Available online at www.ewijst.org ISSN: 0975-7112 (Print) ISSN: 0975-7120 (Online) Environ. We Int. J. Sci. Tech. 5 (2010) 65-78
Environment & We An International Journal of Science & Technology
Pathogenicity of Some Avian Salmonella Serovars in Two Different Animal Models: SPFChickens and BALB/c Mice Kamelia M. Osman1, Ihab M. I. Moussa2*, Ashgan M.M. Yousef 3, Mona M. Aly4, Moustafa I. Radwan4 and H. A. Alwathnani5 1 Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Egypt 2 Center of Excellence in Biotechnology Research, King Saud University P. O. Box 2460, Riyadh, King Saudi Arabia 3 College of Applied studies and Community Services, King Saud University, KSA 4 Animal Health Research Institute, Dokki, Egypt 5 Department of Botany and Microbiology, College of Science King Saud University *E-mail:
[email protected];
[email protected] Fax: 00966-14678456; Tel.: 00966560749553
Abstract The pathogenicity of Salmonella Kentucky, Salmonella Typhimurium, Salmonella Shubra and Salmonella Enteritidis were investigated in specific-pathogenfree chicks (SPF) and BALB/c mice. Mortality rates after the 1st week of infection with the four serovars, only S. Typhimurium and S. Enteritidis produced a fulminate infection (88%) in each of the chicks and mice. The lowest mortality was seen in chicks and mice inoculated with S. Kentucky (40% and 44% respectively,). Over 40% mortality was observed in SPF chicks (45.8%) and BALB/c mice (60%) inoculated with Salmonella Shubra. The lethality of S. Typhimurium in mice was 66% while S. Enteritidis was lethal to chicks by 72%. Analysis of the level of fecal shedding of the four serovars from both infected SPF chicks and infected BALB/c mice revealed that, S. Typhimurium was consistent in being the highest in re-isolation rate throughout the 7 days of observation while, S. Enteritidis comes second in re-isolation rate. A variable re-isolation picture was noticed from S. Kentucky and serovar Shubra throughout the experimental period. The highest level of re-isolation was on the 1st day post infection (DPI) recorded as 60%, 40% and 20% from the serovars Enteritidis, Typhimurium, Shubra and Kentucky, respectively. Key words: S. Shubra, S. Enteritidis, S. Typhimurium, S. Kentucky, Mortality
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 Introduction In Egypt, consumption of poultry products has risen during the past two decades. Parallel Salmonella Enteritidis infections in poultry have increased in recent years in Egypt with significant economic impact on the poultry industry and public health. The genus Salmonella consists of more than 2,500 serovars (Bopp et al., 1999) and most human cases of salmonellosis in the United States are caused by 4 serovars. The US Centers for Disease Control and Prevention (CDC) reported that approximately 60% of human cases were caused by S. Enteritidis (24.7%), S. Typhimurium (represented 46.4% of the isolates from nonhuman sources that year), S. Newport (6.2%), and S. Heidelberg (5.1%) (CDC., 2002). Human cases of salmonellosis were found to be caused by some 100 serotypes of which S. Shubra, S. Enteritidis, S. Typhimurium and S. Kentucky were included in that report and which were reported to be widespread in a study carried out by Galanis et al., (2006) . In addition, S. Enteritidis, S. Typhimurium, S. Newport and S. Agona, were among the twenty most frequently isolated Salmonella serovars in the EU from poultry laying flocks (Task Force on Zoonoses Data Collection TFZDC 2007). Many of the existing serovars of Salmonella enterica have unique characteristics regarding their host specificity, epidemiology, and pathogenesis (McClelland et al., 2001, Parkhill et al., 2001). S. 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 (Edwards et al., 2000, Dhillon et al., 2001, Kogut et al., 2003, Takata et al., 2003, Deng et al., 2008). In a susceptible host, S. 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). As indicated previously (Galanis et al. 2006) and because of the increased prevalence of Salmonella and its complex life cycle, it is important to understand the correlation between the progression of the infection and mortality. This has not been described previously with S. Shubra and S. Kentucky in correlation to the most frequently studied S. Enteritidis and S. Typhimurium. Understanding this correlation will help gain further insight into the pathogenesis of Salmonella different serovars infections. In addition, previous work with the chick and murine models has not utilized the serovars S. Shubra and S. Kentucky isolates making direct comparison of results previously obtained in the chick and murine models with the large amount of data obtained in vitro and in chick and murine models over the last 30 years challenging. In our study, we highlight important differences in the virulence between four different serovars of Salmonella (S. Shubra, S. Enteritidis, S. Typhimurium and S. Kentucky) infections on chick and murine models, and provide evidence that suggests that the role of HSP90, SPI1, TCI, rfbS and RRP may not be the same during colonization of both animal models. We believe that this analysis will help provide valuable insights into the etiology of some of the Salmonella serovars infections especially if they were implicated with foodborne illness.
