About all

Strain of salmonella: The Big Five: Most Common Salmonella Strains in Foodborne Illness Outbreaks

Содержание

The Big Five: Most Common Salmonella Strains in Foodborne Illness Outbreaks

This article was originally published on August 2 by The Midwest Center for Investigative Reporting as part of a series titled “Cracks in the System.” An estimated 1.2 million Salmonella-related illnesses occur each year in the United States. Approximately 400 people die. While Salmonella is most often associated with poultry products, outbreaks are linked to a wide variety of sources, including contaminated ground beef, fruits and vegetables, dog food, turtles and hedgehogs. Scientists first identified Salmonella as a human pathogen in the late 19th Century. While monitoring and tracking methods have improved, the bacteria continue to cause significant issues and foodborne disease outbreaks. There are more than 2,000 strains of Salmonella. The different strains of Salmonella are categorized based on the specific antigen set of each. Antigens are substances that stimulate the body to fight pathogens. These antigen-based subsets are called serotypes. Here is a list of the five most common serotypes in order of prevalence in relation to foodborne illnesses. Salmonella Enteritidis Enteritidis is the most common strain of Salmonella in our food supply. The increased prevalence in poultry products made Salmonella Enteritidis a food-safety issue in the 1970s. It is the serotype most often associated with poultry. Prior to that, Salmonella Pullorum and Salmonella Gallinarum were endemic in poultry flocks, according to research by Steven Ricke at the University of Arkansas. However, these strains were minimized through aggressive eradication programs. Salmonella Enteritidis infects the gastrointestinal tract of poultry. Salmonella is passed from bird to bird in several ways, most commonly through fecal matter. Poultry have a tendency to scratch through dirt and manure, and hen they do this, they can become infected if the manure has live Salmonella bacteria in it. When poultry are slaughtered, Salmonella is spread from the intestinal tract onto the meat. Salmonella is also found in the ovaries of laying hens and thus passed on through shell eggs. There are several testing programs in place to detect and reduce the threat of this strain in the poultry meat and shell egg supply. Based on U.S. Centers for Disease Control and Prevention data, Enteritidis outbreaks since 2010 were linked to shell eggs, alfalfa sprouts, pine nuts and ground beef. Salmonella Typhimurium Typhimurium is the second most common serotype associated with foodborne illness and the third most frequently identified with chicken. This serotype is also linked to ground beef, pork and other poultry products. Beef researchers say that Salmonella Typhimurium in ground beef could be the biggest food safety issue facing the beef industry today. Typhimurium has proven to be antibiotic-resistant, which makes eliminating the pathogen from food products very challenging. Beef researchers are looking into pre-harvest interventions such as vaccinations and probiotics to reduce Typhimurium in cattle. Unlike other serotypes that populated the intestinal tract of animals, Typhimurium might be in the lymph system of cattle. Research is ongoing. The CDC list of outbreaks associated with Typhimurium since 2006 show the following as sources: Ground beef, hedge hogs, cantaloupes, peanut butter, tomatoes and African dwarf and water frogs. Salmonella Newport Newport is currently the third most common Salmonella serotype associated with foodborne illness. This strain is most often associated with turkey products. Like Typhimurium, it has been determined to be antibiotic-resistant. In the fall of 2012, Salmonella Newport and Typhimurium were found in cantaloupe. The outbreak led to three deaths and more than 250 illnesses in 24 states. In addition to cantaloupe, live poultry and alfalfa sprouts have been linked to Newport outbreaks since 2010. Salmonella Javiana Javiana is the fourth most common serotype associated with foodborne illness. A report on Salmonella serotypes from the Food Safety and Inspection Service (FSIS), the food safety arm of the U.S. Department of Agriculture, indicated this strain is not often associated with products regulated by the agency. This serotype is associated with exposure to amphibians in the Southeast U.S. It has also been linked to contaminated mozzarella cheese, watermelon, bass, poultry, lettuce and tomatoes. CDC has not reported a multistate outbreak associated with Javiana since 2006. However, in January 2011 there was a death at a retirement home in Maine attributed to Javiana in a food product. Salmonella Heidelberg Heidelberg is the fifth most common Salmonella serotype associated with foodborne illness and the second most frequently associated with human health issues and poultry, according to a recent report from FSIS. Salmonella Heidelberg has caused recent poultry recalls and foodborne illness outbreaks. In March of this year, 128 illnesses in 13 states were linked to Heidelberg in chicken meat. It is also found in shell eggs. However, current FDA guidelines are designed to limited Salmonella Enteritidis and do not specifically address Heidelberg. “Heidelberg [in eggs] is a new threat for the CDC and FDA to deal with,” said Paul Patterson, professor of poultry science at Pennsylvania State University. “Testing isn’t specifically designed for this strain, but if a farm is testing and has knowledge it is present, they are obligated to act.” John Sheehan, director of FDA’s Division of Plant and Dairy Food Safety, said that Heidelberg is not a new issue for the agency. He noted it was mentioned in 2004 as a major challenge. While the new egg safety rule that went into effect in 2010 primarily addresses Enteritidis issues, he said inspectors are trained to look for Heidelberg as well. “The egg safety rule is all about Salmonella Enteritidis, and our goal is to eliminate Salmonella Enteritidis as a source of foodborne illness,” Sheehan said. “But if we learn that Heidelberg is present, we cannot ignore it. There is transference potential, and it can’t be ignored in an egg-production environment.” Sheehan noted that FDA sent a warning letter to an egg producer whose facilities tested positive for Heidelberg in the fall of 2012. The letter went to Centrum Valley Farms in Iowa. Centrum just happens to be the new owner of Wright Country Egg and Hillandale Farms, the sources of more than 500 million Enteritidis-contaminated eggs involved in the largest egg recall in U.S. history. FDA officials noted that two of Centrum Valley’s hen houses tested positive for Heidelberg during an inspection. Eggs were tested and came back negative for Heidelberg. No eggs were distributed until the negative results were received and the farm received the go-ahead from FDA. The Midwest Center for Investigative Reporting is an independent, nonprofit newsroom devoted to coverage of agribusiness and related topics such as government programs, environment and energy. Visit them at www.investigatemidwest.org.

The Five Most Common Salmonella Strains

The Five Most Common Salmonella Strains

Salmonella infections (similar to e. coli infections) are caused by a group of infectious bacteria that is responsible for at least 1.2 million illnesses and about 400 deaths annually in the United States. More often than not, people associated the infectious salmonella bacteria with poultry products, but the truth is that people can be infected by contact with the bacteria through beef products, fruits and vegetables, and even contact with pets, namely turtles and hedgehogs.

 

More often than not, people associated the infectious salmonella bacteria with poultry products, but the truth is that people can be infected by contact with the bacteria through beef products, fruits and vegetables, and even contact with pets, namely turtles and hedgehogs

There are thousands of unique strains of salmonella, though there are some that are more prevalent and cause more sicknesses than others each year. These are:

  • Salmonella Enteritidis. This is the most common strain, and is the strain most often associated with poultry. It manifests in the digestive tract and ovaries of chickens and laying hens, so when eggs are laid or the chickens are slaughtered or the chicken defecate, the infectious bacteria can be transferred to the meat and eggs through the fecal matter of the chickens.
  • Salmonella Typhimurium. This is the second most common strain associated with food poisoning, and is related to chicken and poultry, ground beef, and pork – but like all salmonella, can contaminate most any food product. This strain is showing recent properties that are disturbing, being particularly dangerous and hard to eliminate because it a number of its variants are resistant to antibiotics. Recent reported sources of infections from this strain include “hedge hogs, cantaloupes, peanut butter, tomatoes, and frogs”, says the Center for Disease Control and Prevention (CDC).
  • Salmonella Newport. This strain is third most common in cases of food-related illness. This strain is often resistant, like typhimurium, to one or more types of antibiotic.  It has been identified in recent outbreak associated with turkey and poultry products, cantaloupe, and alfalfa.
  • Salmonella Javiana. This strain is the fourth most often salmonella serotype associated with sickness from food, and the sources of infection often include products not regulated by the United States Department of Agriculture (USDA). Reported cases have included contact with amphibians in the Southeast United States, as well as mozzarella cheese, watermelon, bass, poultry, lettuce, and tomatoes – each of which (unlike most meats) fall under the authority of the Food and Drug Administration.
  • Salmonella Heidelberg. This strain is often antibiotic resistant as well, and has most often been associated with poultry and egg products, as well as cattle and livestock.  In recent years, many consumers  have been sickened when fed prepared chicken that was not heated to an internal temperature of at least 165 degrees.

Salmonella infections present symptoms that include “diarrhea, fever, and abdominal cramps six hours to four days after infection,” says the CDC. The sickness often passes (in healthy individuals) within a week without medical attention, but in other cases symptoms last longer and even worsen in severity.  In these cases, serious medical attention should be sought, as there can be more serious complications like post-infectious IBS or Post-infectious arthritis.

The best ways to avoid salmonella infections are to practice good hand-washing before and after consuming or preparing foods (especially raw meats), avoid contact with fecal matter or preparing food where there can be contact with fecal matter, and stay up to date with food safety and outbreak news.

Salmonella Enteritidis Strains from Poultry Exhibit Differential Responses to Acid Stress, Oxidative Stress, and Survival in the Egg Albumen

Abstract

Salmonella Enteritidis is the major foodborne pathogen that is primarily transmitted by contaminated chicken meat and eggs. We recently demonstrated that Salmonella Enteritidis strains from poultry differ in their ability to invade human intestinal cells and cause disease in orally challenged mice. Here we hypothesized that the differential virulence of Salmonella Enteritidis strains is due to the differential fitness in the adverse environments that may be encountered during infection in the host. The responses of a panel of six Salmonella Enteritidis strains to acid stress, oxidative stress, survival in egg albumen, and the ability to cause infection in chickens were analyzed. This analysis allowed classification of strains into two categories, stress-sensitive and stress-resistant, with the former showing significantly (p<0.05) reduced survival in acidic (gastric phase of infection) and oxidative (intestinal and systemic phase of infection) stress. Stress-sensitive strains also showed impaired intestinal colonization and systemic dissemination in orally inoculated chickens and failed to survive/grow in egg albumen. Comparative genomic hybridization microarray analysis revealed no differences at the discriminatory level of the whole gene content between stress-sensitive and stress-resistant strains. However, sequencing of rpoS, a stress-regulatory gene, revealed that one of the three stress-sensitive strains carried an insertion mutation in the rpoS resulting in truncation of σS. Finding that one of the stress-sensitive strains carried an easily identifiable small polymorphism within a stress-response gene suggests that the other strains may also have small polymorphisms elsewhere in the genome, which likely impact regulation of stress or virulence associated genes in some manner.

Introduction

Salmonella enterica
serovar Enteritidis is the most common serotype of Salmonella isolated from cases of foodborne gastroenteritis throughout the world (EFSA, 2007; WHO, 2008). Chickens are the single largest reservoir host for Salmonella Enteritidis, and source attribution studies have determined that contaminated poultry and poultry products are the major sources of human infection (Kimura et al.,
2004; Patrick et al.,
2004). In the chicken host, Salmonella Enteritidis is transmitted via a fecal-oral route. Young chickens <2 weeks of age often develop gastroenteritis and systemic disease with varying degrees of mortality. In contrast, most adult hens become colonized with Salmonella Enteritidis, but typically remain asymptomatic carriers with intermittent fecal shedding of Salmonella. During intestinal colonization, inflammation ensues and is characterized by an early influx of heterophils (avian counterpart to mammalian neutrophils) and macrophages that play an important role in host defense (Henderson et al.,
1999; Chappell et al.,
2009). Salmonella Enteritidis has the ability to survive within these cells (Henderson et al.,
1999; Stabler et al.,
1994; Okamura et al.,
2005) and also penetrate the mucosal epithelium, resulting in dissemination to organs, including spleen, liver, and reproductive tract (Desmidt et al.,
1997; Gantois et al.,
2009). Due to the specialized ability of Salmonella Enteritidis to colonize the avian reproductive tract and to contaminate internal contents of eggs, contaminated eggs or egg products have been implicated in the majority of foodborne outbreaks worldwide (Kimura et al.,
2004; Patrick et al.,
2004).

The specialized ability of Salmonella Enteritidis to contaminate and survive within the egg is important from the public health perspective. The potential for Salmonella Enteritidis to be deposited in egg contents primarily depends on the “virulence” of a particular strain, i.e., the ability of the bacterium to invade avian intestinal epithelium with subsequent dissemination to internal tissues (Gast and Benson, 1996; Gast, 1994; Gast and Beard, 1990). It has been reported that naturally occurring Salmonella Enteritidis strains vary in their virulence in chickens in terms of organ invasiveness (Gast and Benson 1995, 1996; Dhillon et al.,
1999), and the ability to contaminate and survive within the egg contents (Petter, 1993; Clavijo et al.,
2006; Yim et al.,
2010). These differences in virulence exist irrespective of the phage type (Poppe, et al.,
1993) or clonal lineages (Yim et al.,
2010; Olsen et al.,
1999). The basis for this differential virulence of Salmonella Enteritidis in the chicken is somewhat understood, because phenotypic variation has been traced to the level of the single nucleotide polymorphism rather than to differences in whole gene content (Morales et al.,
2007).

