Poult. Sci.
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Poult Sci 2008. 87:1768-1772. doi:10.3382/ps.2008-00166
© 2008 Poultry Science Association
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IMMUNOLOGY, HEALTH, AND DISEASE: Research Note

The Pathogenesis of Salmonella Enteritidis in Experimentally Infected Ducks: A Quantitative Time-Course Study Using TaqMan Polymerase Chain Reaction1

S. X. Deng*, A. C. Cheng*,{dagger},2, M. S. Wang*,{dagger},{ddagger}, B. Yan*, N. C. Yin*, S. Y. Cao*, Z. H. Zhang* and P. Cao*

* Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Yaan, Sichuan, 625014, China; {dagger} Key Laboratory of Animal Diseases and Human Health of Sichuan Province, Yaan, Sichuan, 625014, China; and {ddagger} College of Life Science and Technology of Southwest University for Nationalities, Chengdu, Sichuan, 610041, China

2 Corresponding author: chenganchun{at}vip.163.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Ducks were subcutaneously infected with a high-virulence strain of Salmonella enterica ssp. enterica serovar Enteritidis (Salmonella Enteritidis). The kinetics of the Salmonella Enteritidis genomic DNA loads, the immunohistochemical localization of the bacterial antigens, and the histopathological examination in various tissues were investigated. The results showed that the time course of the appearance of the Salmonella Enteritidis bacterial antigens and the lesions in various tissues was coincident with the bacterial load of the organism in various infected tissues. This suggests that Salmonella Enteritidis loads in systemic organs are closely correlated with the progression of the infection.

Key Words: Salmonella Enteritidis • pathogenesis • quantitative study • duck


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
A significant proportion of all cases of human salmonellosis are known to be caused by the consumption of raw or partially cooked eggs (Hope et al., 2002; Massi et al., 2006; Xu et al., 2007). Salmonella enterica ssp. enterica serovar Enteritidis (Salmonella Enteritidis) is responsible for most causes of gastrointestinal infection in the world. The incidence of this infection is the greatest in children, the elderly, and immunosuppressed individuals (Agron et al., 2001). In China, consumption of poultry products is high; Salmonella Enteritidis infections in poultry have increased in recent years in China and have had a significant economic impact on the poultry industry, especially the egg industry (Deng et al., 2007, 2008). Salmonella Enteritidis infections in poultry are characterized by vascular damage, eruptions at specific locations on the mucosal surface of the gastrointestinal tract, lesions in the lymphoid organs, and degenerative sequelae involving the parenchymatous organs (Turnbull and Richmond, 1978; Edwards et al., 2000; Dhillon et al., 2001; Kogut et al., 2003; Takata et al., 2003; Deng et al., 2008). In a susceptible host, Salmonella Enteritidis replicates primarily in the mucosa of the digestive tract after oral challenge and then spreads to the spleen, liver, and various other organs and tissues (Dibb-Fuller et al., 1999).

Because of the increased prevalence of Salmonella Enteritidis and its complex life cycle, it is important to understand the correlation between the levels of this bacterium in systemic organs and the progression of the infection; this has not been described previously. Understanding this correlation will help gain further insight into the pathogenesis of Salmonella Enteritidis infections. The objectives of the present study were to determine the pathogenesis of a high-virulence strain of Salmonella Enteritidis (No. MY1; phage type 4, isolated from Peking ducks and maintained by the Key Laboratory of Animal Disease and Human Health of Sichuan Province, China) in ducks by a time-course study using a quantitative TaqMan assay (Deng et al., 2008) and to correlate these findings with the results obtained from the immunohistochemical localization and histopathological examinations of selected Salmonella Enteritidis-infected tissues; we believe that this analysis will help provide valuable insights into the etiology of Salmonella Enteritidis infections.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Bacterial Strains

A high-virulence strain of Salmonella Enteritidis (No. MY1, phage type 4) was isolated from Peking ducks and maintained by the Key Laboratory of Animal Disease and Human Health of Sichuan Province (Deng et al., 2008). The serotype and phage type of the isolates were determined by the National Center for Medical Culture Collection (Beijing, China).