66
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 Materials and Methods Animals Newly hatched 1 day old SPF White Leghorn chicks were wing banded and held in a safety cabinet at constant humidity and temperature and received food and water ad libitum. The chicks originated from Salmonella-free flocks. In addition, six week old BALB/c mice were obtained from Animal Health Research Institute, Dokki, Egypt. Animals were housed in standard polycarbonate microisolator cages in groups of three to four. Mice were fed a 4% standard rodent chow with free access to food and water. The procedures used for animal care and housing were in accordance with the U.S. Department of Agriculture through the Animal Welfare Act (7USC 2131) 1985 and Animal Welfare Standards incorporated in 9 CFR Part 3, 1991. Bacterial strains and growth conditions A total of four Salmonella serovars isolated from the fowls and maintained by the Animal Health Research Institute, Dokki, Egypt, were studied (S. Shubra, S. Enteritidis, S. Typhimurium and S. Kentucky). They were isolated from different governorates in Egypt from infected poultry. Samples collected for Salmonella isolation were taken to the laboratory under ambient conditions on the day of collection, were pre-enriched in 225 ml sterile buffered peptone water (BPW, Merck, Darmstadt, Germany) in a dilution of 1:10, and incubated at 37oC for 18 h. One milliliter of incubated broth was transferred to 9 ml of Muller-Kauffmann Tetrathionate novobiocin broth (MKTTn broth, Oxoid, England) and incubated at 37oC for 18 h. In addition, a second transfer of incubated broth was transferred to 9 ml of Rappaport-Vassiliadis broth (RV broth; Difco, USA), which was prepared according to the instructions on the package. The RV broth was incubated for 24 h at 41.5oC. One milliliter of the MKTTn broth and 0.1 ml of the RV broth samples were streaked onto Xylose Lysine Desoxycholate agar, Brilliant Green Agar and Hektoen Enteric Agar (Oxoid, England) and incubated at 37oC for 24 h. Black or red colonies (nonlactose fermenters) were inoculated onto triple-sugar iron agar by using a sterile inoculating needle and were incubated for 24 h at 37oC. Tubes with red slants and black or yellow butts were identified to be due to salmonellae. Reagents and media All Salmonella, non-Salmonella strains and chicken samples used in this study were tested for Salmonella by the ISO 6579:2002 European International Standard Method. The purity of Salmonella strains was verified, and biochemical and antigenic identifications were performed. Biochemical testing was performed according to Quinn et al., (2002). Further confirmation was done based on agglutination reaction with somatic (O), flagellar (F) and virulence (Vi) antisera. The isolates were serogrouped using specific somatic antisera. Serotyping was performed according to the Kauffmann-White typing scheme (OIE, 2004) using slide agglutination with standard antisera (Difco, USA). One milliliter from each serovar was taken into a sterile Eppendorf tube and was stored at -20°C until used. 67
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 Quantitative real-time RT-PCR screening Methods for bacterial DNA preparation, primer sequences and reaction conditions have been described previously (Osman et al.,2009). All strains were tested in duplicate with appropriate positive and negative controls. In our previous study, we established a serovar specific Quantitative Real-Time PCR Assay for detection of Salmonella DNA of S. Enteritidis, S. Typhimurium, S. Newport, S. Agona, S. Kentucky, S. Saintpaul (Osman et al. ,2009). Real-time PCR reactions were performed in triplicates using iCycler iQ real-time detection system (Bio-Rad, Hercules, CA, USA). The reaction mixture consisted of 1 µl cDNA, 0.5 mM of each primer, iQ SYBR GREEN PERMIX (BIO-RAD) in a total volume of 20 µl. The FastStart polymerase was activated and cDNA denatured by a preincubation for 10 min at 95°C, the template was amplified for 50 cycles of denaturation programmed for 20 s at 95°C, annealing of primers at 62°C programmed for 20 s, and extension at 72°C programmed for 30 s. Fluorescent data were acquired during each extension phase. After 50 cycles, a melting curve was generated by heating the sample to 95 °C followed by cooling down to 55°C for 7 s and slowly heating the samples at 0.1°C/s to 95°C while measuring fluorescence continuously.
Virulence studies The infective doses of the strains S. Shubra, S. Enteritidis, S. Typhimurium and S. Kentucky were estimated by plating the appropriate dilution of the stock suspension in sterile saline on tryptic soy agar (TSA). Colonies were counted after incubation for 1 day at 37°C. The chickens were randomized into seven groups and were infected by crop gavage with 0.5 ml of broth culture containing 1 x 106 colonyforming units (CFU) of the corresponding strain using 20-ga x 1½ in. dosing needles (Dhillon et al.,2001). To calculate the 50% lethal dose (LD50) of each strain, the number of dead chickens was recorded every 24 h. The LD50 was calculated at day 3 post infection by using the Grafit computer program (version 3.0; Erithacus Software Limited). Chicks were observed twice daily for clinical signs of illness and mortality. Dead chicks were removed from the units and necropsied. Two chicks were taken randomly from each treatment group. These animals were euthanized after different time intervals (1-7 days DPI) and samples of the liver, lung and an approximately 3 cm piece of the cecal pouch (wall and content) was recovered aseptically from each bird and homogenized (PowerGen 125 S1, Fischer Scientific, Pittsburgh, PA) in PBS for culture and isolation of salmonellae. Bacteriological analysis of the liver, cecal pouches, lungs and cloacal swabs was performed as outlined. Six week old BALB/c mice were fasted for food and water for six hours before oral infection with 20 µl of bacterial suspension (~109 CFU) prepared as described above. Food and drink were returned 30 minutes after infection (Dieye et al., 2009). Liver, sections of small and large intestine and lung were collected on days 1-7 DPI. Samples were processed as described for chicks.
