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The Effects of Stress on Respiratory Disease and Transient Colonization of Turkeys with Listeria monocytogenes Scott A V. Dutta,A G. R. Huff,BF W. E. Huff,B M. G. Johnson,C R. Nannapaneni,D and R. J. SaylerE A
Department of Poultry Science, University of Arkansas, Fayetteville, AR 72704 USDA, Agricultural Research Service, Poultry Production and Product Safety Research, Poultry Science Center, University of Arkansas, Fayetteville, AR 72701 C Department of Food Science, University of Arkansas, Fayetteville, AR 72704 D Department of Food Science, Nutrition & Health Promotion, Mississippi State University, Mississippi State, MS, 39762 E Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72704 Received 31 March 2008; Accepted and published ahead of print 19 June 2008 SUMMARY. Listeria monocytogenes contaminates poultry processing plants due to its ubiquitous nature and high resistance to disinfection. The sources of the persistent, biofilm-forming strains that colonize processing plants are unknown. The purpose of this study is to determine if intrinsic colonization of turkeys is a possible source of contamination. Male poults were subjected to cold stress from 4–12 days of age; poults were unchallenged or were exposed to an aerosol and oral challenge of either an avian pathogenic strain of Escherichia coli (Ec), the Scott A strain of L. monocytogenes (Lm), or to a combination (Ec–Lm). At 7 wk, all cold-stressed poults were treated with an immunosuppressive dose of dexamethasone (Dex) and exposed to the same bacterial challenges. Birds were necropsied at 1 wk and 2 wk post-Dex treatment. Percent mortality, body and organ weights, and airsacculitis scores were determined. Liver and knee synovial tissues were sampled, using transport swabs, and cultured by direct plating, pre-enrichment, and TaqmanH real-time PCR. There were no significant differences in cumulative mortality, and airsacculitis scores were variable but tended to be decreased by cold stress. Relative weights of liver and heart were increased, whereas body weights, and spleen and bursa relative weights, were decreased following all challenges. Listeria monocytogenes was isolated from up to 50% of liver or knee synovial tissues, using pre-enrichment followed by culturing methods, and from up to 67% of knee synovial tissues using pre-enrichment followed by real-time PCR at 1 wk postchallenge. The higher percentages of positive birds, detected by real-time PCR rather than by culture, and the negative results at 2 wk post-challenge suggest that the birds experienced a transient infection that was cleared by the immune system. These results suggest that environmentally acquired L. monocytogenes can transiently colonize the liver and synovial tissues of stressed turkeys and may be a sporadic source of contamination of processing plants. RESUMEN. Efecto del estre´s en la enfermedad respiratoria y colonizacio´n transitoria con Listeria monocytogenes Scott A en pavos. La Listeria monocytogenes contamina las plantas de procesamiento avı´cola debido a su naturaleza ubicua y alta resistencia a la desinfeccio´n,. Se desconocen las fuentes de las cepas persistentes formadoras de biocapas que colonizan las plantas de procesamiento. El propo´sito de este estudio fue determinar si la colonizacio´n intrı´nseca de los pavos representa una fuente posible de contaminacio´n. Pavos machos jo´venes se expusieron a estre´s por frı´o desde los cuatro hasta los 12 dı´as de edad, las aves se mantuvieron sin desafı´o o fueron expuestas a un desafı´o oral y por aerosol con una cepa aviar de Escherichia coli pato´gena, la cepa Scott A de L. monocytogenes o a una combinacio´n de ambas. A las siete semanas, todas las aves estresadas con frı´o se trataron con una dosis inmunosupresora de dexametasona y se expusieron a los mismos desafı´os bacterianos. La necropsia de las aves se realizo´ una y dos semanas posteriores al tratamiento con dexametasona. Se determino´ el porcentaje de mortalidad, el peso corporal y de los o´rganos y los valores registrados de aerosaculitis. El hı´gado y los tejidos sinoviales de la articulacio´n de la rodilla se muestrearon utilizando hisopos y se procesaron por cultivo directo, preenriquecimiento y la prueba de reaccio´n en cadena por la polimerasa transcriptasa reversa en tiempo real TaqmanH. No se observaron diferencias significativas en la mortalidad acumulada y los valores registrados de aerosaculitis fueron variables pero con tendencia a ser disminuidos por el estre´s por frı´o. Se incrementaron los pesos relativos del hı´gado y del corazo´n, mientras que los pesos corporales y los pesos relativos del bazo y la bolsa disminuyeron posterior a los desafı´os. Una semana posterior al desafı´o, se aislo´ Listeria monocytogenes de hasta el 50% de los hı´gados o tejidos sinoviales utilizando preenriquecimiento seguido de me´todos de cultivo, mientras utilizando preenriquecimiento seguido de la prueba de reaccio´n en cadena por la polimerasa en tiempo real, se identifico´ la bacteria en hasta un 67% de los tejidos sinoviales. Los mayores porcentajes de deteccio´n de aves positivas mediante la prueba de reaccio´n en cadena por la polimerasa en tiempo real en lugar de mediante cultivo, y los resultados negativos a las dos semanas posteriores al desafı´o, sugieren que las aves experimentaron una infeccio´n transitoria que fue eliminada por el sistema inmunolo´gico. Estos resultados sugieren que la L. monocytogenes adquirida del ambiente puede colonizar el hı´gado y los tejidos sinoviales de pavos estresados y pueden constituirse en fuentes espora´dicas de contaminacio´n de las plantas de procesamiento. Key words: turkeys, Listeria monocytogenes, dexamethasone, cold stress, real-time PCR Abbreviations: CBA 5 Columbia blood agar; CFU 5 colony forming unit(s); Dex 5 dexamethasone; Ec 5 Escherichia coli challenge; Ec–Lm 5 Listeria monocytogenes and Escherichia coli co-challenge; FAC 5 ferric ammonium citrate; LA 5 Listeria selective agar; Lm 5 Listeria monocytogenes challenge; R-ti-PCR 5 real-time polymerase chain reaction; TOC 5 turkey osteomyelitis complex; UVM 5 University of Vermont modified Listeria medium; UVM–I 5 University of Vermont enrichment media
Corresponding author. E-mail: [email protected] Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.
