceca. At necropsy, cecal sample from each bird was taken asepticall y, weighed, incised, and placed in first tube containing 10 ml of sterile saline solut ion. The tube was shaken thoroughly until cecal contents were washed off from the wall. The cecum was tran sferred to second tube containing 10 ml of saline solution and shaken. An additio nal washing was made in third tube and the cecum was removed. On e ml each from the 1st, 2 nd and 3 rd washings were pooled, mixed, and diluted by serial tenfold steps. From the diluti on of odd numbers, 0.1 ml of the suspension was withdrawn and spread on MLCB agar. A 0.1 ml of the 3rd tube was also spread on MLCB agar. Th e cecum from the third tub e was blotted on a filter paper, weighed again and minced by a pair of scissors in a sterile mort ar. To the minced wall added 9 volume s of sterile saline solutio n and the mixture was homogenated. The homogenate was diluted by serial tenfold steps. From each of the serial dilut ion , including the initial homogenate, 0.1 ml of the suspension was withdrawn and spread on MLCB agar. The agar plates were incubated at 37 DC for 24 h. S. typhim urium was confirmed by agglutination with the commercial polyvalent and 0 group-specific antisera (K i rasaro ), Fluorescent antibody exami nation Fifteen, 5-week-old chickens were given a single oral inocul ation of 101 colony-forming units (CFD) of S. typhimurium . Twenty days later, antisera were collected from 6 surviving birds. The tube agglutinin tit er was 1;12,400. The globulin was precipitated with ammonium sulfate and conjugated with fluorescein isothiocyanate accord ing to the procedures described by Hamashima and Kyogoku (1965). Non-specific antiglobulin was adsorbed with chicken liver powder. Cecal samples for fluorescent anti body examination were washed once with sterile saline solution to remove extraneous materials. Ceca were frozen in n-Hex ane at - 70 DC in a dry-ice aceto ne bath and sectioned in 6 Ilm thick by a cryostat-microtome maintained at - 20 °C. Th ese sections were mounted on cover slips and dried under warm air. They were fixed in aceto ne at - 70 °C for 5 min and dried at 20 °C to 26 °C for 1 h. The sections were reacted with a drop of the labelled antiglobulin and held in a moist chamber at 4 °C over night after which they were washed for 15 min with 3 changes of pho sphate buffered saline solution. Th e sections were mounted on slide with buffered glycerol and examined with a Nikon fluorescence microscope.

Invasion of S. typhimurium with the Aid of E. tenella

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Histological examination Cecal samples for histological examination were fixed with 10% buffered formalin and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. Experimental design Experiment 1: This experiment consisted of three identical trials. In each trial, a total of 30 birds, 4 days old, were divided into 2 groups of 15 chickens each. The group were birds infected with S. typhimurium alone and birds infected with both E. tenella and S. typhimurium. Chickens in the coccidia-infected group received orally a single inoculation of 20,000 sporulated oocysts suspended in 1.0 ml of tap water. Chickens were each exposed to oral inoculation of 1 x 103 to 1 X 105 CFU of S. typhimurium daily 1 through 5 days after coccidia exposure. Five chickens in each group were necropsied 7, 10 and 14 days after coccidial infection and ceca from each bird were provided for bacteriological examination. Experiment 2: In this experiment, a total of 60, 4-day-old birds were divided into 2 groups; one group of 20 birds infected with S. typhimurium alone and another group of 40 birds infected with E. tenella and S. typhimurium. Chickens in the E. tenella infected group received a single oral inoculation of 20,000 oocysts and were inoculated orally with a daily dose of 4 x 103 to 2 X 104 CFU of S. typhimurium daily 1 through 5 days after coccidial infection. Chickens in group exposed to S. typhimurium alone were inoculated orally with a daily dose of 2 x 106 to 2 X LO? CFU of S. typhimurium for 5 consecutive days on a similar schedule. On 7, 10 and 14 (or 15) days after coccidial infection, 11 to 16 birds in both organisms-infected group and 6 to 8 birds in salmonella alone-infected group were necropsied. Two adjacent sections of 4 to 5 mm width each were excised from the middle portion of the right cecum in each bird. One was provided for the fluorescent antibody study and another for histological examination to aid the fluorescent antibody observation. The remaining portions of the right cecum were provided for bacteriological examination Statistical analysis In experiment 1, data on the number of S. typhimurium after square root transformation were subjected to analysis of variance (Steel and Torrie, 1960), and the number of birds positive for salmonella were subjected to Student's t-test.