Avian innate defenses play an important role in the outcome of infection. For instance, the two major avian innate defenses that impose severe stress on an invading Salmonella Enteritidis include the acidic stress from gastric acidity (pH 2.6) (Joyner and Kokas, 1971; Carter and Collins, 1974) and oxidative stress from effects of hydrogen peroxide (H2O2) produced by infected avian heterophils and macrophages (Qureshi et al.,
2000; Withanage et al.,
2005; Kogut et al.,
2002). Furthermore, egg albumen contains several antibacterial substances such as lysozyme, ovatransferrin, and avian B-defensins (Van Immerseel, 2010; Kang et al.,
2006), thereby presenting another detrimental environment for the survival and growth of this bacterium. Assessing the ability of Salmonella Enteritidis strains to survive under different stress conditions encountered early during infection in chickens may facilitate better understanding of the pathogenesis of Salmonella Enteritidis. Nevertheless, limited studies have been conducted to determine the responses of Salmonella Enteritidis to various stress conditions (Yim et al.,
2010; Humphrey et al.,
1993, 1995). For instance, Humphrey et al. (1995) reported that Salmonella Enteritidis strains isolated from poultry had reduced tolerance to acid and oxidative stress as compared with the strains isolated from human clinical cases. In another study, Yim et al. (2010) reported that vast majority of Salmonella Enteritidis strains isolated from food sources showed diminished survival in egg albumen as compared with the clinical strains. In addition, Salmonella Enteritidis produces a specialized capsular-like lipopolysaccharide O-antigen that contributes to the survival of the pathogen in the egg, but its production varies between strains (Guard-Bouldin et al.,
2004). We hypothesized that differential virulence of Salmonella Enteritidis strains is due to the differential fitness in the stressful environments encountered in the host during infection. To address this hypothesis, we tested the ability of several Salmonella Enteritidis strains to survive under various stressful conditions that may be encountered in the chicken host such as acidic-stress, oxidative-stress, growth in egg albumen, and in vivo virulence in orally inoculated chickens.

Methods

Bacterial strains

A panel of six Salmonella Enteritidis strains (UK, G1, BC8, C19, C45, and G45) isolated from poultry or poultry-associated environments were analyzed in this study. Previous work from our laboratory showed that the UK (corresponds to the sequenced phage type-4 P125109 strain), G1 (phage type-4), and BC8 (phage type-8) strains differ from C19 (phage type-13), C45 (phage type-13), and G45 (phage type-13a) strains in terms of their invasiveness into human intestinal epithelial cells (Caco-2), virulence in orally challenged mice, motility, biofilm production, and phage types () (Shah et al.,
2011). Frozen stocks of cultures were grown on Luria-Bertani (LB) agar incubated at 37°C for overnight. For all the experiments, a single colony from the overnight culture was inoculated into LB broth and grown at 37°C or 42°C for 16 h with shaking at 200 rpm.

Table 1.

Phenotypic and Genotypic Characteristics of Poultry-Associated Salmonella Enteritidis Strains Used in This Study (Shah et al.,
2011)

Strain Phage type MLVA type Caco-2 cell invasiveness Survival in chicken macrophages Motility Biofilm
C19 PT13 11 Low Low Low Negative
C45 PT13 11 Low Low Low Negative
BC8 PT8 9a High High High Mixed
G1 PT4 13a Medium High High Positive
G45 PT13a 4a Low Low Low Negative
UK PT4 13a High High High Positive

Survival assays

The stationary phase (16 h) cultures of Salmonella Enteritidis strains were used to measure their survival rates in LB broth adjusted to pH 2.6±0.02 with 1 M HCl (Humphrey et al.,
1993, 1995) and in normal saline (0.9% NaCl) after addition of H2O2 to a final concentration of 15 mM (Robbe-Saule et al.,
2003). Controls included the cultures incubated in the same media but in the absence of stressors. Stationary phase cultures of Salmonella Enteritidis strains were used because this growth phase provides a reliable model to measure the differences in tolerance to acid and oxidative stress (Humphrey et al.,
1995). All the media, tubes and reagents were prewarmed to avoid a sudden shift in temperatures that may otherwise markedly alter stress tolerance in Salmonella Enteritidis (Humphrey et al.,
1993). For the acid stress survival assay, ∼1×108 colony forming units (CFU) of each Salmonella Enteritidis strain was inoculated in LB (pH 2.6) at 37°C or at 42°C followed by incubation at respective temperatures. For the oxidative stress survival assay, ∼1×108 CFU of the stationary phase cultures were resuspended in normal saline at either 37°C or 42°C. Subsequently, H2O2 was added to the final concentration of 15 mM, mixed thoroughly, and the suspension was incubated at 37°C or at 42°C. Aliquots from acid and oxidative stress cultures were collected at 10 min and 1 h post-inoculation. Serial 10-fold dilutions of each culture was prepared using maximum recovery diluent (MRD; Oxoid, Lenexa, KS) and plated on LB agar for enumeration of viable CFU. Each strain was tested in duplicate, and at least three independent experiments were completed.

Growth in egg albumen

The ability of Salmonella Enteritidis strains to survive in egg albumen was quantified as described previously with minor modifications (Lu et al.,
2003). Briefly, organic, unfertilized, antibiotic-free eggs were purchased from Chino Valley Ranchers, CA and stored for less than 1 week at 4°C. For each experiment, two to three eggs were disinfected by immersion into 70% ethanol and aseptically broken to collect egg albumen into a sterile container. Egg albumen was pooled, and 1 mL aliquots were distributed into 96-well blocks (Qiagen, Valencia, CA). An overnight culture of each bacterial strain was added to egg albumen to a final concentration of ∼500 CFU/mL and thoroughly mixed and incubated at 25±2°C for 24 h. After incubation, the bacteria-albumen mixture was diluted in MRD and plated on LB agar to enumerate the viable bacterial counts. Uninoculated egg albumen served as a negative control. Five independent experiments were completed for each strain.

Comparative genomic hybridizations

The microarray was constructed by MYcroarray Inc. (Ann Arbor, MI) using the genomic information for the sequenced Salmonella Enteritidis PT4 P125109 (Thomson et al.,
2008) which is referred to as the UK strain in this study. This microarray consists of a total of 16,320 features. Of these, 4,343 are control features (spike-in positive controls, empty spots, negative control probes, etc). The remaining 11,977 features (∼47-mers) represent 4,200 ORFs as follows: three specific probes/ORF (3,742 genes), two specific probes/ORF (293 genes), and one specific probe/ORF (165 genes). Three arrays per slide were generated by in situ synthesis using a proprietary light-directed oligonucleotide synthesis technology (www.mycroarray.com). Each strain was tested in triplicate (3 arrays/strain) using hybridization protocols as described previously (Call et al.,
2003). Slides were scanned using a Genepix400B scanner (Axon Instruments, Inverurie, Scotland). After acquisition of signal intensity data, each spot was corrected by subtracting local background values and each “corrected” spot intensity was used to calculate median signal intensity for each array. Inferior spot intensities (empty spots or those where signal intensity was less than two times the background) were reset to an arbitrary number (50) and were not used in the median calculation. The array median intensity was then used for within array normalization. The data was log2 transformed and quantile normalized between arrays using SOLO (http://www-microarrays.u-strasbg.fr/Solo/index.html). Finally, the estimated probability of presence (EPP) for each probe was determined using a GACK-transformation as described previously (Kim et al.,
2002). Microarray data and platform information have been submitted to GEO (under accession no. {“type”:”entrez-geo”,”attrs”:{“text”:”GSE33102″,”term_id”:”33102″}}GSE33102).

rpoS gene sequencing

The total genomic DNA from each Salmonella Enteritidis strain was extracted using DNeasy Tissue kit (Qiagen) according to the manufacturer’s protocol. The full length rpoS gene (993 bp) was amplified from all six strains using primers rpoS_F (5’-atgagtcagaatacgctgaaag-3’) and rpoS_R (5’-ttactcgcggaacagcgc-3’). PCR amplification included 28 cycles, each consisting of 30 s of denaturation at 96°C, 30 s of annealing at 50°C, and 45 s of extension at 72°C. PCR products were cloned into pGEM®-T Easy vector (Promega, Madison, WI) according to manufacturer’s instructions. The rpoS gene insert was sequenced using universal reverse (5’-actttatgccggctcgtatgttgt-3’) and a universal forward (5’-atgtgtgcaaggcgattaagttggg-3’) primer at the Washington State University Genomics Core. The rpoS sequences were aligned against rpoS gene from a reference UK strain P125109 (GenBank accession no. {“type”:”entrez-nucleotide”,”attrs”:{“text”:”AM933172″,”term_id”:”206707319″,”term_text”:”AM933172″}}AM933172) (Thomson et al.,
2008). Sequence alignment and single nucleotide polymorphism were detected using Geneious Pro 5.3 (Biomatters Ltd., Auckland, New Zealand). The sequences of rpoS genes were deposited in the GenBank under the following accession numbers: {“type”:”entrez-nucleotide”,”attrs”:{“text”:”JN588998″,”term_id”:”377655119″,”term_text”:”JN588998″}}JN588998 (UK), {“type”:”entrez-nucleotide”,”attrs”:{“text”:”JN588996″,”term_id”:”377655115″,”term_text”:”JN588996″}}JN588996 (G1), {“type”:”entrez-nucleotide”,”attrs”:{“text”:”JN588997″,”term_id”:”377655117″,”term_text”:”JN588997″}}JN588997 (BC8), {“type”:”entrez-nucleotide”,”attrs”:{“text”:”JN588999″,”term_id”:”377655121″,”term_text”:”JN588999″}}JN588999 (C19), {“type”:”entrez-nucleotide”,”attrs”:{“text”:”JN589000″,”term_id”:”377655123″,”term_text”:”JN589000″}}JN589000 (C45), and {“type”:”entrez-nucleotide”,”attrs”:{“text”:”JN588995″,”term_id”:”377655113″,”term_text”:”JN588995″}}JN588995 (G45).

Chicken virulence assay

Twenty-one 1-day-old chickens were obtained from Belt Hatchery (Fresno, CA) and distributed in seven groups (n=3/group) in environmentally controlled isolation cages. Cloacal swabs were taken prior to placement of chicks in cages and examined for the presence of Salmonella by selective enrichment in tetrathionate broth (TTB; Difco, Franklin Lakes, NJ) followed by plating onto XLD agar (Dicfo). Antibiotic-free flock raiser diet (Purina, St. Louis, MO) and water was provided ad libitum throughout the experimental period. At 3 days of age, chickens were challenged orally with stationary phase cultures of Salmonella Enteritidis strains at a concentration of ∼107 CFU. One group served as the uninoculated negative control. The chickens in all groups were euthanized at 4 days post-infection, necropsied, and the small intestine, ceca, liver, and spleen were examined by selective enrichment in TTB followed by plating onto XLD-agar. The viable counts of Salmonella per gram in liver and spleen tissues from all the chickens were obtained by emulsification of weighed tissues in PBS followed by plating of serial dilutions of the ground tissues on XLD-agar plates. Presumptive Salmonella colonies were confirmed by slide agglutination test using serogroup D antiserum (Difco). The animal challenge experiments were conducted in accordance with the protocol approved by the Washington State University Institutional Animal Care and Use Committee (WSU IACUC).

Statistical analysis

Data was analyzed using one-factor analysis of variance (ANOVA) with Tukey’s Kramer post-test in NCSS 2007 (NCSS, LLC, Kaysville, UT).

Results and Discussion

Because the ability of Salmonella to withstand stomach acidity is a recognized virulence attribute, we tested the survivability of stationary phase cultures of six Salmonella Enteritidis strains by substantially mimicking the avian stomach environment (pH 2.6) (Joyner and Kokas, 1971). While there were no differences in the survival of Salmonella Enteritidis strains in the absence of acid-stress (data not shown), significant differences in the tolerance of Salmonella Enteritidis strains to acid-stress were observed (). When 8 log CFU of Salmonella Enteritidis strains were exposed to pH 2.6 at 37°C for 10 min, the survival rate of UK, G1, and BC8 strains (>7 log CFU) was significantly higher (p<0.05, Tukey’s Kramer test) as compared with the C19, C45, and G45 strains (<5 log CFU) (). At 1 h post-incubation, <1 log CFU of C19 and C45 and 1.2 log CFU of G45 strain survived, whereas >1.96 log CFU of the UK, G1, and BC8 strains survived this treatment. These differences were statistically significant (p<0.05). At 42°C (chicken body temperature), the counts of C19, C45, and G45 strains declined from an initial 8 log CFU to ≤1 log within 10 min. Thus, killing of stress-sensitive strains was more pronounced at the higher body temperature of the bird (). In contrast, >4 log CFU of the UK, G1, and BC8 strains survived this treatment, indicating that these strains were resistant to short-term acidic stress at 37°C and 42°C. We were not able to recover any of the Salmonella Enteritidis strains after 1 h of incubation at 42°C, suggesting that none of the strains survived prolonged acidic stress at high temperature. These results indicate that C19, C45, and G45 strains are acid-sensitive, whereas the UK, G1, and BC8 strains are relatively acid-resistant ().