V. Dutta et al.
Listeriosis, a systemic infection with Listeria monocytogenes, causes both sporadic episodes and outbreaks of food-borne human illness globally and is responsible for the highest hospitalization rate (91%) of the known food-borne diseases (34). Listeria monocytogenes does not affect most healthy persons; however, pregnant women and their fetuses, the elderly and the very young, and immunocompromised persons are all vulnerable to infection (51). The prevalence of Listeria spp. in turkey meat (57,65) and the meat processing plant environment (40) is considered a result of the ubiquitous presence of this organism and of an inability to completely sanitize processingplant equipment. The high resistance of L. monocytogenes to decontamination treatments (12,17,34), and its ability to form highly resistant biofilms on surfaces (9), contributes to its ability to persist in the processing plant environment. Reports have shown an increase in L. monocytogenes contamination of processing plants in the later stages of processing of many food commodities (40). Genigeorgis et al. (20,21) reported such an increase in poultry processing plants, even without the isolation of Listeria from any external sources of contamination such as feathers, feces, or incoming chilling and scalding water. It has also been reported that the incidence of Listeria spp. increases on the gloves and hands of workers from the beginning to the end of the cut-up line (12,49,50), suggesting the possibility of intrinsic infection of poultry carcasses as a source of contamination. Listeria monocytogenes is an opportunistic, intracellular pathogen that causes systemic and localized infections, including an uncommon but serious condition of infectious arthritis in humans (43) which has been associated with immunosuppressive therapy (59,63). Listeriosis is also associated with environmental stressors, including cold temperature (6,8,22), which can cause alterations in the immune response of animals, including birds (23,44,61,64). In turkeys, stress-induced immunosuppression has been shown to result in synovial colonization with opportunistic pathogens in apparently healthy birds; this can lead to a processing plant condemnation problem known as turkey osteomyelitis complex (TOC; 32). We have hypothesized that, under severe environmental stress, opportunistic bacteria such as L. monocytogenes can become systemic and can colonize the synovial and other tissues of immunosuppressed birds, and that the resulting subclinical infection may then act as a potential source of contamination in the poultry processing plant (30). We have developed an experimental model for TOC, using immunosuppression with the synthetic glucocorticoid dexamethasone (Dex), which reproduces osteomyelitis, synovitis, arthritis, and colonization of the synovial tissues of turkeys with opportunistic pathogens. In this model, respiratory challenge with low numbers of Escherichia coli (25–100 colony forming units [CFU]) results in soft tissue colonization with E. coli as well as with Staphylococcus aureus and other environmental pathogens (32). The objective of this study was to use a similar model to determine the effects of stress and environmental exposure to a concurrent E. coli challenge on colonization of turkeys with a serotype 4b strain of L. monocytogenes, because 4b serotype strains are associated with food-borne outbreaks and have been isolated from turkey meat processing plants (16,36). MATERIALS AND METHODS Stress and challenge. Two biosecure colony houses were used to house birds in this study. Male turkey poults (n 5 152) were obtained from a commercial hatchery, wing-banded, and placed in floor pens on pine shavings in the control colony house at a room temperature of 26–
29 C; poults were also provided with infrared brooding lamps as an additional heat source (MOR Electric Heating Inc., Cornstock Park, MI). Poults were provided an ad libitum supply of water and a standard turkey starter diet that met or exceeded the nutritional requirements recommended by the National Research Council (48). At 4 days-of-age, 129 birds were moved to the challenge house. These birds were then exposed to different combinations of cold stress and environmental bacterial challenge. From day 4–12, the challenge house temperature was maintained at 15–21 C. Non–cold-stressed pens were provided with brooding lamps as a heating source and cold-stressed pens were not provided with brooding lamps. Challenged birds were exposed to 2 ml of a coarse spray, directed at eyes and nose, containing 8 3 108 CFU/ml E. coli (Ec); or 5 3 108 CFU/ml L. monocytogenes (Lm); or 4 ml of the combination (Ec–Lm) on days 