Results

Experiment 1 Table 1 summarizes the results of S. typhimurium recovery from the cecal washings and cecal wall of chickens killed 7, 10 and 14 days after coccidial infection. The number of chickens positive for S. typhimurium in the cecal washings of birds killed 10 and 14 days after coccidial infection was significantly greater in chickens infected with E. tenella and S. typhimurium than in S. typhimurium alone-infected birds. The number of chickens positive for S. typhimurium in the cecal wall of birds killed on 7, 10 and 14 days after coccidial infection was greater (P 0.05) than those of birds infected with S. typhimurium alone. S. typhimurium counts in the cecal washings of birds killed 10 days after coccidial infection were significantly (P 0.05) greater than the counts in birds infected with S. typhimurium alone. In birds killed 7 and 14 days after coccidial infection the average number of S. typhimurium in the cecal washings were greater than in those infected with S. typhimurium alone, but the differences were not significant (P> 0.05). The counts of S. typhimurium in the cecal wall of birds

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Invasion of S. typhimurium with the Aid of E. tenella

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Experiment 2 On 7 days after coccidial infection, brilliant, specific fluorescence was present around oocysts and epithelial debris in the lumen. Brilliant, specific fluorescence was noted in the surface epithelia and lamina propria ulcerated by E. tenella infection. Specific fluorescence was also seen in the grandular epithelia and lamina propria. There is specific fluorescence in the epithelia destructed by coccidia and around gametocytes and ooc ysts in the grandular epithelial cells (Fig. 1). Bright, specific fluorescence in the epithelia and lamina propria was seen (Fig. 2). Specific fluorescence was also observed in the submucosa and occasionally in the muscularis and serosa.

Fig. 1. Fluorescence micrograph of the cecum from a S. typhimurium infected chicken killed 7 days after coccidial infection. Specific fluorescence of S. typhimurium antigen is seen in the grandular epithelia and lamina propria ulcerated by E. tenella. Coccidialoocysts show yellow coloured autofluorescence. S. typhimurium counts in the cecal washings and cecal wall were 7.3 x 106 /g and 1.1 x 10' /g, respectively. L = lumen, E = epithelium, LP = lamina propria, 0 = oocyst.

On 10 days after coccidial infection, specific fluorescence was seen in the lumen, epithelia, lamina propria and occasionall y submucosa. Brightness of specific fluorescence in the grandular epithelia and lamina propria was lesser in degree than those on 7 days after coccidial infection. Specific fluorescence was seen around degenerated ooc ysts that remained in the epithelia and lamina propria (Fig. 3). 23 Zbl. Bakt. Hyg., I. Abt, Orig, A 253

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Fig. 2. Fluorescence micrograph of the cecumfrom a S. typhimurium infected chickenkilled 7 days after coccidial infection. In the grandular epithelia, specific fluorescence of S. typhimurium antigen is seen around oocysts and in the lamina propria. Coccidial oocysts show yellow coloured autofluorescence. S. typhimurium counts in the cecal washings and cecal wall were 7.3 x 106{g and 1.1 x l0 6/g, respectively. C = crypt, E = epithelium, LP = lamina propria, MM = muscularis mucosa, 0 = oocyst.