Table 2.

Survival Values for High- and Low-Pathogenic Salmonella Enteritidis Strains Exposed to Acidic (pH 2.6) and Oxidative (15 mM H2O2) Stress

 


Number of survivors (mean log10 CFU±SE)a


 


Stress-resistant


Stress-sensitive


Conditions UK BC8 G1 C19 G45 C45
pH 2.6 for 10 min (37°C) 7.33±0.4 7.08±0.43 7.33±0.36 4.05±1.01b 4.84±0.75b 4.14±1.06b
pH 2.6 for 1 h (37°C) 1.96±1.15 2.88±1.67 3.21±1.35 <0.01b 1.2±0.41b 0.09±0.09b
pH 2.6 for 10 min (42°C) 5.35±0.9 4.28±1.29 5.02±1.17 0.66±0.5b 1.02±0.65b 1.01±0.64b
pH 2.6 for 1 h (42°C) 0 0 0 0 0 0
15 mM H2O2 for 10 min (37°C) 8.07±0.04 8.07±0.06 8.06±0.05 6.1±0.18b 5.53±0.19b 6.33±0.19b
15 mM H2O2 for 1 h (37°C) 7.95±0.05 7.81±0.24 7.92±0.12 1.54±1.04b 0.4±0.4b 1.47±0.6b
15 mM H2O2 for 10 min (42°C) 8.06±0.08 8.08±0.1 8.06±0.07 6.4±0.23b 5.33±0.19b 6.5±0.07b
15 mM H2O2 for 1 h (42°C) 7.94±0.09 7.43±0.19 7.85±0.01 0 0 0

Hydrogen peroxide produced by avian macrophages and heterophils plays a crucial role in Salmonella killing because it can penetrate cell membranes and may act on intracellular targets (Desmidt et al.,
1997; Qureshi et al.,
2000; Withanage et al.,
2005; Kogut et al.,
2002). Therefore, we determined the ability of Salmonella Enteritidis strains to counteract the effects of hydrogen peroxide. Irrespective of the temperature (37°C or 42°C) or time (10 min or 1 h) of incubation, there was no significant decline in the counts of UK, G1, and BC8 strains () indicating that these strains were highly resistant to the oxidative stress induced by hydrogen peroxide. In contrast, the counts of C19, C45, and G45 strains declined by 1.5–2.5 log within 10 min at 37°C or 42°C (). By 1 h, the counts further declined by 6.5–7.5 log, indicating that these strains were highly sensitive to the oxidative stress induced by hydrogen peroxide (). Overall, these results indicate that C19, C45, and G45 strains (stress-sensitive strains) show impaired ability to survive in both acid and oxidative stress when compared with the UK, G1, and BC8 strains (stress-resistant strains).

To assess the virulence (i.e., the ability to invade and systemically spread in the internal organs) of stress-sensitive and stress-resistant Salmonella Enteritidis strains in chickens, 3-day-old chickens were orally inoculated with ∼107 CFU of each strain and sacrificed at 4 days post-infection. For stress-resistant strains, the mean log10 CFU of Salmonella Enteritidis/gram of liver ranged from 1.57±0.79 (UK) to 1.75±0.14 (G1), whereas mean log10 CFU of Salmonella Enteritidis/gram of spleen ranged from 3.22±0.32 (BC8) to 3.96±0.05 (UK) (). In contrast, none of the stress-sensitive strains were recovered from either liver or spleen by either direct plating or by enrichment procedure, indicating that the acid-sensitive strains had impaired ability to invade the internal tissues. Similar to the results obtained in this study, Jorgensen et al., (2000) reported that two acid-sensitive Salmonella Typhimurium strains failed to invade chicken tissues, whereas Humphrey et al. (1996) reported that one acid and H2O2 sensitive Salmonella Enteritidis strain was significantly less invasive in chickens when compared with one acid and H2O2 resistant strain. It was also reported that the fecal carriage rates of stress-sensitive strains of Salmonella Tyhimurium were not significantly different from those of stress-resistant strains (Jorgensen et al.,
2000; Williams et al.,
1998). In this study, the intestinal samples collected from chickens challenged with stress-resistant strains were positive for Salmonella by selective enrichment procedure, indicating that intestinal carriage of these strains was not affected. Nevertheless, none of the intestinal samples collected from chickens infected with stress-sensitive strain were positive for Salmonella, indicating that stress-sensitive strains had an impaired ability to colonize the intestine. Studies on oral infection of Salmonella Enteritidis in mice have shown that only 1% of the inoculum survives the low pH during the passage through the stomach (Carter and Collins, 1974). About 80% of the bacteria that survive the passage through the stomach are passed with the feces within 6–10 h post-infection, whereas 15% remain localized in the lumen of cecum and large intestine, while only 5% manage to penetrate the intestinal wall of the small intestine and reach gut associated lymphoid tissues (Carter and Collins, 1974). Because the stress-sensitive strains tested in this study showed impaired ability to survive in gastric acidity and oxidative stress, these strains appear to have cleared from the intestine before the birds were sacrificed 4 days post-infection. Additional time-course studies to investigate changes in the intestinal carriage and systemic spread of stress-sensitive and stress-resistant strains may be required to fully understand the differences in their kinetics of infection in chickens.

The mean log10 colony forming units (CFU)±standard error (SE) of stress-sensitive and stress-resistant Salmonella Enteritidis strains recovered from liver (gray bars) and spleen (black bars) of chicks. Chicks in each group (n=3) were orally inoculated at number (#) of days of age with ∼1×107 CFU of Salmonella Enteritidis strains, and the challenge strains were recovered from the internal organs at 4 days post-infection.

It has been hypothesized that stress-induced survival mechanisms may enable certain Salmonella Enteritidis strains to cope with the antimicrobial compounds present in the egg albumen (Van Immerseel, 2010). Consequently, we tested all six Salmonella Enteritidis strains for their ability to survive and/or grow in egg albumen. With the starting inoculum of ∼500 CFU, all the stress-resistant strains grew in egg albumen up to 4–5 log after 24 h of incubation at 25°C (). In contrast, two stress-sensitive strains (C19 and C45) did not survive or grow in egg albumen, whereas one strain (G45) grew as well as the stress-resistant strains. These results indicate that some stress-sensitive strains may also have an impaired ability to respond to the hostile environment of egg albumen—an environment that includes iron restriction, high pH, and other enzymatic activities. Further research is required to see if additional factors, such as expression of an O-antigen capsule, also contribute to the survival and growth of some strains Salmonella Enteritidis in albumen.

The mean log10 colony forming units (CFU)±standard error (SE) of stress-sensitive (gray bars) and stress-resistant (black bars) Salmonella Enteritidis strains in egg albumen. An overnight culture of each bacterial strain was added to egg albumen to a final concentration of ∼500 CFU/mL and incubated at 25±2°C for 24 h.

To determine if the differential stress response and virulence observed in this study could be due to the differences in the gene content between different strains, we compared the genomes of all the strains using CGH microarray designed based on the genomic information available for the sequenced strain. The CGH analysis revealed no consistent differences in the genomes between the six strains tested in this study. These results corroborate previous reports indicating that Salmonella Enteritidis strains are relatively genetically homogeneous, despite geographical, temporal, and source differences between the different strains (Porwollik et al.,
2005; Morales et al.,
2005; Olson et al.,
2007; Betancor et al.,
2009). It is important to point out that the microarray used in this study contained genes representing a single Salmonella Enteritidis strain and therefore only genes that are present on the array can be assayed. Some strains tested in this study may contain additional genes (e.g., phage-related genes) that were not represented on the microarray. Overall, these results indicate that the differential stress-response and virulence is less likely due to the variation in the whole gene content, but are more likely due to small polymorphisms that may alter regulation or expression of stress or virulence associated genes. To determine if the phenotypic differences were related to the rpoS gene, we sequenced the full length rpoS from all the six Salmonella Enteritidis strains. Several studies have shown that σS, encoded by the rpoS gene, controls the general stress response of bacteria, in particular the ability to resist hydrogen peroxide and acid stress during stationary phase (Hengge-Aronis, 2002). In addition, it has been reported that RpoS contributes to the virulence of Salmonella (Jorgensen et al.,
2000; Nickerson and Curtiss, 1997). The rpoS gene in two stress-resistant strains (UK and G1) and one stress-sensitive strain (C19) was 100% identical in DNA sequence to the rpoS gene of Salmonella Enteritidis P125109 (GenBank accession no. {“type”:”entrez-nucleotide”,”attrs”:{“text”:”AM933172″,”term_id”:”206707319″,”term_text”:”AM933172″}}AM933172). Strains BC8 contained a synonymous substitution from G to A at nucleotide 375 and a non-synonymous mutation at codon 36 (AGT [Ser] to AGG [Arg]). Strain C45 contained a non-synonymous mutation at codon 126 (GGG [Gly] to GAG [Glu]). Strain G45 contained two mutations, a synonymous substitution from T to C at nucleotide 605 and an insertion of A at nucleotide position 666. The insertion resulted in the introduction of an internal stop codon (TAA). Thus, one of the three stress-sensitive strains analyzed had a discernible mutation that interrupted the ORF of rpoS, consistent with the hypothesis that differential survivorship between strains is related to the single nucleotide polymorphisms in the chromosome. It is possible that the other strains may also have small polymorphisms elsewhere in the genome, which likely impact regulation of stress or virulence associated genes in some manner.

To determine whether any of the mutations altered the production of σS, we measured the activity of rpoS indirectly by analysis of hydrogen peroxidase II (HPII) activity using a semiqualitative catalase test as described previously (Taylor and Achanzar, 1972). Catalase test is based on principle that when 3% H2O2 is added to the bacterial colonies grown on LB plates, HPII breaks down H2O2 with the concomitant release of O2 resulting in bubbling of the colony. Strains producing RpoS bubble vigorously (+++), whereas strains bearing null alleles either do not form bubbles (-) or bubble only slightly (+) (Taylor and Achanzar 1972). We found that UK, G1, BC8, C19 and C45 strains formed vigorous bubbles (+++), suggesting that rpoS mutations in strains BC8 and C45 did not affect the σS function. In contrast, G45 strain with truncated rpoS did not form bubbles (-), indicating impaired σS function and a possibility of impaired KatN, an rpoS regulated non-hem catalase expression (Robbe-Saule, et al.,
2001). It is important to note that the G45 strain is impaired in motility and also lacks a large virulence plasmid (Shah et al.,
2011) and Salmonella plasmid virulence (spv) genes whose expression is controlled by rpoS during systemic infection (Jorgensen et al.,
2000; Nickerson and Curtiss, 1997). Therefore it is likely that the impaired motility or regulation of virulence genes in this particular strain may have led to the reduced pathogenicity in chickens. Interestingly, the growth of G45 strain in the egg albumen was not affected indicating that σS may not be required for the survival of Salmonella Enteritidis in egg albumen (). Previous studies have reported that genes involved in bacterial cell wall structure and function, amino acid and nucleic acid metabolism, motility and stress responses may contribute to the survival of Salmonella Enteritidis in egg albumen (Clavijo et al.,
2006; Lu et al.,
2003; Gantois et al.,
2008).

their excretion by infected chickens.

J Hyg (Lond). 1980 Jun; 84(3): 479–488.

This article has been cited by other articles in PMC.