6, 10, 11, and 12. The same bacterial challenge was also added to the drinking water, adding 10 ml each for Ec and Lm, and 20 ml for Ec–Lm, on days 7 and 11. During week 7, on 48, 49, and 50 days-of-age, each challenged bird was given an injection of Dex (Sigma Chemical Co., St. Louis, MO) into a thigh muscle at a dosage of approximately 2 mg of Dex/kg of body weight as previously described (29), and the same drinking water bacterial challenges were administered on days 50, 52, and 53 (5 ml per bird per day). The negative control was comprised of 15 non-stressed birds that were in the control house and were never exposed to cold stress, Dex, or bacterial challenge. A Dex control was comprised of 14 birds in the challenge house which were not directly challenged with bacteria, but were exposed to cold stress, Dex treatment, and the same environment as the challenged birds. Bacterial strains, culture media, and growth conditions. The L. monocytogenes strain used in this study was a serotype 4b, designated Scott A, a human-epidemic isolate obtained from the U.S. Food and Drug Administration (Cincinnati, OH). The bacterium was maintained on glass beads at 270 C in suspending media composed of brain heart infusion broth with 15% glycerol (Remel, Lenexa, KS). A nonmotile strain of E. coli serotype O2 was used for challenge. This strain had originally been isolated from chickens with colisepticemia and has been used in previous colibacillosis studies (29,32). The inocula were prepared by adding two inoculating loops of an overnight culture on Columbia blood agar (CBA; Remel) to 100 ml of tryptose phosphate broth and incubating for 2.5 hr in a 37 C shaking waterbath to achieve an early log-phase culture. The cultures were held overnight at 20 C while standard plate counts were made. Necropsy. Body weights of poults were determined weekly. A sample of birds was euthanatized by CO2 asphyxiation and necropsied at both 1 wk and 2 wk post-Dex treatment. All of the mortalities and the necropsied birds were weighed and examined for lesions of air sacculitis. The following key, used for scoring airsacculitis lesions, was modified from the one described by Piercy and West (54): 0 5 no inflammation; 1 5 opacity and thickening of the inoculated airsac; 2 5 mild airsacculitis and mild pericarditis; 3 5 moderate air sacculitis/ pericarditis with spread to liver, abdominal cavity (perihepatitis/ peritonitis), or both; 4 5 severe fibrinous air sacculitis and severe pericarditis; and 5 5 severe air sacculitis/pericarditis with spread to liver, abdominal cavity, or both. Liver, heart, spleen, and bursa of Fabricius were excised, weighed, and expressed as a percentage of body weight. Liver and knee synovial tissues were sampled using sterile Bacti-Swabs (Remel), which were then kept at room temperature and cultured within 1–3 hr. All research involving animals was evaluated and approved by the Institutional Animal Care and Use Committee of the University of Arkansas. Isolation of Listeria monocytogenes using cultural methods. The Bacti-Swabs used to culture liver and knee synovial tissues were directly plated onto CBA and Listeria selective agar (LA), a University of Vermont modified Listeria medium (UVM; Difco Laboratories, Detroit, MI) containing Listeria-selective supplement SR140E (Oxoid Ltd., Ogdensburg, NY), moxalactam antimicrobial supplement (BectonDick-
Listeria monocytogenes in turkeys
Fig. 1. Regression graph showing regression equation (R 2 5 0.9922) used to optimize the threshold value (CT value) for unknown samples and to calculate corresponding log CFU/10 ml. inson, Cockeyville, MD), and ferric ammonium citrate (FAC; SigmaAldrich Corp., St. Louis, MO). This was followed by incubation for 24 hr at 37 C. FAC provides an estimate of apparently positive samples, based on an FAC indicator system in which black colonies are formed (13). The identity of putative Lm isolates was confirmed biochemically. Swabs that were negative for Listeria by direct plating were aseptically snipped from their stems and placed in 10 ml of UVM–I medium and incubated for 24 hr at 30 C (Oxoid, Hampshire, United Kingdom). The pre-enrichment step was followed by addition of 1 ml of the UVM–I enriched culture to 10 ml of Fraser broth (Oxoid) for further enrichment at 30 C for 48 hr. Postenrichment samples were plated onto CBA and LA, followed by isolate identification and biochemical confirmation. All isolates from LA plates were identified by Gram’s staining and hemolysis on CBA, and representative isolates were biochemically confirmed, at a species level, using API listeria strips (Biomerieux, Hazelwood, MO) according to manufacturer’s instructions. This was