Experimental Birds and Samples

Nine-day-old white Peking ducks free from Salmonella Enteritidis infection were used in the study. Before challenge with Salmonella Enteritidis, all ducks were found to be negative for Salmonella Enteritidis-specific antibodies and Salmonella Enteritidis-specific antigens by ELISA and PCR, respectively (Gast and Beard, 1990; Deng et al., 2008). The ducks were maintained in isolation units in a biosecure animal building and fed a commercial duck diet ad libitum. In brief, Salmonella Enteritidis cells were grown overnight in a Luria-Bertani broth. The cells were cultured overnight and the presumptive live number of Salmonella Enteritidis cells was determined by the spread-plate method. Thereafter, a group of 48 ducks were subcutaneously injected with a high-virulence S. Enteritidis strain (No. MY1; inoculation site, back). Each bird was inoculated with 4.0 x 105 cells in 0.2 mL of water. Another group of 36 ducks was treated with an equal volume of water and used as a control group. The liver, spleen, lung, kidney, jejunum, ileum, rectum, cecum, bursa of Fabricius, thymus, and Harderian gland were analyzed by a fluorescent quantitative PCR assay at postinoculation (PI) times of 30 min, 1, 2, 4, 8, 12, 24, and 36 h, and 2, 3, 6, and 9 d.

At each time point, 3 ducks were randomly selected from the infection and control groups, and their tissue samples were collected and processed for further analyses. Extraction of DNA from the tissue samples was performed as described previously (Deng et al., 2008).

Quantitative Real-Time PCR Assay for Detection of Salmonella Enteritidis DNA

In our previous study, we established a serovarspecific real-time PCR assay (designed with SdfI, GenBank Accession No. AF370707.1; Deng et al., 2008). A real-time PCR assay was carried out using a real-time PCR core kit (R-PCR version 2.1, Takara, Dalian, China) with an Icycler iQ Real-time PCR Detection System (version 3.1, Bio-Rad, Hercules, CA) and was performed as described previously. The PCR amplification was performed in a 25-µL reaction mixture containing 0.6 µL of each primer (10 µmol/L), 0.75 µL of deoxyribonucleotide triphosphates (10 mmol/L), 1.25 U of Ex Taq DNA Polymerase (Ex Taq Hot Start Version, Takara), 5 µL of 5x PCR buffer (Mg2+ free), 0.8 µL of TaqMan probe (5 µmol/L), 0.5 µL of Mg2+ (250 mmol/L), and 5 µL of templates. The reaction mixture was subsequently made up to a volume of 25 µL with deionized water. Each PCR run consisted of a 5-min hot start at 95°C, which activated the conjugated polymerase, followed by 40 cycles consisting of 30 s of denaturation at 94°C, 30 s of annealing at 55°C, and a fluorescent read step. In this study, we used the real-time PCR assay specific to serovars to study the Salmonella Enteritidis loads in various duck tissues following subcutaneous infection.

Immunohistochemical Localization of Salmonella Enteritidis Antigen

Small pieces of tissues were collected and fixed in 10% neutral buffered formalin, processed for paraffin embedding, and sectioned at a thickness of 5 µm. The sections were stained for Salmonella Enteritidis antigen by using the avidin-biotin-peroxidase complex method as described previously (Islam et al., 1993). In brief, deparaffinized and rehydrated tissue sections were incubated with rabbit anti-Salmonella Enteritidis antiserum. Then, an avidin-biotin-peroxidase staining kit (ExtrAvidin, Sigma, St. Louis, MO) was used to trace the rabbit antibodies bound to the site of antigen localization. Tissue sections from the uninfected control samples were used as negative controls. The sections were mounted with Vectashield mounting medium (version 3.0, Takara), and the slides were examined and photographed using a photomicroscope.