Abstract

Inoculated orally, 16 Salmonella typhimurium strains belonging to 12 phage types varied greatly in their ability to kill 1-day-old chickens; variation was noted even between strains of the same phage type. Fourteen strains belonging to 11 food poisoning serotypes other than S. typhimurium were practically non-lethal when examined in this manner. All of them were lethal by the intramuscular route but some were more so than others. Two were more lethal by this route than one of the S. typhimurium strains that was highly lethal when given orally. With age, chickens rapidly became resistant to fatal infection with the food poisoning strains; given orally, a S. typhimurium strain killed 79% of 1-day-old chickens but only 3% of 2-day-old chickens. Of 2 specific poultry pathogenic strains, one, of S. gallinarum, was lethal by oral inoculation to chickens of all ages but the other, of S. pullorum, was only lethal to very young ones. Some salmonella strains, such as those of S. infantis and S. menston, were more efficient at infecting and colonizing the alimentary tract of chickens than were the more virulent S. typhimurium strains, the S. gallinarum and S. pullorum strains and a S. cholerae-suis strain.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  • SMITH HW. The evaluation of culture media for the isolation of salmonellae from faeces. J Hyg (Lond) 1952 Mar;50(1):21–36. [PMC free article] [PubMed] [Google Scholar]
  • SMITH HW. Observations on experimental fowl typhoid. J Comp Pathol. 1955 Jan;65(1):37–54. [PubMed] [Google Scholar]
  • Smith HW, Tucker JF. The effect of antibiotic therapy on the faecal excretion of Salmonella typhimurium by experimentally infected chickens. J Hyg (Lond) 1975 Oct;75(2):275–292. [PMC free article] [PubMed] [Google Scholar]
  • Smith HW, Tucker JF. The effect of feeding diets containing permitted antibiotics on the faecal excretion of Salmonella typhimurium by experimentally infected chickens. J Hyg (Lond) 1975 Oct;75(2):293–301. [PMC free article] [PubMed] [Google Scholar]
  • Smith HW, Tucker JF. The effect of antimicrobial feed additives on the colonization of the alimentary tract of chickens by Salmonella typhimurium. J Hyg (Lond) 1978 Apr;80(2):217–231. [PMC free article] [PubMed] [Google Scholar]
  • Smith HW, Tucker JF. The effect on the virulence and infectivity of Salmonella typhimurium and Salmonella gallinarum of acquiring antibiotic resistance plasmids from organisms that had caused serious outbreaks of disease. J Hyg (Lond) 1979 Oct;83(2):305–317. [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Hygiene are provided here courtesy of Cambridge University Press


How Does Salmonella Make Us Sick?

Question: What do turtles, casino brunches and psychoactive herbs have in common? (Feel free to get creative and imagine a particularly eventful outing for a herpetology enthusiast club.)

Answer: all are potential sources of Salmonella infection that have been associated with recent outbreaks in the U.S.. While we mostly think of eggs and poultry products as carriers of Salmonella, pets, livestock, produce, and soil can also spread infection.

Salmonella are gram-negative bacteria and common causes of gastrointestinal illness. Infection typically manifests as severe stomach cramps, fever, and diarrhea that can last several days, though other disease presentations, such as sepsis, can also occur. Many of us are unfortunately familiar with the misery that ensues upon eating spoiled food, but what actually happens inside of our bodies during Salmonella infection? What is it about Salmonella that can make us so sick? Salmonella uses a variety of unique virulence mechanisms to invade our intestinal cells and confuse our immune cells, leading to many of the key symptoms associated with food poisoning.

Salmonella Nomenclature: Species, Serotypes, and Typhoid Disease

Before discussing Salmonella pathogenesis in more detail, it’s important to understand the complex and distinct ways in which different strains of Salmonella are characterized. Though there are only 2 species of Salmonella (S. enterica and S. bongori), there are more than 2,500 serotypes, which are groups of related bacteria with similar antigen presentation, across both species. Salmonella enterica is further broken up into 6 subspecies based upon genetic similarity. Of subspecies I, II, IIIa, IIIb, IV, and VI, only members of subspecies I are human pathogens. Despite the large number of identified strains, fewer than 100 Salmonella strains are suspected to be pathogenic.

Though pathogenic serotypes can be very genetically similar, they can have different abilities to cause severe disease. Some serotypes cause a few days of mostly self-limiting illness, whereas others cause life-threatening complications. It is not well understood how specific serotypes alter host immune response and modulate bacterial virulence.
 
The serotype is defined by the immunoreactivity of distinct molecular patterns on bacterial cell surfaces, namely the “O antigen” and the “H antigen” which are the primary antigens present in many coliform bacteria. The “O” antigen is part of the lipopolysaccharide layer of the bacterial membrane, whereas the “H” antigen is located on the organism’s motile tail, known as the flagella. Many molecular variations in the “O” and “H” antigens exist, allowing for a wide range of different serotype combinations. The “H “antigen on the flagella can be described as “phase 1” or “phase II,” which refers to which key flagellar genes (fliC and fliB) are being expressed. The gold-standard system for serotyping Salmonella strains is the Kauffman-White scheme, a complex alphanumerical system that separates “O” antigens into alphanumerical categories (ex. A, B, C1), “H phase 1” antigens into a-z and z1-z99 categories, and numbers “H phase 2” antigens from 1-12. However, the Center for Disease Control and Prevention (CDC) and many scientists refer to different serotypes using a shortened, simplified nomenclature, and refer to strains in a “genus-species-serotype” format (e.g., S. enterica serovar typhimurium).     
 
Serotypes can be further divided into typhoid and non-typhoid serotypes, based on their ability to cause typhoid or paratyphoid fever, a type of severe S. enterica infection that spreads from person-to-person. Typhoid and paratyphoid strains are typed using O and H antigens, but also by their Vi (Virulence) antigens. The Vi antigen is a capsular antigen that contributes significantly to virulence, which may partially explain the disparity in disease severity between typhoid and non-typhoid strains. Typhoid Salmonella infections are more likely to be life-threatening, causing high fevers, headaches, constipation or diarrhea, and rose spots—patches of red discoloration on the skin where bacterial emboli are present.  
 

Typhoid fever remains a global public health threat, but it is not common in the United States and other developing countries. The disease can be prevented by the typhoid vaccine. The primary serotypes associated with non-typhoid, foodborne gastroenteritis are S. enterica serovar typhimurium and S. enterica serovar enteridis, which are also the most prevalent S. enterica serotypes found in poultry products. In contrast to typhoid fever, which affects only 5,700 Americans each year, foodborne Salmonella gastroenteritis is estimated to cause 1 million illnesses per year.

Salmonella Pathogenesis

Let’s assume you ate a questionable, undercooked omelette for breakfast and accidentally exposed yourself to a pathogenic, non-typhoidal S. enterica serovar typhimurium strain—what happens next? Salmonella prefers to replicate and infect host cells intracellularly. Once Salmonella is ingested, it invades the epithelial cells of the intestine, as well as nearby phagocytic immune cells. Salmonella uses a variety of dynamic techniques to impair and confuse host immune cells, including its ability to induce phagocytosis in certain white blood cells, which allows the organism to gain entry into cells more effectively.

Salmonella undermines non-phagocytic immune cells too, by inducing reactive oxygen species (ROS) production from human neutrophils. This defense mechanism is intended to protect the host by damaging bacterial nucleic acids and proteins. However, Salmonella benefits from ROS production, because it has an arsenal of peroxidases and catalases to help it survive ROS exposure. Other resident gut microbes are less equipped to survive this harsh environment, thus creating a selective advantage for Salmonella.
 
Once inside the host cell, Salmonella divides rapidly, and can either enclose itself within membrane-bound vacuoles, or as was recently discovered, replicate within the cytosol of cells. Salmonella’s preference to replicate in vacuoles versus in the cytosol possibly depends upon flagellar motility. Cytosolic Salmonella have more active flagella than those within vacuoles, and have the ability to extrude epithelial cells, meaning that infected epithelial cells that form a membrane, such as those within the intestinal lining, can “squeeze” out of their membrane layer and wander, allowing the infected cells to potentially spread to other organ sites. Cytosolic Salmonella also divide very quickly and have the ability to hyper-replicate in intestinal epithelial cells, gallbladder epithelial cells, and polarized epithelial cells that mimic the internal environment of the intestine. This adaptability to new environments may suggest that the cytosolic subpopulations of Salmonella have the ability to leave and survive outside of the intestine, potentially allowing them to spread to other body sites, leading to much more serious and invasive illness.  
 
Fortunately, the human immune system is equipped to fight Salmonella’s invasive and evasive maneuvers. While innate immune cells, such as neutrophils and macrophages, can be fooled by Salmonella’s immune evasive strategies, adaptive immune cells are vital for host defense. When Salmonella is introduced to the GI tract orally, such as through contaminated food or kissing your cute pet hedgehog, Salmonella-specific immune cells, such as CD4+ T cells, are generated to help fight the invading pathogen. These cells later develop into memory cells following clearance of the infection, which provides some long-term protection against Salmonella. But because different serotypes are recognized by immune cell interaction with their unique patterns, unrelated serotypes can cause a subsequent bout of disease.
 
Other adaptive immune cells, including CD8+ T cells and B cells, also play important roles in fighting off Salmonella infections, with the strongest evidence pointing towards the protective role of CD8+ T cells during later stages of infection. CD8+ T cells are specialized cells that can target intracellular pathogens with the help of B cells, which can serve as antigen-presenting cells that help CD8+ T cells to recognize Salmonella-specific antigens and mount an inflammatory immune response to help clear infection. Production of interferon-gamma, a pro-inflammatory cytokine produced by CD4+ and CD8+ T cells, has demonstrated importance for controlling infection in intestinal epithelia. However, even these savvy adaptive cells aren’t foolproof: Salmonella can suppress T cell and B cell responses by impairing effector cell priming to blunt the necessary inflammatory response.
 
While the immune system is always hard at work, the best ways to prevent Salmonella infection are through careful handwashing, sanitary food preparation, and proper handling of pets and livestock.  If you are traveling abroad, it’s always a good idea to check whether typhoid fever is a potential risk at your destination. Extensively drug-resistant (XDR) typhoid, in particular, is an important issue in certain countries. Pakistan is currently experiencing an XDR typhoid outbreak, which began in 2016 and has sickened 5,200 people, including travelers from the US and UK. This type of typhoid infection is resistant to most antibiotics, making it very difficult to treat. Do your hard-working T cells and B cells a favor, especially in extreme cases such as these – get your recommended vaccinations and use proper hygiene to prevent serious Salmonella infections.

Be sure to check out this additional article in our series on foodborne illness:

Frontiers | Comparative phenotypic and genotypic virulence of Salmonella strains isolated from Australian layer farms

Introduction

Enteric bacterial pathogens are among the most common causes of diarrheal disease world-wide. Consumption of contaminated food items is frequently the source of pathogens responsible for outbreaks of gastroenteritis. With the globalization of food distribution, contaminated or improperly handled food products have the potential to cause disease in multiple countries. As a consequence, foodborne gastrointestinal diseases could have major socioeconomic impacts in both developed and developing countries (Hendriksen et al., 2011).

Salmonella is a diverse group comprised of two major species, Salmonella (S.) bongori and S. enterica. Salmonella enterica is further subdivided into six subspecies and is the largest group containing over 2500 serovars (Guibourdenche et al., 2010). Salmonella is an intracellular pathogen and depending on both the host species and serovar can cause disease in both humans and animals ranging from mild diarrhea to typhoid fever. Humans generally acquire Salmonella through the consumption of contaminated foods, including fruits, vegetables, nuts, dairy, meat, eggs and poultry meat (reviewed in Carrasco et al., 2012). In particular, contaminated raw eggs or improperly handled egg-related products are common sources of Salmonella infection (Chen and Jiang, 2014; Threlfall et al., 2014). The incidence of Salmonella infection as a consequence of egg or egg product consumption is 23% in the US (Jackson et al., 2013), 39% in Australia (Moffat and Musto, 2013) and has been estimated at 32% in Europe (Pires et al., 2010).

Many Salmonella spp. have established a unique niche within poultry environments. The bacteria are able to colonize the gastrointestinal tract of chickens and ultimately spread horizontally and vertically within a flock (reviewed in Foley et al., 2011; Howard et al., 2012). Chicks within the first few days of life are more susceptible than older chickens to Salmonella colonization through horizontal transmission of bacteria from a contaminated environment (Foley et al., 2011). Some Salmonella serovars such as S. Enteritidis can infect chicks through vertical transmission from infected parents (Howard et al., 2012; Sivaramalingam et al., 2013). In addition, infection with S. Typhimurium or S. Enteritidis can result in a persistent infection or colonization of vital organs in chickens (Wales and Davies, 2011; Gast et al., 2013). Intermittent shedding of Salmonella spp. in fecal material can occur as a consequence of physiological and/or environmental stress (Nakamura et al., 1994; Quinteiro et al., 2012; Gole et al., 2014a). Longitudinal epidemiological investigation of Salmonella in layer flocks has correlated point-of-lay with peak bacterial loads in feces (Gole et al., 2014a). During these periods, it is likely that egg contamination can occur.

Globally, the two most common Salmonella serovars associated with gastrointestinal disease of humans are S. Enteriditis and S. Typhimurium (Hendriksen et al., 2011). Other non-typhoidal Salmonella (NTS) serovars are also responsible for causing considerable disease but they do not have widespread global distribution and their prevalence is location dependent (Hendriksen et al., 2011). Previous virulence studies utilizing primarily European or North American serovars have described considerable variation in their in vitro invasive capacity (Suez et al., 2013) as well as their ability to cause disease in mouse models (Swearingen et al., 2012). In Australia, NTS particularly S. Typhimurium definitive types are also frequently responsible for egg product related food poisoning outbreaks (Ozfoodnet Working Group, 2012) but there is currently limited characterization of their virulence.