followed by isolate identification and confirmation using the Biolog Microbial Identification System (Biolog Inc., Hayward, CA). Isolates and the control Scott A strain were inoculated onto GP microplates (Biolog) and analyzed and compared with the MicroLog database, version DE, release 3.5, according to manufacturer’s instructions. TaqManH real-time PCR (R-ti PCR) assay. Bacterial DNA was extracted from 2 ml of the UVM–I-enriched culture using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations. Enriched UVM–I samples were incubated with an enzymatic lysis buffer (1 M EDTA, 1 M Tris-Cl, Triton X-100, 20 mg/ ml lysozyme). DNA concentrations were determined by Nano Drop (ND-1000 Spectrophotometer V-3.1.0, Wilmington, DE). UV fluorescence emission was recorded and quantified using ND-1000 V 3.1.0 software (Nanodrop). Table 1. CFU dilution
1:10 1:100 1:1000 1:10,000 1:100,000 1:1,000,000 A
Oligonucleotide primers targeting the L. monocytogenes 64 bp hly gene (GenBank accession no. M24199) were the same as used by Rodrı´quezLa´zaro et al. (56). The oligonucleotide primers were purchased from Applied Biosystems (Foster City, CA). TaqManH R-ti PCR assay was performed as described by Rodrı´quezLa´zaro et al. (56) using TaqMan PCR core reagents (Applied Biosystems–Roche Molecular Systems, Alameda, CA). The 20-ml reaction volume contained 13 PCR TaqMan buffer A (including 5carboxy-X-rhodamine as a passive reference dye); 6 mM MgCl2; 200 mM each dATP, dCTP, and dGTP; 400 mM dUTP; 50 nM primers; 100 nM probe; 1 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA) 0.2 U of AmpErase uracil Nglycosylase (Applied Biosystems); and 2 ml of the target DNA solution. Reactions were performed using the Stratagene MX-3000 P Q-PCR system (La Jolla, CA) with the following program: 2 min at 50 C, 10 min at 95 C, 50 cycles of 15 sec at 95 C, and 1 min at 63 C. For every reaction, a regression equation was devised from a regression graph plotted for threshold (CT) values of a known concentration of DNA that had been diluted over the full range from 1021 to 1025 as positive controls. The lowest value of regression coefficient accepted for each reaction was R2 5 0.98. Log CFUs, obtained for unknown samples, were based on the regression equation from a regression graph drawn between log CFU/ 10 ml and CT values for known concentrations of DNA (Fig. 1). Known concentrations of DNA were obtained by serially diluting the DNA extracted from known CFU of the Scott A strain used in this study, and the corresponding CT values were obtained for respective DNA dilutions by amplification and extrapolation (Table 1). A threshold line was drawn at mean + SEM of the negative-control samples, and samples having a higher number of CFU/10 ml than this threshold were considered positive. Statistics. Treatment means were analyzed for ANOVA using the general linear models procedure of SAS software SAS Institute Inc., Cary, NC; (58). All percentage data were subjected to arcsine transformation. Significant mean differences among the treatments were separated using the least square means procedure. CFU determined by extrapolation from R-ti PCR CT values were analyzed by treatment using the Fit Y by X procedure of Jmp 7.0.1 SAS Institute Inc.; (35). Unless otherwise mentioned, a P value of #0.05 was considered significant.
There were no significant differences in mean cumulative percent mortality; however, the mean of bacteria-challenged pens (19.3%) was more than twice the mean of non-challenged pens (7.9%; Table 2). Air sacculitis scores at 1 wk post-Dex treatment were higher relative to the negative control in non–cold-stressed birds that were challenged with Ec and in cold-stressed birds in either the Dex control or those challenged with Ec–Lm (Table 3). Cold-stressed
Optimization table giving an estimate of log CFU/10 ml and the corresponding CT value for known concentrations of DNA. A
Estimated log CFU/10 ml
550,000 55,000 5500 550 55 5.5
10.1 1.01 0.101 0.00101 0.000101 0.0000101
5.74 4.74 3.74 2.74 1.74 0.74
16.43 18.65 22.82 25.20 28.11 30.31
6 6 6 6 6 6
0.20 0.31 0.12 0.12 0.15 0.13
Corresponds to the serial dilution of pure cultures of Listeria monocytogenes Scott A. Values for 1:10,000, 1:100,000, and 1:1,000,000 are actual plated values in 10 ml and the rest were extrapolated to calculate the theoretical colony forming units (CFU) per reaction. C Concentrations of DNA for the known number of CFU for serial dilution of 1:10 were measured by Nano-Drop (ND-1000 Spectrophotometer V 3.1.0, Wilmington, DE) and the rest were extrapolated. D Mean 6 SE of threshold value for corresponding known concentrations of DNA subjected to amplification in triplicate. B