68
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 78 Statistical Analysis Data were analyzed by SAS general models (SAS Institute, 1984) and SYSTAT (Wilkinson and Coward et al., 1992) procedures. The survival surviv analysis evaluated the effect of each Salmlonella isolate to the survivability impact from each group (Wilkinson et al., 1992). Results Serum samples collected from 11-d-old SPF chicks and six week old BALB/c mice were negative for S. Enteritidis and S. Typhimurium antibodies. Gross pathological lesions were not present at necropsy in 11-d-old old SPF chicks and the six week old BALB/c mice. Liver, lung, and ceca samples collected from 20 SPF chicks at 1 d of age were also negative for Salmonella isolation. These ese results indicated that the chicks and BALB/c mice were healthy and free of Salmonella. The mortality rate during the pathogenicity study Mortality rates after the 1st week among the different inoculated groups after infection by crop gavage into the day old chicks and mice, are presented in Figure 1. Of the four serovars tested for pathogenicity, only S. Enteritidis and S.. Typhimurium produced a fulminant infection (88 (88%) %) in each of the chicks and mice. The lowest mortality was seen in chicks and mice inoculated with S. Kentucky (40% and 44% respectively). Over 40% mortality was observed in SPF chicks (45.8%) and BALB/c mice (60%) inoculated with S.Shubra. The lethality of S.. Typhimurium in mice was 66% while S.. Enteritidis was lethal to chicks by 72%. No mortality occurred in the uninoculated controls. 40 44
Serovars
S. Kentucky
66 S.Typhimurium
88 88
S. Entertidis
72 60
S. Shubra
45.8 0
10
20
30
40
50
60
70
80
90
100
Percentage
mice
Day old chicks
Figure 1 Mortality rate of Salmonella serovars 7 DPI following oral inoculation in Day old SPF chicks and BALB/c mice. Re-isolation of Salmonella from the feces of experimentally infected day old SPF chicks and BALB/c mice. mice We analyzed the level of fecal shedding of serovars S. Shubra, S. S Enteritidis, S. Typhimurium and S.. Kentucky from both infected SPF chicks and infected BALB/c 69
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 78 mice. Re-isolation isolation of the different inoculated Salmonella serovars in the day day-old chicks were checked using cloacal swabs at 1, 2, 3 and 7 DPI (Fig (Figure 2). S. Typhimurium rium was consistent in being the highest in re re-isolation isolation rate throughout the 7 st th days of observation (100% on the 1 and 7 DPI, 60% on the 2nd and 80% on the 3rd DPI). S. Enteritidis comes second in re re-isolation rate (60% on the 1st, 2nd and 3rd DPI th to reach 80% on the 7 DPI). A variable re-isolation isolation picture was noticed from S. Kentucky the re-isolation isolation rate on the 1st, 2nd, 3rd and 7th DPI recorded a rate of 60%, 20%, to rise to 60% again but to drop to 20% again, respectively. Throughout the experimental tal period serovar Shubra was consistent at a rate 60% re re-isolation isolation rate fro day 1 post infection to day 3 post infection to drop to 40% on the 7th DPI. 100 90 80 70 60 50 40 30 20 10 0
S. Shubra
S. Entertains
S. Typhimurium
S. Kentucky
Figure 2 Re-isolation isolation rate of Salmonella serovars from cecal pouches of experimentally infected Day Day-old old SPF chicks (DPI=Days Post Infection ). Re-isolation isolation of the different inoculated Salmonella serovars in the BALB/c mice (Figure 3) indicated a decrease in the re-isolation isolation ability to reach 0% on o the 7th DPI. The highest level of re re-isolation was on the 1st DPI recorded as 60%, 40%, 40% 20% and 20% for from the serovars Entertidis, Typhimurium, Shubra and Kentucky, respectively. We show that the overall level of fecal shedding of serotype Typhimuriu Typhimurium is greater from infected chicks than from infected mice. mice Re-isolation of Salmonella from the internal organs of experimentally infected day old SPF chicks and BALB/c mice We determined the level of re re-isolation isolation of serovar Typhimurium which reached 100% from the liver, cecal pouch and lung of infected chicks (Figure 4) and 70
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 78 mice (Figure 5) over 7 days of infection. In both chicks and mice S.. Enteritidis was reisolated by 100% from the liver. In the SPF chicks, S. Shubra was re-isolated isolated from the cecal pouch (66.7%) only (Figure (Fig 3), while serovar Shubra infected BALB/c mice were re-isolated isolated from the 3 organs, by 100% from the lung and intestine and 75% from the liver (Figure 5). In contrast in this case, we noted that there was a greater overall level oof reisolation of the different serovars from infected mice than from infected chicks. 60 50 40 30 20 10 0
S. Shubra
S. Entertidis
S. Typhimurium
S. Kentucky
Figure 3 Re-isolation isolation rate of Salmonella serovars from intestine of experimentally infected BALB/c mice (DPI=Days Post Infection ).