Comparative genomic analyses of Salmonella enterica has revealed that there is considerable variation in virulence elements across the species as a whole (Jacobsen et al., 2011). The genome of S. Enteriditis, for example, possesses “regions of difference” (ROD) or clusters of coding sequences not present in all serovars, in particular S. Typhimurium (Thomson et al., 2008). Several of these RODs including two coding sequences within SPI-19 (SEN1001 and SEN1002), a cluster of genes within ROD21 linked to tRNA-asnT (SEN1970 to SEN1999), the peg fimbrial operon (SEN2144A to SEN2145B) as well as genes within ROD40 that are components of a type I restriction modification system (SEN4290 to SEN4292) are responsible for conferring increased in vivo virulence to S. Enteriditis (Silva et al., 2012). Characterization of phenotypic virulence and genotypic variability across multiple NTS serovars has, however, not been fully explored. In the present study, we have selected Salmonella serovars that are commonly isolated from layer hen environments that are also associated with human salmonellosis as well as other serovars whose incidence of disease is low (in Australia). Although it is likely that these strains share virulence mechanisms, our aim is to generate a virulence profile and identify genetic differences that lead to variation in overall pathogenicity.

For this study, 10 NTS strains isolated directly from various point sources (e.g., dust, feces, litter) in a layer hen environment were selected. Salmonella Typhimurium definitive types (DT) 44, DT135, DT170, DT193 and Virchow are frequently isolated from contaminated egg products during human salmonellosis outbreaks in Australia while the others are less commonly associated with disease (South Australian Salmonella Reference Laboratory, 2010, 2011, 2012, 2013). The invasive ability of NTS strains was investigated using both an in vitro human intestinal epithelial cell model as well as an in vivo mouse model. Whole genome sequencing of the selected 10 strains was also performed and the sequences of five specific pathogenicity islands with roles in both in vitro invasion and infection in vivo were analyzed and compared with the published reference strain S. Typhimurium LT2. LT2 was selected as a reference strain because the mechanisms of its in vitro and in vivo pathogenicity have been widely studied and the entire annotated genome is publicly available from the National Center for Biotechnology Information (NCBI).

Materials and Methods

Bacterial Strains

Salmonella enterica strains S. Adelaide, S. Bredeney, S. Cerro, S. Orion, S. Senftenberg, S. Virchow, and S. Typhimurium definitive types 44 (DT 44), 170=108 (DT170=108), 135 (DT135), and 193 (DT193) were selected for this study. All Salmonella strains were originally isolated from chicken fecal samples, dust or litter and were obtained from the Salmonella Reference Laboratory (Adelaide, South Australia). Table 1 lists the serovars selected for this study, the source and the number of times over a 4 year period that they were isolated from South Australian egg farms. Individual strains were stored long term at −80°C. Bacteria were recovered from freezing by streaking onto nutrient agar plates and incubated overnight at 37°C. Bacterial suspensions for experiments were prepared using either 0.9% saline or Luria Bertani broth (10 g tryptone, 5 g yeast extract, 10 NaCl per 1 L).

Table 1. The frequency of annual detection of the 10 strains selected for this study.

Cell Culture

The human intestinal epithelial cell line, Caco2 (ATCC HTB-37), was selected for the gentamicin protection invasion assay. Cells were cultured in Dulbecco’s Modified Eagle media (DMEM) (HyClone, Australia) containing 4 mM glutamine, glucose, 10% (vol/vol) fetal bovine serum (Hyclone, Australia), and 100 U/ml penicillin and 100 μg/ml streptomycin (ThermoScientific, Australia) at 37°C with 5% CO2. Cells were used between passages 5 and 10.

In Vitro Bacterial Invasion Assay

The invasive capacity of each Salmonella strain selected for this study was characterized using the gentamicin protection assay on polarized Caco2 cells. Briefly, Caco2 cells were first expanded in growth media (DMEM containing 10% FBS and 100 U/ml penicillin and 100 μg/ml streptomycin). Cells were then sub-cultured and placed into wells of a 48 well tissue culture tray (NUNC) at a concentration of 104 cells per well. A polarized cell monolayer was obtained by maintaining the culture in growth media and monitored for the production of alkaline phosphatase using a SensoLyte pNPP detection kit following manufacturer instructions (AnaSpec, USA). Once alkaline phosphatase production stabilized for 48 h (generally after 13–15 days), invasion experiments were conducted. Tissue culture media was changed every 48 h during polarization.

The gentamicin minimum inhibitory concentration (MIC) was determined for all strains included in this study using the Clinical and Laboratory Standard Institute (CLSI) guidelines (Clinical and Laboratory Standards Institues, 2013). The MIC for all strains was less than 0.25 μg/ml gentamycin. Prior to invasion experiments, bacteria were recovered from freezing by plating on to nutrient agar plates and incubating at 37°C overnight. Bacterial suspensions were created by suspending individual colonies in normal saline to an OD600 between 0.15 and 0.20 (corresponding to 108 bacteria cells/ml). Individual Salmonella strains were added separately to wells of the tissue culture tray to a multiplicity of infection (MOI) of 100 in DMEM containing no supplements. Prior to the addition of bacteria, the polarized Caco2 monolayer was washed three times with DMEM containing no supplements. Bacteria were incubated with the cell monolayer for 2 h and then removed by aspiration. Caco2 cells were then washed two times with DMEM containing 400 μg/ml gentamicin as per (Mickael et al., 2010) and incubated at 37°C for 15 min. The gentamicin was removed and the cell monolayers were washed three times with DMEM. Cells were lysed in 10% Triton X for 30 min at 37°C. The cell lysate was collected and serial 10-fold dilutions were prepared. Dilutions were plated onto Xylose lysine deoxycholate agar (XLD) (Oxoid, Australia) plates and incubated at 37°C overnight. Bacterial colonies were enumerated. Data are represented at mean percent recovery. Experiments were performed with duplicate replications and were repeated five times.

Additional invasion experiments were performed using bacteria grown to stationary phase in LB. Twenty-four hours prior to the invasion assay, a single colony was taken from each plate and placed into separate tubes containing 3 ml of LB broth. Tubes were incubated with shaking (100 rpm) for 6 h at 37°C. After 6 h, 10 μl of the starter culture was added to tubes containing 5 ml of LB. Bacteria were incubated with shaking overnight at 37°C. Suspensions were diluted to an OD600 between 0.15 and 0.2. Invasion assays proceeded as described above and were repeated five times.

In Vivo Pathogenicity

Inbred female BALB/c mice were obtained from Laboratory Animal Services (Adelaide, South Australia). All mice were between 6 and 9 weeks of age at the time of infection and were maintained under specific pathogen free conditions prior to and during experiments. All experiments were performed with the approval of the University of Adelaide Ethics Committee and in accordance with the guidelines of the National Health and Medical Research Council.

Inocula were prepared by growing bacteria to stationary phase in LB broth. Inoculum concentration was confirmed by plating serial 10-fold dilutions. Mice were inoculated with either 103 or 105 CFU bacteria by oral gavage. Following infection, mice were monitored for signs of infection. Scores were given to disease parameters such as coat ruffling, change in behavior, hunching, dehydration, consumption of food, and percent loss of mass. Changes in coat appearance or posture had a scoring range of zero to three; zero indicating that the animal was normal and three that a severe effect was observed. Scores were also given for behavior, evidence of dehydration, feed intake and the presence of tremors. Scores for these clinical parameters ranged from zero to two, zero for normal behavior and two for a severe behavior. If an animal exhibited a clinical score of 5 at any stage during the experiment it was humanely euthanized. Clinical scoring was performed by a single individual for consistency and objectivity.

Collection of Fecal Samples and Culture Methods

Fecal pellets were collected from each mouse at day 3, 6, 9, 12, 15, and 18 days post infection (p.i.) and processed for Salmonella isolation by culture method described previously (Gole et al., 2014a). Briefly, 100 mg of fecal material was incubated in 1 ml of buffered peptone water (1:10) and incubated at 37°C overnight. 100 μl of this sample was added to Rappaport-Vassiliadis Soya peptone broth (RVS, Oxoid, Australia) and incubated at 42°C overnight. A 100 μl of the RVS culture was then spread onto XLD agar plates (Oxoid, Australia) and a Brilliance Salmonella agar (Oxoid, Australia).

DNA extraction from fecal samples

Fecal samples were collected from 0 to 18 days p.i. from each treatment group. DNA from feces (0.2 g) was extracted using a QIAamp DNA stool mini kit (Qiagen, Australia) according to manufacturer instructions. Extracted DNA was quantified using a Nanodrop ND1000 (ThermoScientific, Australia) and stored at −80°C until used for real-time (RT)-PCR. Five nanograms of fecal DNA were used for the RT-PCR reaction.

Q-PCR (Real Time PCR)

Salmonella shedding in fecal material was quantified using real time PCR (RT-PCR). RT-PCR was performed using a Rotor Gene 3000 real time PCR machine (Qiagen, Australia) and a TaqMan®Salmonella enterica detection Kit (Applied Biosystems, Australia). Each reaction contained 9 μl of qPCR supermix and 6 μl of DNA template (5 ng) in a total reaction volume of 15 μl. The cycling parameters were 95°C for 10 min, then 40 cycles at 95°C for 15 s followed by 60°C for 60 s. All real time PCR runs included a negative and positive control. The data was analyzed by Two-Way analysis of variance (ANOVA).

A standard curve was generated by preparing a serial dilution of the Salmonella Typhimurium DT135 strain used in this study. Bacteria were resuscitated on nutrient agar overnight at 37°C. The individual isolated colonies were then suspended in 2 mL of phosphate buffered saline (PBS) and matched with a 0.5 McFarland standard (bioMerieux Australia). Serial dilutions were performed to achieve 108 CFU/ml. The CFUs were confirmed by spreading serial dilutions on XLD agar plates. In order to determine, the limit of detection of Q-PCR, fecal samples were spiked with various concentrations (108–100 CFU/mL) of Salmonella Typhimurium DT135. qPCR was performed on serial dilutions (108–100) of genomic DNA and a proportionality relationship was produced by plotting the Ct value against the logarithm CFU number. Salmonella copies were calculated using a standard curve prepared by serial 10 fold dilution of a cultured Salmonella spp. During each reaction, internal standards comprised of serial dilutions of genomic DNA of Salmonella Typhimurium DT135 were included. A negative control was also included in each reaction.

Whole Genome Sequencing

Whole genome sequencing was performed on the 10 Salmonella strains selected for this study. Salmonella were cultured on nutrient agar. A single colony was selected and grown overnight at 37°C with shaking (100 rpm) in brain heart infusion broth. Bacterial DNA was purified from the overnight culture using the Promega Wizard Genomic DNA purification Kit (Promega, USA). Quality of DNA was assessed by Nanodrop ND1000 (ThermoScientific, Australia) and agarose gel. Sequencing was performed by the Australian Genomic Research Facility using the Illumina MiSeq platform.

Sequence analysis

Sequences were analyzed using CLC Genomics Workbench (version 7.0.4). Sequences were trimmed and de novo assembly was performed to obtain large contigs no less than 1000 base pairs. Single gene analysis of five Salmonella specific pathogenicity islands (SPI) 1, 2, 3, 4, and 5 was performed using Salmonella Typhimurium LT2 (NC_003197) as a reference strain. Sequences have been uploaded to NCBI and accession numbers are listed in Table 2.

Table 2. NCBI accession numbers for pathogenicity island sequences.

Statistical Analysis

Kruskal-Wallis ANOVA with post hoc analysis utilizing Dunn’s multiple comparisons test was used to determine statistical significance of invasive capacity of Salmonella strains in Caco2 cells. All statistical analyses were performed using GraphPad Prism version 6.0. P-values less than 0.05 were considered statistically significant.

Results

Comparative Invasive Capacity of Multiple

Salmonella Strains

The invasive capacity of the 10 NTS strains was first investigated using bacteria suspended in normal saline. Data are presented as mean percent recovery of individual Salmonella strains in relation to the original inoculum (Figure 1A). Overall cell invasion was limited for all strains, with mean percent recoveries ranging from 0.03 to 0.75. There were, however, significant differences detected amongst Salmonella strains tested. S. Typhimurium DT 170=108 exhibited the greatest invasive capacity (mean percent recovery, 0.75 ± 0.16) and was found to be significantly more invasive compared to all other strains tested (p < 0.05) (Figure 1A). S. Typhimurium DT DT44, DT135, DT193, and S. Virchow exhibited moderate invasion while S. Bredeney, S. Orion and S. Senftenberg all exhibited low invasive capacity. S. Adelaide and S. Cerro exhibited negligible invasion (Figure 1A).