V. Dutta et al.
Table 2. Cumulative percent mortality of turkeys at 2 wk postdexamethasone (Dex) treatment and challenge with Escherichia coli (Ec), Listeria monocytogenes (Lm), or their combination (Ec–Lm).A Treatment
Neg controlC Dex controlD Ec Lm Ec–Lm
Non–cold stress % Mortality
8.7 6 – 20.0 6 26.7 6 13.3 6
6.0 7.1 18.2 16.0 21.7
8.4 11.8 9.1
– 6 6 6 6
7.1 8.4 7.4 8.8
Values indicate the mean 6 SE of all mortalities and necropsies. Poults were exposed to cold stress (15–21 C) from day 4–12 posthatch. C Neg control 5 control pen having non–cold-stressed, non-Dex treated, and non-challenged birds maintained in a biosecure colony house. D Dex control 5 control pen with non-challenged, but Dex-treated, birds kept in the challenge house and not provided with infrared heaters. B
birds challenged with Ec had significantly lower air sacculitis scores as compared to non–cold-stressed birds challenged with Ec (Table 3). At 2 wk post-Dex treatment, the Lm challenge of non– cold-stressed poults, and the Ec–Lm challenge of both non–coldstressed and cold-stressed birds, led to significantly increased air sacculitis scores compared to both negative- and Dex-control poults (Table 3). Cold-stressed birds challenged with Lm had significantly lower air sacculitis scores compared to non–cold-stressed birds challenged with Lm (Table 3). All treatments significantly decreased body weight relative to the negative control at both 1 wk and 2 wk post-Dex treatment (Table 4). The Ec–Lm challenge of cold-stressed poults significantly decreased body weight at 2 wk post-Dex treatment relative to coldstressed birds challenged with Ec or Lm alone, or with Dex treatment alone (Table 4). Relative liver weights were significantly increased by all treatments at both 1 wk and 2 wk post-Dex treatment, relative to the negative control (Table 5). At 1 wk post-Dex treatment, the Ec–Lm challenge had higher liver relative weight than Ec challenge alone, both in cold-stressed and non–cold-stressed poults. At 2 wk postDex treatment, the Ec–Lm challenge of cold-stressed birds resulted in higher liver weights relative to all other treatments of both the cold-stressed birds and the Lm challenge of non–cold-stressed birds. Cold-stressed birds challenged with Ec had significantly lower liver weight relative to non–cold-stressed birds challenged with Ec (Table 5). At 1 wk post-Dex treatment, heart relative weights were increased by all treatments compared to the negative control popults, and the
Ec–Lm challenge significantly increased the relative heart weight compared to the Ec or Lm challenge alone in cold-stressed birds (Table 5). At 2 wk post-Dex treatment, relative to the negative control, heart relative weights were significantly increased due to all challenges of non–cold-stressed birds and to the Dex, Lm, and Ec– Lm challenges on–cold-stressed birds, (Table 5). Two weeks postDex treatment, cold-stressed birds challenged with Ec had significantly lower relative heart weights compared to non–coldstressed birds challenged with Ec, while cold-stressed birds challenged with Ec–Lm had significantly higher relative heart weights compared to the respective non–cold-stressed challenge (Table 5). Spleen relative weights were significantly reduced by all bacterial challenges, but not by Dex control at 1 wk post-Dex treatment, and there were no significant differences in spleen relative weights at 2 wk post-Dex treatment or due to cold stress (Table 5). Bursa relative weights were significantly decreased at both 1 wk and 2 wk post-Dex treatment by all treatments relative to negative control, and there were no other consistent differences (Table 5). Listeria monocytogenes was not isolated from knee joints using direct plating of transport swabs on LA, at either 1 wk or 2 wk postDex treatment, and only one liver sample was positive using direct plating (data not shown). At 1 wk post-Dex treatment, after pre-enrichment in UVM–I and Fraser broth, L. monocytogenes was isolated from the liver of 50%, 20%, and 40% of non–cold-stressed poults challenged with Ec, Lm, and Ec–Lm, respectively; and from 14.3%, 38%, and 50% of coldstressed birds challenged with Ec, Lm, and Ec–Lm, respectively (Table 6). Cold-stressed poults challenged with Ec–Lm had significantly higher liver isolation compared to the negative control poults (Table 6). There was no L. monocytogenes isolated from liver samples at 2 wk post-Dex treatment (data not shown). Using culturing methods after pre-enrichment in UVM and Fraser broth, L. monocytogenes was isolated from knee synovial tissues of 50%, 20%, and 20% of non–cold-stressed poults challenged with Ec, Lm, and Ec–Lm, respectively; and from 43%, 25%, and 33.3% of cold-stressed poults challenged with Ec, Lm, and Ec–Lm, respectively. However, there were no significant differences between treatments (Table 7). There were no L. monocytogenes isolated from knee synovial tissues at 2 wk post-Dex treatment. Using R-ti PCR, L. monocytogenes was detected in knee synovial tissues of 37.5%, 33.3%, and 28.6% of non–cold-stressed poults challenged with Ec, Lm, and Ec–Lm, respectively; from 12.5% of the Dex control poults; and from 7.7%, 16.7%, and 7.7% of coldstressed poults challenged with Ec, Lm, and Ec–Lm, respectively (Table 7).