80
Serovars
S. Kentucky
40
0
100 100 100
S.Typhimurium
50 50
S. Entertidis
100
0 S. Shubra
66.7
0 0
20
40
60
80
100
Percentage
Lung
Cecal pouch
Liver
Figure 4. Recovery rate of Salmonella serovars 7 DPI from the liver, cecal pouch and lung following oral inoculation in Day old SPF chicks. 71
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 78
30
Serovars
S. Kentucky
60
30
100 100 100
S.Typhimurium
30 60
S. Entertidis
100 75 100 100
S. Shubra
0
20
40
60
80
100
Percentage
Lung
intestine
Liver
Figure 5 Recovery rate of Salmonella serovars 7 DPI from the liver, intestine and lung following oral inoculation in BALB/c mice. Discussion Salmonellae are Gram Gram-negative negative bacteria that cause a variety of disease syndromes in humans and animals. Salmonella is a facultative, intracellular pathogen capable of infecting a variety of hosts, resulting in several manifestations of the disease (Goldberg et al., 1988) Salmonella enterica serotype Typhimurium is a nonnon pathogenic commensal organism in chicks greater than 3 days of age and can colonize the intestinal tract subclinically for 8–9 8 9 weeks in >90% of birds after experimental infection (Beal et al., 2004). This subclinical carriage has consequences for human health, as broilers are currently brought to slaughter slaughter at about 40 days of age, well within this period. Typhimurium remains an important concern to the poultry industry (Babu and Raybournem al., 2008) causing a systemic infection in newly hatched chicks, often resulting in death (Barrow et al.,1987). In older lder birds infection by Typhimurium leads to an asymptomatic carriage state with colonization of the digestive tract and continuous shedding (Barrow et al.,1988, Withanage et al.,2005). al., In this study four different Salmonella serovars were selected to study stud their pathogenicity in SPF White Leghorn chicks and mice. Mortality was present with the four Salmonella serovars in SPF chicks. Several investigators investigated the pathology of different phage types of S. Enteritidis (Dhillon et al., 2001, Alisantosa et al.,2000, Roy et al., 2001). There is also variation among the various phage types of SE in their virulence for chickens and PT4 was considered to be the most pathogenic (Alisantosa et al.,2000, 2000, Gast et al.,1995). 1995). it was also reported that significant differences exist in virulence between S. Enteritidis PT4 isolates (Gast et al.,1995). However, there is also evidence that variation in virulence exists among various isolates of S.. Enteritidis of the same PT (Barrow et al.,1991, 1991, Gast et al.,1995). Chickens of different ages vary in their susceptibility to S. Enteritidis,, young chickens being more susceptible than adults. Limited numbers of reports have been published on the pathogenicity of different PTs of S. Enteritidis in both broiler and layer chicks 72
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 lesions and mortality in both experimental and natural infection of S. Enteritidis (Alisantosa et al.,2000, Barrow et al.,1991, Gast et al.,1995, O’ Brien, 1988, Turnbull et al.,1974). Most of these reports indicate that the PT4 is more pathogenic than other PTs such as PT6, PT7, PT8, PT13, and PT13a. Limited studies of the pathogenicity of S. Enteritidis in layer type chicks indicate that 1-day-old chicks are more susceptible than 7-day-old or 4-week-old chicks when challenged with S. Enteritidis PT13a or PT4 (Gorham et al.,1994, Desmidt et al., 1997). The difference in mortality might be explained by the type of animal model, the dose of inoculum or the phase of inoculated bacterial growth. In view of the earlier reports ( Barrow et al.,1991,Gast et al.,1995, Shivaprasad et al.,1990), we were not surprised to find there was a variation in the virulence between the isolates of different S. Enteritidis. The reason for such variation within and between serovars could be hypothetically explained and correlated on the basis that, HSP90, SPI1, SPI2, TCI, rfbS and RRP differed in their expression levels between the four Salmonella serovars (Osman et al.,1999). Colonization of salmonellae in different organs was supported in this study by isolating salmonellae from the liver, lung and cecal pouches at 7 DPI which indicated that the chicks were subclinically infected, although they were clinically normal. The cecal pouch was identified as an ideal organ for Salmonella isolation during acute and chronic infection, compared with other organs, and it poses a potential source for environmental contamination (Dhillon et al.,2001). In the intestinal tract itself, the cecal contents are the major site for recovery of serotype Typhimurium in the cecum of 1-week-old chicks and Salmonella-resistant mice (Sivula et al.,2008). Many studies of Salmonella pathogenesis have been conducted by comparing a specific phenotype measured in an in vitro assay of a mutant and its parental strain. This approach has generated much useful information about the possible functions of individual genes in pathogenesis. Lu et al., (1999) suggested that there are as-yetuncharacterized features of clinical isolates of S. Enteritidis that determine their lethality in