On 15 days after coccidial infection, intense, specific fluorescence was evidenced on the surface of epithelial linings and in the surface epithelia and lamina propria (Fig. 4). Specific fluorescence was also seen in the grandular epithelia and lamina propria. Specific fluorescence was seen occasionally in the submucosa, muscularis and serosa. In the caseous core, brilliant, specific fluorescence was observed around the oocysts (Fig. 5). Oocysts showed yellow coloured autofluorescence. In birds killed 7 days after S. typhimurium infection alone, specific fluorescence was seen in the lumen, epithelia, lamina propria and occasionally submucosa (Fig. 6).

Discussion The number of S. typhimurium in the ceca, both washings and wall, was markedly increased by E. tene/La infection, and these findings were in agreement with those of previous reports (Arakawa et a!', 1981; Baba et a!', 1982). Greater numbers of S. typhimurium in cecal walls of birds infected with E. tene/La than those of S. typhimurium alone clearly demonstrate that E. tene/La has increased S. typhi-

Invasion of S. ryphirnurium with the Aid of E. tenella

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Fig. 3. Fluorescence micrograph of the cecum from a S. typhimurium infectedchicken killed 10 days after coccidial infection. Bright, specific fluorescence of S. typhimurium antigen is seen on the surface of grandular epithelial linings, in the epithelia, and lamina propria. Specific fluorescenceis also seen around the coccidial oocysts that remained in the epithelial cellsand lamina propria. Coccidial oocysts show yellow coloured autofluorescence. S. typhimurium counts in the cecal washings and cecal wall were 2.5 x 108/g and 4.8 x los/g, respectively. C = crypt, E = epithelium, LP = lamina propria, MM = muscularis mucosa, O = oocyst.

murium population in th e contents and thereby enh an ced S. typhimurium invasion into the wall. The fluore scent an tibody study was aimed to dete rm ine if the invasion of S. typhimurium into the cecal wall was ind eed assoc iated with E. tenella infection and identify the location and degree of invasion. T o compare between birds infected with E. tenella and S. typhimurium and those infected with S. typhimurium alone, similar population of the organ ism should be maintained in the cecal contents in both groups of birds. Therefore each chicken in the group receiving S. typh imur ium alone was expose d to daily do se of 107 CFU of the organ ism. Bright , specific fluorescence in th e epithelia, lam ina propria, subm ucosa, and occasionally muscularis and sero sa of th e E. tene lla infected ceca indicated th e invasion of S. typhimurium. Furthermor e, extensive invasion of S. typhimurium was observed in the epithelia ulcer ated by E. tenella infection an d aro und the coccidial ooc ysts in the epithelial cells. H owever, even th ough the populat ion of S. typhimurium in the cecal washings of birds expose d to the organism alone was as high as 10 6 CFU

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Fig. 6. Fluorescence micrograph of the cecum from a chicken infected with S. typhimurium alone and killed 7 days after S. typhimurium infection. Specific fluorescence of S. typhimurium antigen is present along the surface of epithelial linings and in the epithelia. S. typhimur ium counts in the cecal washings and cecal wall were 8.0 x lOs/g and 8.2 x l03 /g, respectively. L = lumen, LP = lamina propria, MM = muscularis mucosa.

per g, intense, specific fluorescence was on ly limi ted in the lumen and on th e epithelial surface of epithelia l linings. S. typhimurium population in the wa ll was tend to be fewer th an those of E. tenella-S. typhim urium infected chickens . Although a few non-specific fluore scence was noted in the cecal tissues of chickens infected with E. tenella alone, it wa s pale yellow in colo r and readily differenciated from brilliant specific fluorescence. These observations demonstrate that E. tenella infection was responsible for the increased po pulation of S. typhimurium in th e cecal contents and enhanced the invasion of S. typhimurium into the cecal wa ll by eroding the epithelial linings. As observed by the fluo rescent antibody study, a large population of S. typhimurium were seen around coccidial ooc ysts in the caseou s core fo rme d in the lumen after coccidial infection. T he core in the ceca may rema in for weeks or

Fig. 5. Fluorescence micrograph of the caseous core in the cecum from a S. typhimurium infected chicken killed 10 days after coccidial infection. Specific fluorescence of S. typhimurium antigen is present around oocysts in the caseous core. Coccidial oocysts show yellow coloured autofluorescence. S. typhimurium counts in the cecal washings were 2.5 X l08/g. 0 = oocyst.