Table 3. Air sacculitis scores of birds necropsied at 1 wk and 2 wk post-dexamethasone (Dex) treatment and challenge with Escherichia coli (Ec), Listeria monocytogenes (Lm), or their combination (Ec–Lm).A Air sacculitis score One week post-Dex Treatment
Neg controlC Dex controlD Ec Lm Ec–Lm A
0.0 6 – 2.0 6 0.5 6 1.0 6
Two weeks post-Dex Cold stressB
0.7 0.7ab 1.0ab
2.0 0.3 0.7 1.8
– 6 0.7a 6 0.3b 6 0.3ab 6 0.6a
0.0 6 – 1.3 6 2.5 6 1.6 6
1.3 0.3a 0.7ab
0.0 0.8 0.7 2.5
– 6 6 6 6
0.0c 0.5bc 0.5bc 0.5a
Values indicate the mean 6 SE of all mortalities and necropsies. Poults were exposed to cold stress (15–21 C) from day 4–12 post-hatch. C Neg control 5 control pen having non–cold-stressed, non-Dex treated, and non-challenged birds maintained in a biosecure colony house. D Dex control 5 control pen with non-challenged, but Dex-treated, birds kept in the challenge house and not provided with infrared heaters. a–c Means within each day, with no common superscript, differ significantly (P # 0.05). B
Listeria monocytogenes in turkeys
Table 4. Body weight of turkeys necropsied 1 wk and 2 wk post-dexamethasone (Dex) treatment and challenge with Escherichia coli (Ec), Listeria monocytogenes (Lm), or their combination (Ec–Lm).A Body weight (g) One week post-Dex Treatment
Non–cold stress C
Neg control Dex controlD Ec Lm Ec–Lm
5075 6 – 2816 6 3145 6 2945 6
Two weeks post-Dex Cold stress
– 6 6 6 6
3117 3081 2887 2743
190b 169b 157b
5674 6 – 2933 6 2912 6 3106 6
93b 157b 155b 259b
275bc 332bc 286bc
3509 3597 3317 2556
6 6 6 6
185 b 297b 207b 86c
Values indicate the mean 6 SE of all mortalities and necropsies. Poults were exposed to cold stress (15–21uC) from day 4–12 post-hatch. C Neg control 5 control pen having non–cold-stressed, non-Dex treated, and non-challenged birds maintained in a biosecure colony house. D Dex control 5 control pen with non-challenged, but Dex-treated, birds kept in the challenge house and not provided with infrared heaters. a–c Means within each day, with no common superscript, differ significantly (P # 0.05). B
Using R-ti PCR, the percentage of birds positive for L. monocytogenes from challenged, non–cold-stressed birds was significantly higher than from challenged, cold-stressed birds (P 5 0.04). The mean L. monocytogenes CFU, calculated using CT values obtained from R-ti PCR for the Ec treatment (579 CFU), was significantly higher as compared to the negative control, Ec–Lm warm, Ec cold, Lm cold, and Ec–Lm cold treatments (Fig. 2B). The main effect mean CFU for cold-stressed birds (73 CFU) was significantly lower than that of birds not exposed to cold stress (334; P 5 0.009; Fig. 2B).
There was no L. monocytogenes detected in knee joints using realtime PCR at 2 wk post-Dex treatment. DISCUSSION
The results of this study demonstrate that environmentally acquired L. monocytogenes can transiently colonize the liver and synovial tissues of Dex-treated turkeys. These data also provide insight into the effects of concurrent E. coli exposure on L.
Table 5. Percent body weight of liver, heart, spleen, and bursa of Fabricius at 1 wk and 2 wk post-dexamethasone (Dex) treatment and challenge with Escherichia. coli (Ec), Listeria. monocytogenes (Lm), or their combination (Ec–Lm).A Percent Body Weight One week post-Dex Organ
Liver Neg controlC Dex controlD Ec Lm E –Lm
2.1 6 – 2.8 6 3.2 6 3.7 6
Heart Neg control Dex control Ec Lm Ec–Lm
0.51 6 – 0.65 6 0.65 6 0.69 6
Spleen Neg control Dex control Ec Lm Ec–Lm
0.15 6 – 0.11 6 0.08 6 0.11 6
Bursa of Fabricius Neg control Dex control Ec Lm Ec–Lm
Values indicate the mean 6 SE of all mortalities and necropsies. Poults were exposed to cold stress (15–21 C) from day 4–12 post-hatch. C Neg control 5 control pen having non–cold-stressed, non-Dex treated, and non-challenged birds maintained in a biosecure colony house. D Dex control 5 control pen with non-challenged, but Dex-treated, birds kept in the challenge house and not provided with infrared heaters. a–d Organ weight means within each column, with no common superscript, differ significantly (P # 0.05). B
V. Dutta et al.
Table 6. Isolation of Listeria monocytogenes from liver, using culturing methods, at 1 wk post-dexamethasone (Dex) treatment and challenge with Escherichia coli (Ec), Listeria monocytogenes (Lm), or their combination (Ec–Lm).A Percent of birds with positive isolation Treatment
Neg controlC Dex controlD Ec Lm Ec–Lm
00.0 6 – 50.0 6 20.0 6 40.0 6
29.0 20.0ab 25.0ab
00.0 14.3 38.0 50.0
– 6 6 6 6
00.0ab 14.2ab 18.3ab 19.0a
Values indicate the mean 6 SE of all mortalities and necropsies. Birds positive for Lm upon direct plating and after pre-enrichment are included. B Poults were exposed to cold stress (15–21 C) from day 4–12 posthatch. C Neg control 5 control pen having non–cold-stressed, non-Dex treated, and non-challenged birds maintained in a biosecure colony house. D Dex control 5 control pen with non-challenged, but Dex-treated, birds kept in the challenge house and not provided with infrared heaters. a,b Means within each column, with no common superscript, differ significantly (P # 0.05).