mice. Lu et al., (1999) indicated that clinical isolates of S. Enteritidis are highly heterogeneous in their ability to cause death in mice and that, there was no apparent correlation between virulence in mice and phage type. In addition, it has been demonstrated previously that virulence of mice varied with the strain of mouse used (Chart and Rowe, 1991, Cox and Woolcock, 1994, Sad et al., 2008). We also studied the congruence between DPI and reisolation rate of the four Salmonella serovars in both chicks and mice. The bacterial reisolation in the cecal pouches of chicks were consistently of the same orders of magnitude except during the 2nd and 7th DPI where there was a drop in the rate with S. Shubra and S. Kentucky. In addition, the bacterial reisolation of the four serovars in the intestine of mice reflected complete inability for reisolation by the 7th DPI. Salmonellae invade and destroy specialized epithelial cells in the host intestine, and migrate to the mesenteric lymph nodes, where they encounter, subsequently survive and replicate within macrophages. Many virulence phenotypes of Salmonella enterica are encoded by genes located on pathogenicity islands. Specific virulence factors encoded within the Salmonella pathogenicity island (SPI) are required at various stages of Salmonella infection (Groisman et al.,1999) 73
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 Salmonella enterica serovar Typhimurium, which causes gastroenteritis in humans and a systemic disease in mice similar to human typhoid fever, harbours two kinds of type III protein export systems: one for flagellar proteins and one for virulence factors. Several genes from the two systems share sequences in common (Aizawa, 2001, Macnab, 2004). Some important virulence factors are directly delivered into the host environment by two different type III secretion systems (TTSSs) encoded on SPI1 and SPI-2. The SPI-1 TTSS mediates bacterial entry into non-phagocytic cells (Galan, 2001) and the SPI-2 TTSS is required for survival and replication in the intracellular environment of host cells and for systemic infection in mice ( Hensel et al.,1998, Ochman et al.,1996, Shea et al., 1996). Dieye et al., (2009) found that Typhimurium SPI1 contributes to colonization of the cecum in the chicken and important for successful infection in the murine model (Sivula et al., 2008).The infection by Typhimurium in these two animal models leads to different outcomes. In mice, Typhimurium causes an acute systemic infection, frequently resulting in death, while in one-week or older chickens, the infection leads to heavy colonization of the intestinal track and asymptomatic carriage. It is interesting to note that in animal models where Salmonella infection results in acute systemic disease, SPI2 is a major player in the systemic infection. These include the infection of mice by Typhimurium (Haraga et al., 2008) and the systemic disease in chickens infected by serovars Pullorum (Wigley et al., 2002) and Gallinarum (Jones et al.,2001). In contrast, in animals where infection results in healthy carriage, such as in chickens, SPI2 plays a minor role in the persistence of the bacteria in the systemic compartment. This is demonstrated for Typhimurium in pigs (Boyen et al.,2008) and for serovar Enteritidis in chicken (Bohez et al., 2006). Dam methylation is an essential factor involved in the virulence of an increasing number of bacterial pathogens including Salmonella enterica. Lack of Dam methylation causes severe attenuation in animal models (mice, chickens, cattle) (García-del-Portillo et al., 1999, Dueger et al., 2003). S. enterica employ a type III secretion system (TTSS) to translocate virulence determinants, called effector proteins, from the bacterial cytoplasm into the host cell cytoplasm thus increasing their pathogenic potential and the outcome of infection. TTSS involves the coordinated expression of approximately 20 proteins and is therefore regulated by complex mechanisms. Effects of Dam (DNA adenine methylase) on TTSS have been observed in S. Typhimurium, although the basic regulatory mechanisms have not been elucidated. Several authors observed that both bacteria-associated and secreted proteins are affected by the loss of Dam regulation (García-del-Portillo et al., 1999, Giacomodonato et al.,2009b, Heithoff et al.,2001).A report by Balbontín et al., (2006) provided evidence that Dam methylation regulates the invasion genes of the pathogenicity island 1 (SPI-1). The need for Dam methylation to activate the expression of invasion genes could explain the reduced secretion of SPI-1 effectors such as SipA, SipB and SipC reported earlier (García-del-Portillo et al., 1999) and the impaired invasion of epithelial cells (García-del-Portillo et al., 1999, Giacomodonato et al., 2009b). Recently, Giacomodonato et al., (2009a) demonstrated that Dam methylation regulates synthesis and secretion of the Salmonella virulence effector SopA (Giacomodonato et al., 2009b). This finding extends to an effector located outside SPI-1 (and secreted via the SPI-1 TTSS) described in previous reports, indicating that Dam methylation regulates Salmonella SPI-1.