Figure 1. The invasive potential of Salmonella strains was characterized using polarized Caco2 cells. Data are represented as percent invasion. (A) Invasion potential of bacteria suspended in normal saline. S. Typhimurium DT170=108 suspended in normal saline was significantly more invasive than all other strains (p < 0.05). (B) Growth to stationary phage in LB substantially increased the invasive capacity of most strains. A significant effect of serovar was detected for bacteria cultured in LB (p < 0.01). S. Typhimurium DT44, DT135, DT170=108 and S. Virchow exhibited the highest mean percent invasion.

In vitro growth media substantially affects the expression of genes required for in vitro invasion (Mills and Finlay, 1994; Ibarra et al., 2010). Therefore, the gentamicin invasion protection experiment was repeated with bacteria cultured to stationary phase in Luria Bertani (LB) broth. A significant increase in invasive capacity (p < 0.001) was observed for all strains grown in LB broth as compared with suspensions in physiological saline. Increase in invasive capacity ranged from 6.9 to 375 fold, with S. Cerro exhibiting the greatest change. The S. Typhimurium definitive types DT44, DT135, DT170=108, DT193 as well as S. Virchow exhibited the greatest overall invasive potential. DT135 and S. Virchow had the highest mean percent recoveries, 6.6 ± 1.5 and 5.9 ± 1.8 respectively, and were significantly more invasive than either S. Adelaide or S. Bredeney (p < 0.05) (Figure 1B). Interestingly, despite growth in LB both S. Adelaide and S. Bredeney retained low invasion capacities (Figure 1B).

Comparative

In Vivo Pathogenicity of Salmonella Strains

To further investigate the pathogenicity of the selected Salmonella strains, groups of seven BALB/c mice were inoculated with either 103 or 105 colony forming unit (CFU) of individual strains. These doses were selected as they represent a range of bacterial contamination detected on the surface of an egg shell (Gole et al., 2014b).

Morbidity of mice was characterized using a clinical scoring system. Data obtained for the morbidity scores are presented as mean clinical score ranging from zero to five taken for an entire experimental group. Results are summarized in Figure 2. Animals inoculated with 103 or 105 CFU of S. Adelaide, S. Bredeney, S. Cerro, S. Orion, and S. Senftenberg did not exhibit any clinical signs of infection over the course of the entire 21 day experiment. As such, their clinical scores were zero and not included in the morbidity analysis. Mice inoculated with both doses of S. Virchow exhibited very mild clinical symptoms during the first 72 h of the experiment. Clinical scores for animals infected with S. Virchow did not rise above one during this period (data not shown).

Figure 2. Morbidity in mice infected with Salmonella Typhimurium definitive types. Clinical signs of infection appeared from day 2 with 103 CFU of individual serovars (A). Peak morbidity in low dose animals ranged between day 6 and 10 post infection. Morbidity was observed in animals inoculated with 105 CFU from day and peaked between days 5 and 9 post infection (p.i.) (B). DT44 (red), DT135 (yellow), DT170=108 (green) and DT193 (blue).

Mice inoculated with either 103 or 105 CFU of S. Typhimurium DT44, DT135, DT170=108, or DT193 exhibited the greatest overall degree of morbidity (Figure 2). Mice inoculated with 103 CFU of the S. Typhimurium strains exhibited a range of clinical symptoms over the course of the experiment. In the low dose group, DT44 caused the least morbidity, very mild clinical symptoms from day 3 to 5 were observed but by day 6 post-infection (p.i.) all animals had recovered. The greatest morbidity in the 103 CFU group was observed in both DT135 and DT193. Hunching behavior, coat ruffling and weight loss were observed from day 2 p.i. for animals infected with 103 CFU of DT135. Morbidity for this group increased rapidly from day 3 to day 5 and by day 7 p.i. all animals were euthanized. Similarly, animals infected with DT193 exhibited morbidity from day 5 p.i. which increased till day 7 p.i.; by day 9 p.i., all animals were euthanized. Animals infected with DT170=108 exhibited clinical symptoms from day 4 p.i. Morbidity in this group peaked between days 7 and 9 p.i.. Only one animal remained in this group till the end of experiment at day 21 p.i..

As in the low dose group, clinical symptoms were first observed at day 3 p.i. in mice inoculated with 105 CFU of the S. Typhimurium strains. The degree of morbidity however was substantially increased in mice receiving the high dose of bacteria. Four mice in the DT44 group exhibited severe morbidity between days 6–9 but the other three animals did not display any clinical signs of infection over the course of the experiment. Animals inoculated with 105 CFU of both DT135 and DT193 exhibited the greatest amount of morbidity over the course of the experiment. For both of these strains, clinical symptoms were observed from day 3 and morbidity scores peaked at day 7 and 8 respectively. The majority of animals in the DT170=108 group exhibited minor morbidity over the course of the experiment.

Survivability of NTS infection was also assessed (Figure 3). Throughout the experiment, no mortalities were recorded in animals infected with either 103 or 105 CFU of S. Adelaide, S. Bredeney, S. Cerro, S. Orion, S. Senftenberg, or S. Virchow. Survival of mice infected with all S. Typhimurium definitive types had significantly different survival curves (Mantel-Cox log rank test, p < 0.001) than all other strains at both doses. Interestingly, the mortality of DT170=108 at the 103 dose was higher than 105 dose. Significant differences were detected in survivability between S. Typhimurium groups (p < 0.01). At the low dose DT135, DT170=108, and DT193 were significantly more likely to cause mortality than DT44 (p < 0.01). At the 105 dose DT135 was significantly more virulent than all other S. Typhimurium definitive types included in this study (p < 0.01) (Figure 3).

Figure 3. Survival curves for mice inoculated with either 103 or 105 CFU of S. Typhimurium definitive types. S. Typhimurium definitive types DT44 (red), DT135 (yellow), DT170=08 (green) and DT193 (blue) exhibited significantly greater mortality at both the 103(A) and 105(B) dose, than all other strains (black line) tested in this study. Mice inoculated with 103 CFU of either DT135, DT170=108 or DT193 all had significantly greater mortality than DT44 (p < 0.01). The greatest mortality was observed for mice inoculated with 105 CFU of DT135.

Fecal Shedding of

Salmonella Strains

The shedding of Salmonella in feces is an important mechanism of transmission of the bacteria from host to host. In this study, Salmonella shedding was monitored both by culture isolation as well as by a qPCR method. The TaqMan Salmonella enterica PCR assay does not enable the quantification of positive fecal samples. Therefore, a standard curve generated by preparing a serial 10-fold dilution of a known concentration of Salmonella spp. (108–100 CFU) was used. The standard curve produced a slope of −3.2, a y intercept of 39.4 and R2 of 0.91. A cut-off Ct of 33.7 was used to exclude detection of false positives. A Ct of 33.7 corresponded to 50 CFU of Salmonella. Amplification was not recorded in the negative control (LB) samples or any of the treatment groups at day 0 of infection. Shedding of Salmonella into the feces was consistently observed by both culture and qPCR methods during the course of the experiment for all strains at both doses (Table 3). For S. Typhimurium DT135, S. Orion, S. Virchow and S. Bredeney treatment groups, a significant difference in Salmonella shedding between the dose and days p.i. was detected (p < 0.0001). In addition, significant interaction between dose and days p.i. was observed (p < 0.0001). A significant effect between serovar and day p.i. was observed for S. Typhimurium DT44, DT193 (p < 0.0005), DT108=170, S. Adelaide, S. Senftenberg and S. Cerro (p < 0.0001). A significant interaction between day p.i. and dose was observed for all groups except for DT44, DT 193, and S. Cerro. The highest amount of Salmonella detected in feces was detected in mice inoculated with 103 CFU S. Senftenberg with a mean of 1.3 × 108 ± 1.3 × 108 CFU/g fecal material.

Table 3. Real time qPCR and culture detection of Salmonella in fecal samples collected from BALB/c mice inoculated with 103 and 103CFU.

Comparative Genomics of SPIs of Multiple Non-Typhoidal

Salmonella Strains

To determine whether genotypic variability contributed to pathogenic verses non-pathogenic phenotypes observed in this study, whole genome sequencing was performed on each of the 10 Salmonella strains. Single gene analysis of specific pathogenicity islands (SPI) 1, 2, 3, 4, and 5 was performed. Amino acid sequences for each gene were generated in silico and compared with the corresponding sequence of S. Typhimurium LT2.

SPI-1 contains 39 genes involved in the formation of a Type III transmembrane secretion system (T3SS) as well as multiple effector proteins. The greatest sequence variation was observed in avrA, srpB, orgC, prgI, sptP, and sipA for S. Adelaide, S. Bredeney, S. Cerro, S. Orion, and S. Senftenberg (Table 4). S. Adelaide and S. Orion both lacked the avrA gene. For S. Bredeney, a single base pair deletion shifts the open reading frame causing a premature stop codon at 292. Base pair substitution in the avrA coding sequence of S. Virchow causes a premature stop at amino acid 251. S. Typhimurium DT44 possesses a triple base pair deletion in avrA that truncates the protein by one amino acid but does not affect the open reading frame.

Table 4. Comparative analysis of SPI-1 sequences from 10 NTS strains.

Amino acid variability was also observed for orgB, orgC and prgI in all strains except the four Typhimurium definitive types. An insertion of 10 base pairs at the 3′ end of the orgB sequence was detected in S. Adelaide, S. Bredeney, S. Cerro, S. Orion, and S. Senftenberg. This leads to a premature stop at amino acid 224 disrupting the open reading frame. Considerable amino acid variability was also observed for prgI in S. Adelaide, S. Cerro and S. Orion. S. Bredeney had a nine base pair deletion in sipD causing a three amino acid truncation of the protein. Minor amino acid variability was observed for the sptP sequence of S. Adelaide, S. Bredeney, and S. Senftenberg. A single base pair substitution was detected in the sptP sequence of S. Virchow creating a premature stop at position 128 of a 544 amino acid protein.

A second T3SS is encoded by SPI-2 has 31 genes in multiple operons. Sequence variability amongst the 10 Salmonella strains examined in this study was found in the ssa and sse operons in S. Adelaide, S. Bredeney, S. Cerro, S. Orion, S. Senftenberg, and S. Virchow (Table 5).

Table 5. Sequence variability within SPI-2.

Analysis of SPI-3 genes revealed that, 69.2% of the strains analyzed in this study, had greater than 98% amino acid homology with the LT2 strain. Sequence variation was primarily observed in the region between STM3752 and rhuM. S. Adelaide, S. Cerro and S. Senftenberg were found to lack multiple genes within the STM372-rhuM region. STM3754 was absent in S. Adelaide, S. Cerro, and S. Senftenberg (Table 6). S. Senftenberg lacked the gene sugR. S. Virchow and S. Bredeney lacked the entire region (Table 6).

Table 6. Amino acid variability of SPI-3, -4 and -5.

SPI-4 contains a single operon, siiABCDE (McClelland et al., 2001). In general, all 10 Salmonella strains were highly conserved for SPI-4; 70% of the genes exhibited greater than 98% homology to LT2 (Table 6). The highest amino acid variability was observed in siiA, B and E for S. Bredeney, S. Cerro, S. Orion, and S. Senftenberg.

The pathogenicity island, SPI-5, encodes six genes involved in enteropathogenesis (Wood et al., 1998). Genes within SPI-5 code for effector proteins translocated by the T3SSs of both SPI-1 and SPI-2 (Knodler et al., 2002). Sequence variability was observed primarily in the pipABC operon (Table 6). A large deletion in S. Bredeney pipA alters the open reading frame. Considerable amino acid variation was also observed in the pipA sequence of S. Adelaide, S. Cerro, and S. Orion. Minor amino acid variability was also observed for pipC and sopB in S. Adelaide, S. Cerro, S. Orion, and S. Senftenberg.

Discussion

Surface contamination of an egg shell with Salmonella is an important contributing factor to outbreaks of human salmonellosis. Higher loads of bacteria on the egg shell can contribute to contaminated hands in a kitchen environment as well as contaminated utensils (Humphrey et al., 1994). Viable bacteria can be isolated from an egg shell up to 21 days post-lay depending on storage conditions (Messens et al., 2006). Furthermore, S. Enteriditis, S. Typhimurium, and S. Heidelberg present in chicken feces are able penetrate into the interior of eggs and subsequently multiply in storage (reviewed in Chen and Jiang, 2014). The in vitro and in vivo experiments described here were designed to determine whether Salmonella strains represent an equal risk to public health.