Table 7. Percentage of birds with positive isolation of Listeria monocytogenes from knee synovial tissues, using culture methods and TaqManH real-time PCR, at 1 wk post-dexamethasone treatment and challenge with Escherichia coli (Ec), Listeria monocytogenes (Lm), or their combination (Ec–Lm).A Percent of birds with positive isolation Non–cold-stress
Culture methods Neg controlD Dex controlE Ec Lm Ec – Lm Real-time PCRF Neg control Dex control Ec Lm Ec–Lm A
Values indicate the mean 6 SE of percent positive isolation from all mortalities and necropsies. B Poults were exposed to cold stress (15–21 C) from day 4–12 posthatch. C Swabs from each bird were directly plated on University of Vermont modified Listeria-specific medium (UVM). Apparently positive samples were confirmed by biochemical tests. Negative samples were preenriched in UVM–I for 48 hr at 30 C and further enriched in Fraser broth at 30 C for 24–48 hr. D Neg control 5 control pen having non–cold-stressed, non-Dex treated, and non-challenged birds were maintained in a bio-secure colony house. E Dex control 5 control pen with non-challenged, but Dex-treated, birds kept in the challenge house and not provided with infrared heaters. F Samples for DNA extraction were obtained from UVM–I enriched swabs that were apparently positive for Lm in Fraser broth. a,b Means within each method and each column, with no common superscript, differ significantly (P # 0.02).
Fig. 2. (A) Colony forming units (CFU) of L. monocytogenes in enriched culture of knee synovial tissues from individual control and cold-stressed turkeys at 1 wk post-dexamethasone treatment and challenge with Escherichia coli (Ec), Listeria monocytogenes (Lm), or their combination (Ec–Lm). Diamonds indicate the 95% confidence interval for each sample. Numbers identify individual birds. Solid line indicates the mean + SEM for the negative control (Neg con) samples. (B) Data represent the mean 6 SEM CFU for each treatment. Means with no common superscript differ significantly (P # 0.05).
monocytogenes colonization of stressed turkeys. Because this was an environmental challenge, and all challenged birds were confined to the same house and were in adjacent pens, it can be assumed that all birds were exposed to both the L. monocytogenes and the E. coli challenge through the air, flies, litter beetles, and particularly through the dust that was a problem in this facility, and settled on litter, water, and feed. In this study, E. coli challenge was used as a physiologic stressor to simulate the exposure to this organism that is prevalent in commercial production and that has been shown to compromise immunity. The ability of E. coli endotoxin to behave as a stressor has been previously reported (7,38). Colibacillosis, a systemic infection with E. coli, is the most frequently reported disease in poultry production (3), and avian pathogenic E. coli have been shown to
Listeria monocytogenes in turkeys
reduce resistance to the bactericidal effects of serum and to decrease the killing capacity of heterophils and macrophages (4). Cold stress can act as an immunomodulator for poultry; it has been reported to both suppress (55) and enhance (24) the cell-mediated immune response, depending upon experimental conditions (25). Furthermore, what constitutes too little or too much stress is determined at the individual level by genetics, environment, and prior experience (5,61). We had hypothesized that exposure to cold stress during the first 2 wk of life would decrease resistance to bacterial challenge in this model. However, the results seen in the present study suggest that cold stress may have had a protective role in modulating the inflammatory response of some individuals, because at 1 wk postDex treatment, cold stress decreased air sacculitis scores in birds challenged with Ec. At 2 wk post-Dex treatment, cold stress decreased air sacculitis scores in birds challenged with Lm. The detection of L. moncytogenes DNA was significantly lower in cold-stressed birds as compared to non–cold-stressed, and significantly high CFU levels were detected in non–cold-stressed birds. While the effects of cold stress seen in this study were not consistent, they suggest an opportunity to use a controlled, early cold stress procedure to modulate and improve the general stress response of turkey poults. Experiments in mice have suggested that cold acclimation may result in attenuated glucocorticoid response to an acute cold stressor and in decreased production of suppressor macrophages (39). The first, and most dramatic, response of turkeys treated with Dex injection was increased water consumption and diuresis, resulting in extremely wet litter. This very wet litter condition continued as long as Dex was administered and stopped within several days after treatment stopped. Diuresis is a normal reaction to stress in birds. Hester et al. (28) and Hart and Essex (26) reported that the handling and cannulation procedures used to estimate urine output of chickens were, in themselves, increasing output greater than five times. Siegel and Van Kampen (62) reported that injection of corticosterone resulted in a fourfold increase in water excretion. Poultry production is sometimes associated with a wet litter condition, which can act as a breeding ground for many pathogens (27,45). Recently, a major risk study of the causes of wet litter in broiler chickens reported that stressors such as cold temperatures, feed equipment failures, and coccidiosis were correlated with the wet-litter problem (27). The interaction between the decrease in disease resistance that can result from production stressors, and the increase in pathogen load resulting from wet litter, makes this of particular importance to food safety. The ability of production stressors to impair resistance of