74
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 Authors' contributions: Dr. Kamelia M. Osman (Professor), Project leader, contributed in experiment design and final editing of the manuscript; Dr. Ihab M. I. Moussa (Associate Professor), coinvestigator for the project, contributed in experiment design, preparation, wrote and corresponding author of manuscript; Dr. Ashgan M.M. Yousef (Assistant Professor) ) performed some of the experiment ;Dr. Mona M. Aly (Researcher), Project leader, contributed in experiment design; Moustafa I. Radwan (Research Assistant) ) performed some of the experiment; H. A. Alwathnani, (Assistant Professor) ) co-investigator for the project, contributed in experiment design.
References Aizawa, S.I., 2001. Bacterial flagella and type III secretion systems. FEMS Microbiology Letters 202,157–164. Alisantosa, B., Shivaprasad, H.L., Dhillon, A.S., Jack, O., Schaberg, D., Bandli, D., 2000. Pathogenicity of Salmonella Enteritidis phage types 4, 8 and 23 in specific pathogen free chicks. Avian Pathology 29,583– 592 Babu, U.S., Raybournem, R.B., 2008. Impact of dietary components on chicken immune system and Salmonella infection. Expert Review of Anti-infective Therapy 6,121–135. Balbontin, R., Rowley, G., Pucciarelli, M.G., Lopez-Garrido, J., Wormstone, Y., Lucchini, S., Garcia-Del Portillo, F., Hinton, J.C., Casadesús, J.,2006. DNA adenine methylation regulates virulence gene expression in Salmonella enterica serovar Typhimurium. Journal of Bacteriology 188, 8160-8168. Barrow, P.A. 1991. Experimental infection of chickens with Salmlonella Enteritidis. Avian Pathology 20,145–153. Barrow, P.A., Huggins, M.B., Lovell, M.A., Simpson, J.M., 1987. Observations on the pathogenesis of experimental Salmonella Typhimurium infection in chickens. Research Veterinary Science 42,194–199. Barrow, P.A., Simpson, J., Lovell, M., 1988. Intestinal colonization in the chicken by food-poisoning Salmonella serotypes; Microbial characteristics associated with faecal excretion. Avian Pathology 17,571–588. Beal, R.K., Wigley, P., Powers, C., Hulme, S.D., Barrow, P.A., Smith, A.L., 2004. Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to rechallenge. Veterinary Immunology and Immunopathology 100,151–164. Bohez, L., Ducatelle, R., Pasmans, F., Botteldoorn, N., Haesebrouck, F., Van Immerseel, F., 2006. Salmonella enterica serovar Enteritidis colonization of the chicken caecum requires the HilA regulatory protein. Veterinary Microbiology 116,202–210. Bopp, C.A., Brenner, F.W., Wells, J.G., 1999. Escherichia, Shigella, and Salmonella. In: Murray, P., Baron, E.J., Pfaller, M.A., Tenover, F., Yolken, R. Manual of clinical microbiology 1, 459–474. Boyen, F., Pasmans, F., Van Immerseel, F., Morgan, E., Botteldoorn, N., Heyndrickx, M., Volf, J., Favoreel, H., Hernalsteens, J.P., Ducatelle, R., Haesebrouck, F.,2008. A limited role for SsrA/B in persistent Salmonella Typhimurium infections in pigs. Veterinary Microbiology 128,364–373. CDC., 2002. Centers for Disease Control and Prevention. Salmonella surveillance: annual tabulation summaries. Available at: http://www.cdc.gov/ncidod/dbmd/phlisdata/Salmonella.htm. Accessed May 21, 2002. Chart, H., Rowe, B., 1991. Antibodies to lipopoljsaccharide and outer membrane proteins of Salmonella Enteritidis PT4 are not involved in protection from experimental infection. FEMS Microbiology Letters 84,345- 350. 75
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 Cox, J.M., Woolcock, J.B., 1994. Lipopolysaccharide expression and virulence in mice of Australian isolates of Salmonella Enteritidis. FEMS Microbiology Letters 19, 95-98. Deng, S.X., Cheng, A.C., Wang, M.S., Cao P., 2008 Serovar-specific real-time quantitative detection of Salmonella Enteritidis in the gastrointestinal tract of ducks after oral challenge. Avian Diseases 52, 88–93. Desmidt, M., Ducatelle, R., Haesebrouck, F., 1997. Pathogenesis of Salmonella Enteritidis phage type 4 after experimental infection of young chickens. Veterinary Microbiology 56,99–109. Dhillon, A.S., Shivaprasad, H.L., Roy, T.P., Alisantosa, B., Schaberq, D., Bandli, D., Johnson, S., 2001. Pathogenicity of environmental origin salmonellas in specific-pathogen-free chicks. Poultry Science 80, 1323–1328. Dibb-Fuller, M.P., Allen-Vercoe, E., Thorns, C.J., Woodward, M.J., 