354

Y.Hikasa, E.Baba, T.Fukata, and A.Arakawa

months (Reid, 1978). Thus, the presence of core could serve as a source of the extended excretion of S. typhimurium from birds infected with E. tenella. It should be taken into consideration that under field conditions the concurrent infections of chickens with E. tenella and S. typhimurium, and probably with other coccidia and salmonella as well, could readily contaminate their environment for extended period of time. Acknowledgments. The authors thank Dr. K. Tsunoda and Dr. S.Satoh of the National Institute of Animal Health in Tsukuba for suppling a strain of E. tene/la and S. typhimurium, L. 55, respectively.

References 1. Arakawa, A., E. Baba, and T. Fukata: Eimeria tene/la infection enhances Salmonella typhimurium infection in chickens. Poult. Sci. 60 (1981) 2203-2209 2. Arakawa, A. and O. Ohe: Reduction of Clostridium perfringens by feed additive antibiotics in the ceca of chickens infected with Eimeria tene/la. Poult. Sci. 54 (1975) 10001007 3. Baba, E., T. Fukata, and A. Arakawa: Establishment and persistence of Salmonella typhimurium infection stimulated by Eimeria tene/la in chickens. Res. Vet. Sci. 33 (1982) 95-98 4. Brown, D. D., ]. G. Ross, and A. F. G. Smith: Experimental infection of cockerels with Salmonella typhimurium. Res. Vet. Sci. 18 (1975) 165-170 5. Brownell, f.R., W. W.Sadler, and M.].Fanelli: Factors influencing the intestinal infection of chickens with Salmonella typhimurium. Avian Dis. 13 (1969) 804-816 6. Brownell, ]. R., W. W. Sadler, and M.]. Fanelli: Role of ceca in intestinal infection of chickens with Salmonella typhimurium. Avian Dis. 14 (1970) 106-116 7. Fanelli, M. l., W. W. Sadler, C. E. Franti, and]. R. Brownell: Localization of salmonellae within the intestinal tract of chickens. Avian Dis. 15 (1971) 366-375 8. Hamashima, T. and M. Kyogoku: Immunohistology, pp.64-81. Igaku Shoin Ltd., Tokyo (1965) 9. Olesiuk, O.M., V.L.Carlson, G.H.Snoeyenbos, and C.F.Smyser: Experimental Salmonella typhimurium infection in two chicken flocks. Avian Dis. 13 (1969) 500-508 10. Reid, W. M.: Coccidiosis. In: M.]. Hofstad (ed.), Diseases of poultry, pp. 784-815. Iowa State University Press, Ames/IA. (1978) 11. Sadler, W. W., ]. R. Brownell, and M.]. Fanelli: Influence of age and inoculum level of shed pattern of Salmonella typhimurium in chickens. Avian Dis. 13 (1969) 793-803 12. Steel, R. G. D. and f. H. Torrie: Principles and procedures of statistics, pp. 161-205. McGraw-Hill Book co. Inc., New York (1960) 13. Stephens, ]. F., B. D. Barnett, and D. F.Holtman: Concurrent Salmonella typhimurium and Eimeria necatrix infections in chicks. Poult. Sci. 43 (1964) 352-356 14. Stephens, ]. F. and O. H. Vestal: Effects of intestinal coccidiosis upon the course of Salmonella typhimurium infection in chicks. Poult. Sci. 45 (1966) 446-450 Dr. Y. Hikasa, Dept. of Veterinary Medicine, University of Osaka Prefecture, College of Agriculture, Mozuumemachi, Sakaishi, Osaka 591, Japan