For histopathological examination, the paraffin-embedded sections were cut at 5 µm thickness and stained with hematoxylin and eosin as described previously (Mutinelli et al., 2003).

Statistical Analysis

The PCR and data acquisition and analysis were performed using the iCycler iQ Optical system software (version 3.1; Bio-Rad). The number of target copies in the reaction was deduced from the threshold cycle (Ct) values. The threshold cycle value corresponds to the fractional cycle number at which the fluorescence emission exceeds the standard deviation of the mean baseline emission by 15-fold. Plasmid DNA containing the target amplicon was diluted to contain 7.0 x 102 to 7.0 x 108 copies of the target DNA per test tube and used as the plasmid standard series. All samples were analyzed 3 times by the fluorescent quantitative PCR assay, and concentrations of the target DNA detected were expressed as the mean log10 of the bacterial genome copy number per gram of tissue tested. The real-time PCR data were analyzed using version 11 of the SPSS software (SPSS Inc., Chicago, IL). The comparison of means was performed using Duncan’s multiple-range test. A P-value < 0.05 was considered statistically significant.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
In this study, the acute stage of an Salmonella Enteritidis infection in ducks caused by a high-virulence strain of the pathogen was investigated with regard to the kinetics of the bacterial DNA loads, the localization of the bacterial antigens, and histopathological examinations in various tissues. At 12 h PI, high Salmonella Enteritidis DNA loads were observed in various organs of the infected ducks. Thereafter, the bacterial DNA loads increased by various amounts until 36 h PI, and the ducks exhibited typical clinical signs of the infection (Table 1Go); after 36 h PI, some ducks were still alive. At 2 d PI, the remaining 12 infected birds died as a result of the infection. Three dead ducks were randomly selected for tissue sample analysis. Severe hemorrhage, some necrotic foci in the parenchymatous organs, and hemorrhagic enteritis were the predominant gross pathological findings in the dead ducks. Extremely high bacterial DNA loads were observed in the ducks at 36 and 48 h PI compared with those observed at 8 to 24 h PI (Table 1Go). The results of quantitative real-time PCR, immunohistochemical localization, and histopathological examination revealed that the time course of the appearance of bacterial antigens and tissue lesions in various tissues was coincident with the levels of the bacterial DNA loads at the infection sites. These results demonstrated that the widespread dissemination of Salmonella Enteritidis to infection in various organs increased with progression of the infection.


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  		Table 1. Kinetics of Salmonella Enteritidis DNA loads in ducks subcutaneously injected with a high-virulence strain determined by quantitative real-time PCR1
 
An important finding of this study was that during the course of infection, the lymphoid and intestinal organs, especially the spleen, jejunum, and ileum, contained relatively greater bacterial DNA loads than the other organs investigated (Table 1Go). Immunohistochemical analysis revealed high levels of expression of the Salmonella Enteritidis antigen in the epithelial cells and lymphocytes of the jejunum, ileum, Harderian gland, and bursa of Fabricius. High levels of this antigen were also noted in some lymphocytes of the spleen. Histological examination revealed lymphoid organ lesions, including lymphoid depletion and necrosis, and epithelial hypertrophy of the bursa. In the intestine, the Salmonella Enteritidis antigen was localized to the surface epithelium and the epithelial crypt cells and also to the macrophages and fibroblasts of the lamina propria. Sheets of epithelial cells displaced from the villi surface were observed in the infected ileum. Furthermore, all the examined infected organs demonstrated vascular damage, severe hyperemia, and hemorrhage. These results suggest the probability that the lymphoid and intestinal organs are the major target organs of Salmonella Enteritidis replication and that the epithelial cells and lymphocytes probably serve as the principal sites of bacterial replication. Furthermore, the high levels of bacteria DNA in these tissues might reflect the presence of abundant target epithelial and lymphoid cells in these tissues (Dunlap et al., 1991; Abshire and Neidhardt, 1993; Cirillo et al., 1998; Dibb-Fuller et al., 1999; Takata et al., 2003). Therefore, it can be inferred that these cells play a key role in the pathogenesis of acute Salmonella Enteritidis infections, which manifest as severe lesions in the lymphoid organs and small intestine.