1999. Fimbriaeand flagella-mediated association with and invasion of cultured epithelial cells by Salmonella Enteritidis. Microbiology 145, 1023–1031. Dieye, Y., Ameiss, K., Mellata, M., Curtiss, R., 2009. The Salmonella Pathogenicity Island (SPI) 1 contributes more than SPI2 to the colonization of the chicken by Salmonella enterica serovar Typhimurium. BMC Microbiology Published online 10.1186/1471-2180-9-3. Dueger, E.L., House, J.K., Heithoff, D.M., Mahan, M.J., 2003. Salmonella DNA adenine methylase mutants elicit early onset protective immune response in calves. Vaccine 21, 3249-3258. Edwards, R.A., Schifferli, D.M., Maloy, S.R., 2000. A role for Salmonella fimbriae in intraperitoneal infection. Proceeding of Natural Academic Science USA 97, 1258–1262. Galan, J.E., 2001. Salmonella interactions with host cells: type III secretion at work. Annual review of cell and developmental biology 17, 53–86. Galanis, E., Lo Fo Wong, D.M.A, Patrick, M.E., Binsztein, N., Cieslik, A., Chalermchaikit, T., Aidara-Kane, A., Ellis, A., Angulo, F.J., Wegener, H.C., 2006. Web-based Surveillance and Global Salmonella Distribution, 2000– 2002. Emerging Infectious Disease 12,381-388. García-del-Portillo, F., Pucciarelli, M.G., Casadesús, J., 1999. DNA adenine methylase mutants of Salmonella Typhimurium are deficient in protein secretion, cell invasion and M cell cytotoxicity. Proceeding of Natural Academic Science USA 96, 11584–11588. Gast, R.K., Benson, S.T., 1995. The comparative virulence for chicks of Salmonella Enteritidis phage type 4 isolates and isolates of phage types commonly found in poultry in the United States. Avian Diseases 39,567–574. Giacomodonato, M.N., Sarnacki, S.H., Llana, M.N., Cerquetti, M.C., 2009a. Dam and its role in pathogenicity of Salmonella enterica. Journal Infect Dev Ctries 3,484-490. Review Article Giacomodonato, M.N., Sarnacki, S.H., Llana, M.N., García Cattaneo, A.S., Uzzau, S., Rubino, S., Cerquetti, M.C., 2009b. Impaired synthesis and secretion of SopA in Salmonella Typhimurium dam mutants. FEMS Microbiology Letters 292, 71-77. Goldberg, M.B., Rubin, R.H., 1988 The spectrum of Salmonella infection. Infectious disease clinic. North America 2, 57–98. Gorham, S.L., Kadavil, K., Vaughn, E., Lambert, H., Abel, J., Pert, B.,1994. Gross and microscopic lesions in young chickens experimentally infected with Salmonella Enteritidis. Avian Diseases 38, 816–821. 76
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 Groisman, E.A., Blanc-Portard, A.B., Uchiya, K., 1999. Pathogenicity islands and the evolution of Salmonella virulence. In Pathogenicity Islands and Other Mobile Virulence Elements, pp. 127–150. Haraga, A., Ohlson, M.B., Miller, S.I.,2008. Salmonellae interplay with host cells. Nature Review of Microbiology 6,53–66. Heithoff, D.M., Enioutina, E.I., Daynes, R.A., Sinsheimer, R.L., Low, D.A., Mahan, M.J., 2001. Salmonella DNA adenine methylase mutants confer crossprotective immunity. Infectious and Immunity 69,6725–6730. Hensel, M., Shea, J.E., Waterman, S.R., Mundy, R., Nikolaus, T., Banks, G., Vazquez-Torres, A., Gleeson, C., Fang, F.C., Holden, D.W., 1998. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages. Molecular Microbiology 30,163–174. Jones, M.A., Wigley, P., Page, K.L., Hulme, S.D., Barrow, P.A., 2001. Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens. Infectious and Immunity 69,5471– 5476. Kogut, M.H., Rothwell, L., Kaiser, P., 2003. Differential regulation of cytokine gene expression by avian heterophils during receptor-mediated phagocytosis of opsonized and nonopsonized Salmonella Enteritidis. Journal of Interferon Cytokines Research 23,319–327. Lu, S., Manges, A., Xu, Y., Fang, F.C., Riley, L.W., 1999. Analysis of Virulence of Clinical Isolates of Salmonella Enteritidis In Vivo and In Vitro. Infectious and Immunity 67, 5651–5657 Macnab, R.M,.2004. Type III flagellar protein export and flagellar assembly. International Journal of Biochemistry, Biophysics and Molecular Biology 1694, 207–217. McClelland, M., Sanderson, K.E., Spieth, J., Clifton, S.W., Latreille, P., Courtney, L., Porwollik, S., Ali, J., Dante, M., Du, F., Hou, S., Layman, D., Leonard, S., Nguyen, C., Scott, K., Holmes, A., Grewal, N., Mulvaney, E., Ryan, E., Sun, H., Florea, L., Miller, W., Stoneking, T., Nhan, M., Waterston, R., Wilson, R.K., 