The in vitro invasion results highlight clear differences in the invasive capacity of the 10 selected Salmonella strains. These invasion data also indicate that some Salmonella strains may have constitutive expression of genes enabling them to invade even at low levels under non-nutritive conditions, ultimately providing them with a competitive advantage. Pathogenic bacteria are able to sense their environment and respond through the up regulation of genes that facilitate their within-host survival (Ellermeier and Slauch, 2007). Furthermore, it has been reported that in vitro growth media stimulates the expression of genes required for Salmonella invasion in cultured epithelial cells (Mills and Finlay, 1994; Ibarra et al., 2010). As such, it is not surprising that substantial increases in invasion were observed amongst the selected strains post-enrichment.

Current in vivo pathogenicity data are largely limited to Salmonella serovars that most commonly cause disease. It has recently been shown that not all NTS serovars possess the same in vivo virulence (Swearingen et al., 2012). The BALB/c mouse strain was selected for this study as it has been previously been shown to be susceptible to infection with Salmonella (Plant and Glynn, 1976). The selection of a susceptible mouse strain was warranted as human cases of salmonellosis occur most commonly amongst the young, elderly or immunocompromised (Jones et al., 2008b). S. Adelaide, S. Bredeney, S. Cerro, S. Orion, and S. Senftenberg exhibited limited or moderate invasive capacity during our in vitro studies hence their lack of in vivo virulence was not unexpected. S. Virchow is consistently associated with cases of human salmonellosis yet the strain used in this study lacked the ability to cause disease in mice. This result was somewhat surprising as the S. Virchow strain was among the most invasive in our in vitro invasion experiments. This discrepancy between cell invasiveness and in vivo virulence has also been described for strains of S. Abortusovis and S. Montevideo (Swearingen et al., 2012) as well as S. Enteriditis (Shah et al., 2011) but the mechanisms responsible for this outcome were not investigated.

The pathogenicity of S. Typhimurium has been studied extensively over the past decade as a model for systemic typhoid fever. As a consequence, the virulence mechanisms that S. Typhimurium employs during in vivo infection have been well characterized (reviewed in Fabrega and Vila, 2013). In this study, we found that mice inoculated with either 103 or 105 CFU of S. Typhimurium DT44, DT135, DT170=108, or DT193 exhibited the greatest overall degree of morbidity, yet there were significant differences between their disease capacity. These findings were consistent with our in-vitro cell invasive data. A multitude of factors including fimbriae, virulence plasmids and the normal expression of virulence genes likely contribute to this variation in virulence and has recently been demonstrated for S. Enteriditis (Shah et al., 2011). The pathogenicity of S. Typhimurium has been studied extensively over the past decade as a model for systemic typhoid fever. As a consequence, the virulence mechanisms that S. Typhimurium employs during in vivo infection have been well characterized (reviewed in Fabrega and Vila, 2013). There is, however, limited evidence characterizing variation in in vivo virulence amongst different Australian S. Typhimurium definitive types. Interestingly, mice infected with 103 CFU of DT170=108 strain exhibited greater mortality than those inoculated with 105 CFU. It has been shown that Salmonella can be more pathogenic at lower doses by evading CD4+ T cell activation (Srinivasan et al., 2004). In this study, increased mortality was, however, observed only for DT170=108. Thus, it could be hypothesized that such a dose dependent impact on T cell responsiveness could vary between serotypes.

Host to host transmission of Salmonella occurs predominantly via the oral-fecal route. As a consequence, the duration of shedding post-infection as well as the total bacterial load within feces of an infected individual can contribute to the transmission of disease. It is interesting to note that despite a lack of evidence of disease, fecal shedding was also observed over the duration of this study for S. Bredeney, S. Cerro, S. Orion, S. Senftenberg, and S. Virchow. S. Bredeney and S. Cerro were detected using both Salmonella detection methods at both doses over the course of the entire experiment. These results are consistent with other virulence studies of NTS serovars demonstrating that avirulent strains are capable of establishing persistent intestinal infection that results in the consistent shedding of bacteria (Swearingen et al., 2012). Mice infected with 103 CFU of S. Adelaide, however, stopped shedding bacteria after day 9 p.i.. Some disparity between culture and qPCR results was observed, in particular for mice infected with DT44. It should be noted that PCR methods detect both viable and non-viable bacteria within a sample. A double enrichment protocol was used to culture Salmonella from the mouse fecal samples. Thus, it is likely that the bacteria detected by qPCR in the 103 DT44 treatment groups were non-viable.

The genotypic variability observed within the five Salmonella pathogenicity islands for the 10 strains included in this study was largely limited to S. Adelaide, S. Bredeney, S. Cerro, S. Orion, S. Senftenberg and to a lesser extent S. Virchow. SPIs are highly conserved across Salmonella enterica yet it is variations within these genes that have the potential to affect virulence. Genes within these pathogenicity islands enable Salmonella to invade host cells, replicate and evade the immune response (reviewed in Fabrega and Vila, 2013). Genomic variability within these pathogenicity islands may contribute to the wide range of virulence observed for members of S. enterica. Sequence analysis of SPI1, 2, 3, 4, and 5 was performed to determine if genotypic variability contributed to the observed range of pathogenicity. Amino acid variation was largely limited to S. Adelaide, S. Bredeney, S. Cerro, S. Orion, S. Senftenberg and to a lesser extent S. Virchow.

In SPI-1, amino acid variation was observed in avrA, srpB, orgC, prgI, sptP, and sipA. AvrA encodes 33 kDa protein that is translocated into host intestinal epithelial cells during infection (Hardt and Galán, 1997) and is important for modulating the host immune response (Wu et al., 2012) as well as intracellular survival of the bacterium (Jones et al., 2008a; Wu et al., 2012). In mice, infection with an avrA deletion mutant has been shown to increase disease (Jones et al., 2008a; Wu et al., 2012). No evidence of aggravated disease was observed for S. Adelaide or S. Orion.

The prg, org, inv and spa operons encode the needle complex of the T3SS while the sic and sip operons encode effector proteins (Fabrega and Vila, 2013). OrgB and orgA are part of the effector sorting platform of the SPI-1 T3SS (Kawamoto et al., 2013). It is unclear whether the premature stop observed for S. Adelaide, S. Bredeney, S. Cerro, S. Orion, and S. Senftenberg would have any functional effect. The needle complex is formed by 120 copies of prgI which interacts with sipD to form the pore structure of the T3SS that embeds in host cell membranes (Rathinavelan et al., 2014). The amino acid sequence variability observed for prgI may affect how the needle complex in these strains is formed. The formation of the pore structure may also be affected if sipD is not able to interact normally with prgI.

sptP encodes a protein tyrosine phosphatase that is translocated across the SPI-1 T3SS and is involved in the initial disruption of the actin cytoskeleton (Fu and Galán, 1998) as well as the modulation of the host immune response (Choi et al., 2013). Minor amino acid variability was observed for several strains but without further functional studies, it is unclear whether the variability observed in sptP would have functional effects on the protein.

A second T3SS is encoded by SPI-2 (T3SS-2) is involved in the translocation of effector proteins across the Salmonella-containing vacuole (Figueira and Holden, 2012). The greatest sequence variability was observed in the ssa and sse operons. Genes within the ssa operon encode a portion of the T3SS-2 that forms within the membrane of the bacteria. Proteins encoded by ssaG and ssaI form part of the needle complex that extends beyond the Salmonella outer membrane (Kuhle and Hensel, 2004). Mutant Salmonella lacking either ssaG or ssaI have previously been shown to be unable to replicate intracellularly or translocate effector proteins through T3SS-2 (Chakravortty et al., 2005). While the strains included in this study possessed a complete open reading frame, it is possible that the amino acid variability observed could prevent normal formation of the needle complex. The sseB, C and D proteins of SPI-2 are secreted onto the surface of the bacterium and are required for the translocation of effectors (Nikolaus et al., 2001; Ruiz-Albert et al., 2003; Fabrega and Vila, 2013). SseC and sseD form the pore structure across the vacuole membrane (Kuhle and Hensel, 2004). Mutations in sseC and sseD have a significant impact on the virulence in mice (Klein and Jones, 2001). Serovars that have mutations in these genes were severely attenuated and unable to secrete effectors through the T3SS (reviewed in Ruiz-Albert et al., 2003). SseB forms part of the translocon through the vacuole membrane. Mutants of sseB were able to replicate within host cells but unable to escape, thus limiting their dispersal (Grant et al., 2012).

SPI-3 has 10 open reading frames that encode virulence determinants with highly diverse functions. Sequence variation was largely observed in the region between STM3752 and rhuM. Sequence diversity has also been demonstrated for this region across S. enterica by several groups (reviewed in Fabrega and Vila, 2013).

SPI-4 is a 27 KB region within the Salmonella genome (Wong et al., 1998). During infection, SPI-4 acts in consort with SPI-1 to initiate invasion in to host epithelial cells (Gerlach et al., 2008). Deletion of SPI-4 attenuated the virulence of both S. Typhimurium and S. Enteriditis in mice (Kiss et al., 2007). siiA, B and E exhibited the greatest variability in amino acid sequence for S. Bredeney, S. Cerro, S. Orion, and S. Senftenberg. The gene siiE encodes a giant, non-fimbrial adhesion protein that enables the bacterium to adhere the apical surface of a host cell a process that is required for SPI-1 T3SS mediated invasion (Gerlach et al., 2007, 2008; Main-Hester et al., 2008). It has recently been shown that siiA and siiB form a proton channel within the inner membrane of the bacteria and that they are regulatory proteins for siiE (Wille et al., 2014). The amino acid variability observed in the siiA and siiB sequences of the Salmonella strains included in this study may have an effect on the regulation of siiE in these strains.

SPI-5 encodes six genes that play a role in the enteropathogenesis of Salmonella spp. (Wood et al., 1998). The highest variability was observed in the pipABC operon. pipA is an effector protein that is translocated across the Salmonella vacuole by the SPI-2 T3SS and is important for the development of systemic disease in mice (Knodler et al., 2002). The pipC protein functions as a chaperone and is also involved in the stabilization of SopB (Darwin et al., 2001).

At this stage, the relationship between genotypic variability and reduced cellular invasion is not clear and requires further functional study. It is also important to note that there are additional pathogenicity islands encoded within the Salmonella genome that are not included in this study. Genes from these SPIs may also contribute to functional variation. Furthermore, we have not discussed the potential contribution of the variation in fimbriae and flagella genes.

The experiments described here were targeted at identifying whether different Salmonella strains represent an equal risk to public health. We have shown that there are clear differences in the overall virulence capacity of the 10 selected strains and that the mechanisms driving this variation are multifactorial and appear to be serovar dependent. A caveat is, however, that only a single strain from each serovar was included in this study. It is highly probable that the differences observed within the SPIs are specific for the strains selected for this study. Within serovar analyses are necessary to define the overall pathogenic potential of that serovar.

Author Contributions

Andrea R. McWhorter and Kapil K. Chousalkar conceived and designed the experiments. Andrea R. McWhorter performed all invasion assays and sequence analysis. The in vivo infection trial was performed by both Andrea R. McWhorter and Kapil K. Chousalkar. Quantitative-PCR was performed by Kapil K. Chousalkar. Both authors contributed equally to statistical analysis and manuscript preparation.

Conflict of Interest Statement

Neither author has a conflict of interest.

Acknowledgments

This work was supported by the Australian Egg Corporation Limited. The authors would like to thank Vivek Pande, Geraldine Laven-Law and Nikita Nevrekar for their technical support during this project. We would also like to acknowledge Dr. Pat Blackall for critically proofreading this manuscript and providing constructive comments.