The kinetics of the bacterial DNA loads in the other parenchymatous organs varied. At 8 h PI, before the appearance of clinical signs of the infection, it was observed that the liver tissues contained significantly greater levels of Salmonella Enteritidis than the lungs and the kidneys (Table 1Go). However, in dead ducks, the bacterial concentration in the lungs and kidneys was greater than that in the liver tissues. In the liver, bacterial antigen-containing hepatocytes were mainly observed within necrotic foci or around blood vessels. The Salmonella Enteritidis antigen was also found in the epithelial cells of the alveoli and the tubular epithelial cells of the kidney. The following pathological features were observed in the organs examined: swollen tubular epithelial cells and nephrosis in the kidney; necrotic foci and varying degrees of hepatocyte fat degeneration of hepatocytes in the liver; and slightly hyperemic and hemorrhagic cribriform changes in the brain. Severe hyperemia, hemorrhages, and heterophil infiltration in the lungs were also observed. The overall pattern of antigen distribution and microscopic lesions in the organs examined were similar to those reported in previous studies (Holt and Porter, 1992; Akaki et al., 1997; Deng et al., 2008; Yan et al., 2008). The factors that determine the levels of Salmonella Enteritidis load in various tissues have not been fully understood; however, the high Salmonella Enteritidis DNA loads in multiple systemic organs results in severe tissue pathology, which accelerates the progression of the infection.

The present study provides a detailed description of the patterns of the Salmonella Enteritidis load, the immunohistochemical localization of the Salmonella Enteritidis antigen, and the histopathological examinations in various organs. The Salmonella Enteritidis load in systemic organs was observed to be closely correlated with the progression of the infection. The high bacterial loads and the high levels of replication in the lymphoid and small-intestinal tissues might reflect the presence of abundant target epithelial and lymphoid cells in these tissues. The control group did not generate any positive results, at any time point of the study, at any location. This study provided valuable insights into the etiology and pathogenesis of Salmonella Enteritidis infections in ducks.

Fluorescent quantitative PCR has become a potentially powerful tool in microbiological diagnostics because of its simplicity, rapidity, reproducibility, and accuracy. However, PCR inhibitors or a large amount of DNA from background organisms may result in variable results. In our studies, standard precautions were followed to prevent PCR contamination by adhering to strict laboratory practices (Jothikumar et al., 2005). Furthermore, in preliminary experiments, we used the phenol-chloroform-isoamyl alcohol method to extract DNA from control group tissues (described above) and added 2.0 x 106 copies of the plasmid DNA standards for each control group DNA sample. Finally, fluorometric cycler measurements were performed as described above. The results were consistent with our expectations, and the variability in the results was statistically low at <4.3%. We believe that the methodology used in this study provides an important tool for studying the progression of Salmonella Enteritidis infections.


   ACKNOWLEDGMENTS
 
This work was supported by the National Key Technology R&D Program of China (No. 2004BA901A03); National Scientific and Technical Support Program (No. 2007Z06–017); The Cultivation Fund of the Key Scientific and Technical Innovation Project and Ministry of Education of China (No. 706050); Program for New Century Excellent Talents in University (No. NCET-04–0906/NCET-06–0818); Sichuan Province Basic Research Program (No. 04JY0290061); and Program for Key Disciplines Construction of Sichuan Province (No. SZD0418).


   FOOTNOTES
 
1 The first 4 authors contributed equally to this work. Back

Received for publication April 22, 2008. Accepted for publication May 19, 2008.


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 MATERIALS AND METHODS
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