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413, 852-856. O’ Brien, J.D.P.,1988. Salmonella Enteritidis infection in broiler chickens. Veterinary Record 122- 214. Ochman, H., Soncini, F.C., Solomon, F., Groisman, E.A.,1996. Identification of a pathogenicity island required for Salmonella survival in host cells. Proceeding of the Natural Academic Science USA 93,7800–7804. OIE World Organization for Animal Health. )OIE manual of diagnostic tests and vaccines for terrestrial animals Available at: http://www.oie.int/eng/normes/mmanual/A_summry.htm. Osman, K.M., Ali, M.M., Radwan, M.I., Kim, H.K., Han, J. 2009. Comparative Proteomic analysis on Salmonella Gallinarum and Salmonella Enteritidis exploring proteins that may incorporate host adaptation in poultry. Journal of Proteomics 7, 2815–2821. Parkhill, J., Dougan, G., James, K.D., Thomson, N.R., Pickard, D., Wain, J., Churcher, C., Mungall, K.L., Bentley, S.D., Holden, M.T., Sebaihia, M., Baker, S., Basham, D., Brooks, K., Chillingworth, T., Connerton, P., Cronin, A., Davis, P., Davies, R.M., Dowd, L., White, N., Farrar, J., Feltwell, T., 77
Osman et al. / Environ. We Int. J. Sci. Tech. 5 (2010) 65-78 78 Hamlin N., Haque A., Hien T.T., Holroyd, S., Jagels, K., Krogh, A., Larsen, T.S., Leather, S., Moule, S., O'Gaora, P., Parry, C., Quail, M., Rutherford, K., Simmonds, M., Skelton, J., Stevens, K., Whitehead, S., Barrell, B B.G., 2001. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 413:848-852. Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J., 2002. Leonard FC. Veterinary Microbiology and Microbial Diseases. Black we well scientific publications, Oxford, London. Roy, P., Dhillon, A.S., Shivaprasad, H.L., Schaberg, D.M., Bandi, D., Johnson,S., 2001. Pathogenicity of different serogroups of avian salmonellae in specific specificpathogen-free free chickens. Avian Diseases 45,922-937. Sad, S., Dudani, R., Gurnani, K., Russell, M., van Faassen, H., Finlay, B., Krishnan, L., 2008. Pathogen proliferation governs the magnitude but compromises the function of CD8 T cells. Journal of Immunology 180,5853–5861. 5861. Shea, J.E., Hensel, M., Gleeson, C., Holden, D.W.,1996 Identification of a virulence locus encoding a second type III secretion system in Salmonella Typhimurium. Proceeding of the Natural Academic Science USA 93,2593 93,2593– 2597. Shivaprasad, H.L., Timoney, J.F., Morales, S., Lucio, B., Baker, R.C.,1990. Pathogenesis of Salmonella Enteritidis infection in laying chickens, I. Studies on egg transmission, clinical signs, fecal shedding, and serological responses. Avian Diseases 34,548 34,548– 557. Sivula, C.P., Bogomolnaya, L.M., Andrews Andrews-Polymenis, H.L., 2008. A comparison of cecal colonization of Salmonella enterica serotype Typhimurium in white leghorn chicks and Salmonella-resistant mice. BMC Microbiology 2008;8:182. Takata, T., Liang, J., Nakano, H., Yoshimura, Y., 2003. Invasion of Salmonella Enteritidis in the tissues of reproductive organs in laying Japanese quail: An immunocytochemical study. Poultry Science 82:1170–1173. TFZDC. Report of the Task Force on Zoonoses Data Collection on the Analysis of the baseline study on the prevalence of Salmonella in holdings of laying hen flocks of Gallus gallus gallus. EFSAJ 2007 97. http://www.efsa.europa.eu Turnbull, P.C., Snoeyenbos, G.H.,1974. Experimental salmonellosis in the chicken. 1. Fate and host response in ali alimentary canal, liver, and spleen. Avian Diseases 18,153–177. Vu emilo, M., Vlahovi, . K., Dov, . A., Mu ini J, Pavlak, M., Jer i J, upan i . Prevalence of Campylobacter jejuni, Salmonella Typhimurium, and avian Paramyxovirus type 1 (PMV-1) 1) in pigeons from different regions in Croatia.. Zeitschrift für Jagdwissenschaft 49,303-313. Wigley, P., Jones, M.A., Barrow, P.A., 2002. Salmonella enterica serovar Pullorum requires the Salmonella pathogenicity island 2 type III secretion system for virulence and carriage in the chicken chicken. Avian Pathology 31,501–506. 506. Wilkinson, L., Coward, M., 1992. Analysis of variance. Pages 119 119–225 225 in: Systat 7.0 225 Statistics. Withanage, G.S., Wigley, P., Kaiser, P., Mastroeni, P., Brooks, H., Powers, C., Beal, R., Barrow, P., Maskell, D., McConnell, I., 2005. Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge. Infectious and IImmunity 73, 5173–5182.
78