poulty to opportunistic infection with bacteria of food safety importance has been recently reviewed (33). Reports have associated L. monocytogenes clinical disease in poultry with wet litter conditions and environmental stress (10,41). While some surveys have failed to find L. monocytogenes in the litter of turkey houses (49), some older studies have reported litter contamination with L. monocytogenes to be a persistent problem (14,15). Common shedding of L. monocytogenes by asymptomatic, healthy poultry (14), and the capability of L. monocytogenes to survive for long periods of time under adverse conditions (18), suggests the probability of its sporadic presence in poultry litter and dust; this might lead to sub-clinical colonization as well as to occasional clinical disease. The lesions of air sacculitis induced in the mixed infection of the present study may have facilitated the airborne transmission of L. monocytogenes, because this pathogen has been shown to cause infection through the respiratory route in rodents (1,37) and turkeys (30). Clinical disease, as evidenced by mortality, air sacculitis scores, and changes in body and organ weights, was a feature of this
challenge model; however, there were no significant differences in cumulative mortalities, and the air sacculitis scores were variable. The increase in relative liver weight seen in this study, due to both Dex treatment and bacterial challenge, is consistent with the role of the liver in both stress and in the inflammatory process (19,47). Previous studies have shown that the Dex–E. coli challenge will result in increased relative weights of liver, heart, and spleen, and decreased weight of the bursa of Fabricius. We have previously reported that Lm challenge also decreases the weight of the bursa of Fabricius and causes lymphocytic depletion in bursal follicles in 1-day-old turkeys (30). Bursal pathology was also seen in challenge studies of 5-wk-old turkeys with L. monocytogenes Scott A (31). Immunity to L. monocytogenes has largely focused on T lymphocytes; however, Menon et al. (46) have demonstrated that L. monocytogenes can infect and kill B cells to initiate infection in a mouse. As the bursa of Fabricius is the primary site of B-cell differentiation and maturation in avian species (60), these results suggest that L. monocytogenes may invade and kill B cells in the turkey. In the present study, the relative weights of the bursa of Fabricius were decreased by all challenges. However, since there were no statistical differences between the effects of Dex alone and Dex and Lm challenge, this result cannot be attributed to the Lm challenge. There was no detection of L. monocytogenes from synovial tissues using direct plating on LA; however, upon enrichment we were able to detect a significant percentage of L. monocytogenes-positive birds. Reports have suggested difficulty in isolation of L. monocytogenes from animal tissues (22), and there is strong evidence from both field and experimental infections that L. monocytogenes fails to grow on initial culture attempts from clinical lesions of poultry. In previous challenge studies in this laboratory, direct plating of tissue swabs from turkeys challenged with L. monocytogenes Scott A were rarely positive, while in vitro infection of macrophages with biofilms produced from synovial tissue swabs, followed by fluorescent antibody detection, revealed internalized bacteria (31). Chronic and persistent bacterial infections, including arthritis and osteomyelitis, have been associated with the ability of causative bacteria to form highly resistant biofilms (11,53), suggesting that the difficulty in culturing the causative bacteria may be due to the biofilm phenotype (2). The ability of L. monocytogenes to form persistent biofilms, particularly under stress (52), and the prevalence of the persistent form of L. monocytogenes in meat processing plants (42), suggests that these unapparent infections may contribute to contamination in the poultry processing plant environment. The isolation of L. monocytogenes from liver and knee synovial tissues at 1 wk post-Dex treatment, with no isolation at 2 wk postDex treatment, indicates that this was a transient systemic infection that was cleared from the bloodstream (as indicated by liver culture) and from the peripheral tissues by 2 wk post-Dex treatment. In conclusion, the results of this study suggest that, in the presence of environmental stressors, L. monocytogenes may cause sub-clinical infection within the tissues of turkeys. Previous studies, using Dex and L. monocytogenes challenge alone, showed that only 5% of turkeys had L. monocytogenes knee synovial tissue colonization (31). The results from the present study are suggestive of a supportive role of E. coli-exposure in colonization with opportunistic pathogens such as L. monocytogenes. Such transient colonization of turkey tissues may contribute to the sporadic contamination of poultry processing plants with pathogenic strains that are already in the biofilm phenotype. Further research is required to study the correlation between bacterial isolates from the soft tissues of turkeys and the persistent biofilm-forming strains of L. monocytogenes that are troublesome in the meat processing plant environment.
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ACKNOWLEDGMENTS This study was funded by the USDA Agricultural Research Service and by a grant from the USDA Food Safety Consortium. We are indebted to Dr. Ken Korth, Associate Professor, Department of Plant Pathology, University of Arkansas, and to his laboratory associates, for the use of their real-time PCR equipment and all of their help in this study. We also appreciate the excellent assistance of USDA-ARS research technicians Dana Bassi, Scott Zornes, and Dr. Sonia Tsai.