References

Carrasco, E., Morales-Rueda, A., and Garcia-Gimeno, R. M. (2012). Cross-contamination and recontamination by Salmonella in foods: a review. Food Res. Int. 45, 545–556. doi: 10.1016/j.foodres.2011.11.004

CrossRef Full Text | Google Scholar

Chakravortty, D., Rohde, M., Jäger, L., Deiwick, J., and Hensel, M. (2005). Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2. EMBO J. 24, 2043–2052. doi: 10.1038/sj.emboj.7600676

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Chen, Z., and Jiang, X. (2014). Microbiological safety of chicken litter or chicken litter-based organic fertilizers: a review. Agriculture 4, 1–29. doi: 10.3390/agriculture4010001

CrossRef Full Text | Google Scholar

Choi, H. W., Brooking-Dixon, R., Neupane, S., Lee, C.-J., Miao, E. A., Staats, H. F., et al. (2013). Salmonella Typhimurium impedes innate immunity with a mast-cell suppressing protein tyrosine phosphatase, SptP. Immunity 39, 1108–1120. doi: 10.1016/j.immuni.2013.11.009

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Foley, S. L., Nayak, R., Hanning, I. B., Johnson, T. J., Han, J., and Ricke, S. C. (2011). Population dynamics of Salmonella Enterica serotypes in commercial egg and poultry production. Appl. Environ. Microbiol. 77, 4273–4279. doi: 10.1128/AEM.00598-11

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Gast, R. K., Guraya, R., Jones, D. R., and Anderson, K. E. (2013). Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages. Poult. Sci. 92, 468–473. doi: 10.3382/ps.2012-02811

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Gerlach, R. G., Cláudio, N., Rohde, M., Jäckel, D., Wagner, C., and Hensel, M. (2008). Cooperation of Salmonella pathogenicity islands 1 and 4 is required to breach epithelial barriers. Cell. Microbiol. 10, 2364–2376. doi: 10.1111/j.1462-5822.2008.01218.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Gerlach, R. G., Jäckel, D., Stecher, B., Wagner, C., Lupas, A., Hardt, W. D., et al. (2007). Salmonella Pathogenicity Island 4 encodes a giant non−fimbrial adhesin and the cognate type 1 secretion system. Cell. Microbiol. 9, 1834–1850. doi: 10.1111/j.1462-5822.2007.00919.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Gole, V. C., Caraguel, C. G., Sexton, M., Fowler, C., and Chousalkar, K. K. (2014a). Shedding of Salmonella in single age caged commercial layer flock at an early stage of lay. Int. J. Food Microbiol. 189, 61–66. doi: 10.1016/j.ijfoodmicro.2014.07.030

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Gole, V. C., Roberts, J. R., Sexton, M., May, D., Kiermeier, A., and Chousalkar, K. K. (2014b). Effect of egg washing and correlation between cuticle and egg penetration by various Salmonella strains. Int. J. Food Microbiol. 182, 18–25. doi: 10.1016/j.ijfoodmicro.2014.04.030

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Grant, A. J., Morgan, F. J., McKinley, T. J., Foster, G. L., Maskell, D. J., and Mastroeni, P. (2012). Attenuated Salmonella Typhimurium lacking the pathogenicity island-2 type 3 secretion system grow to high bacterial numbers inside phagocytes in mice. PLoS Pathog. 8:e1003070. doi: 10.1371/journal.ppat.1003070

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Guibourdenche, M., Roggentin, P., Mikoleit, M., Fields, P. I., Bockemuhl, J., Grimont, P., et al. (2010). Supplement 2003-2007 (No. 47) to the White-Kauffmann-Le Minor scheme. Res. Microbiol. 161, 26–29. doi: 10.1016/j.resmic.2009.10.002

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Hendriksen, R. S., Vieira, A. R., Karlsmose, S., Lo Fo Wong, D. M. A., Jensen, A. B., Wegener, H. C., et al. (2011). Global monitoring of Salmonella serovar distribution from the world health organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathog. Dis. 8, 887–900. doi: 10.1089/fpd.2010.0787

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Howard, Z. R., O’bryan, C. A., Crandall, P. G., and Ricke, S. C. (2012). Salmonella Enteritidis in shell eggs: current issues and prospects for control. Food Res. Int. 45, 755–764. doi: 10.1016/j.foodres.2011.04.030

CrossRef Full Text | Google Scholar

Ibarra, J. A., Knodler, L. A., Sturdevant, D. E., Virtaneva, K., Carmody, A. B., Fischer, E. R., et al. (2010). Induction of Salmonella pathogenicity island 1 under different growth conditions can affect Salmonella-host cell interactions in vitro. Microbiology 156, 1120–1133. doi: 10.1099/mic.0.032896-0

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Jackson, B. R., Griffin, P. M., Cole, D., Walsh, K. A., and Chai, S. J. (2013). Outbreak-associated Salmonella enterica serotypes and food commodities, United States, 1998-2008. Emerg. Infect. Dis. 19, 1239–1244. doi: 10.3201/eid1908.121511

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Jacobsen, A., Hendriksen, R. S., Aaresturp, F. M., Ussery, D. W., and Friis, C. (2011). The Salmonella enterica pan-genome. Microb. Ecol. 62, 487–504. doi: 10.1007/s00248-011-9880-1

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Jones, R. M., Wu, H., Wentworth, C., Luo, L., Collier-Hyams, L., and Neish, A. S. (2008a). Salmonella AvrA coordinates suppression of host immune and apoptotic defenses via JNK pathway blockade. Cell Host Microbe 3, 233–244. doi: 10.1016/j.chom.2008.02.016

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Jones, T. F., Ingram, L. A., Cieslak, P. R., Vugia, D. J., Tobin-D’angelo, M., Hurd, S., et al. (2008b). Salmonellosis outcomes differ substantially by serotype. J. Infect. Dis. 198, 109–114. doi: 10.1086/588823

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Kawamoto, A., Morimoto, Y. V., Miyata, T., Minamino, T., Hughes, K. T., Kato, T., et al. (2013). Common and distinct structural features of Salmonella injectisome and flagellar basal body. Sci. Rep. 3:3369. doi: 10.1038/srep03369

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Klein, J. R., and Jones, B. D. (2001). Salmonella pathogenicity island 2-encoded proteins SseC and SseD are essential for virulence and are substrates of the type III secretion system. Infect. Immun. 69, 737–743. doi: 10.1128/IAI.69.2.737-743.2001

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Knodler, L. A., Celli, J., Hardt, W. D., Vallance, B. A., Yip, C., and Finlay, B. B. (2002). Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems. Mol. Microbiol. 43, 1089–1103. doi: 10.1046/j.1365-2958.2002.02820.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Kuhle, V., and Hensel, M. (2004). Cellular microbiology of intracellular Salmonella enterica: functions of the type III secretion system encoded by Salmonella pathogenicity island 2. Cell. Mol. Life Sci. 61, 2812–2826. doi: 10.1007/s00018-004-4248-z

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Main-Hester, K. L., Colpitts, K. M., Thomas, G. A., Fang, F. C., and Libby, S. J. (2008). Coordinate regulation of Salmonella pathogenicity island 1 (SPI1) and SPI4 in Salmonella enterica serovar Typhimurium. Infect. Immun. 76, 1024–1035. doi: 10.1128/IAI.01224-07

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

McClelland, M., Sanderson, K. E., Spieth, J., Clifton, S. W., Latreille, P., Courtney, L., et al. (2001). Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413, 852–856. doi: 10.1038/35101614

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Mickael, C. S., Lam, P.-K. S., Berberov, E. M., Allan, B., Potter, A. A., and Köster, W. (2010). Salmonella enterica serovar Enteritidis tatB and tatC mutants are impaired in Caco-2 cell invasion in vitro and show reduced systemic spread in chickens. Infect. Immun. 78, 3493–3505. doi: 10.1128/IAI.00090-10

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Moffat, C. R., and Musto, J. (2013). Salmonella and egg-related outbreaks. Microbiol. Aust. 34, 94–98.

Google Scholar

Nakamura, M., Nagamine, N., Takahashi, T., Suzuki, S., Kijima, M., Tamura, Y., et al. (1994). Horizontal transmission of Salmonella Enteriditis and effect of stress on shedding in laying hens. Avian Dis. 38, 282–288. doi: 10.2307/1591950

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Nikolaus, T., Deiwick, J., Rappl, C., Freeman, J. A., Schröder, W., Miller, S. I., et al. (2001). SseBCD proteins are secreted by the type III secretion system of Salmonella pathogenicity island 2 and function as a translocon. J. Bacteriol. 183, 6036–6045. doi: 10.1128/JB.183.20.6036-6045.2001

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Quinteiro, W. M., Gomes, A. V. S., Pinheiro, M. L., Ribeiro, A., Ferraz-De-Paula, V., Astolfi-Ferreira, C. S., et al. (2012). Heat stress impairs performance and induces intestinal inflammation in broiler chickens infected with Salmonella Enteritidis. Avian Pathol. 41, 421–427. doi: 10.1080/03079457.2012.709315

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Rathinavelan, T., Lara-Tejero, M., Lefebre, M., Chatterjee, S., McShan, A. C., Guo, D.-C., et al. (2014). NMR model of PrgI–SipD interaction and its implications in the needle-tip assembly of the Salmonella type III secretions system. J. Mol. Biol. 426, 2958–2969. doi: 10.1016/j.jmb.2014.06.009

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

South Australian Salmonella Reference Laboratory. (2010). Annual Complication of Quarterly Reports. Adelaide, SA: Institute of Medical and Veterinary Sciences.

South Australian Salmonella Reference Laboratory. (2011). Annual Complication of Quarterly Reports. Adelaide, SA: Institute of Medical and Veterinary Sciences.

South Australian Salmonella Reference Laboratory. (2012). Annual Complication of Quarterly Reports. Adelaide, SA: Institute of Medical and Veterinary Sciences.

South Australian Salmonella Reference Laboratory. (2013). Annual Complication of Quarterly Reports. Adelaide, SA: Institute of Medical and Veterinary Sciences.

Ruiz-Albert, J., Mundy, R., Yu, X. J., Beuzon, C. R., and Holden, D. W. (2003). SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2-encoded type III secretion system. Microbiology 149, 1103–1111. doi: 10.1099/mic.0.26190-0

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Shah, D. H., Zhou, X., Addwebi, T., Davis, M. A., and Call, D. R. (2011). In vitro and in vivo pathogenicity of Salmonella Enteritidis clinical strains isolated from North America. Arch. Microbiol. 193, 811–821. doi: 10.1007/s00203-011-0719-4

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Silva, C. A., Blondel, C. J., Quezada, C. P., Porwollik, S., Andrews-Polymenis, H. L., Toro, C. S., et al. (2012). Infection of mice by Salmonella enterica serovar Enteritidis involves additional genes that are absent in the genome of serovar Typhimurium. Infect. Immun. 80, 839–849. doi: 10.1128/IAI.05497-11

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Sivaramalingam, T., Pearl, D. L., McEwen, S. A., Ojkic, D., and Guerin, M. T. (2013). A temporal study of Salmonella serovars from fluff samples from poultry breeder hatcheries in Ontario between 1998 and 2008. Can. J. Vet. Res. 77, 12–23.

Pubmed Abstract | Pubmed Full Text | Google Scholar

Clinical and Laboratory Standards Institues. (2013). CLSI Performance Standards for Antimicrobial Susceptibility Testing; CLSI M100-S23. Wayne, PA: Clinical and Laboratory Standards.

Suez, J., Porwollik, S., Dagan, A., Marzel, A., Schorr, Y. I., Desai, P. T., et al. (2013). Virulence gene profiling and pathogenicity characterization of non-typhoidal Salmonella accounted for invasive disease in humans. PLoS ONE 8:e58449. doi: 10.1371/journal.pone.0058449

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Thomson, N. R., Clayton, D. J., Windhorst, D., Vernikos, G., Davidson, S., Churcher, C., et al. (2008). Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways. Genome Res. 18, 1624–1637. doi: 10.1101/gr.077404.108

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Threlfall, E. J., Wain, J., Peters, T., Lane, C., De Pinna, E., Little, C. L., et al. (2014). Egg-borne infections of humans with Salmonella: not only an S. enteritidis problem. Worlds Poult. Sci. J. 70, 15–26. doi: 10.1017/S0043933914000026

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Wille, T., Wagner, C., Mittelstadt, W., Blank, K., Sommer, E., Malengo, G., et al. (2014). SiiA and SiiB are novel type I secretion system subunits controlling SPI4-mediated adhesion of Salmonella enterica. Cell. Microbiol. 16, 161–178. doi: 10.1111/cmi.12222

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Wong, K.-K., McClelland, M., Stillwell, L. C., Sisk, E. C., Thurston, S. J., and Saffer, J. D. (1998). Identification and sequence analysis of a 27-kilobase chromosomal fragment containing a Salmonella pathogenicity island located at 92 minutes on the chromosome map of Salmonella enterica serovar Typhimurium LT2. Infect. Immun. 66, 3365–3371.

Pubmed Abstract | Pubmed Full Text | Google Scholar

Wood, M. W., Jones, M. A., Watson, P. R., Hedges, S., Wallis, T. S., and Galyov, E. E. (1998). Identification of a pathogenicity island required for Salmonella enteropathogenicity. Mol. Microbiol. 29, 883–891. doi: 10.1046/j.1365-2958.1998.00984.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Ozfoodnet Working Group. (2012). Monitoring the incidence and causes of diseases potentially transmitted by food in Australia: annual report of the ozfoodnet network. Commun. Dis. Intell. Q. Rep. 36, E13–E241.

Pubmed Abstract | Pubmed Full Text | Google Scholar

Researchers Sequence Genome of Drug-Resistant Salmonella Enteritidis Strain That Can Sicken Poultry

Researchers from the NC State College of Veterinary Medicine have sequenced the genome of a virulent Salmonella Enteritidis strain that sickened two poultry flocks in consecutive years and found that it was both antibiotic-resistant and could potentially infect humans.

Characterizing the strain, designated SE_TAU19, will aid public health agencies in tracing outbreaks and preventing exposures.

There are two species of Salmonella, and one of these, Salmonella enterica, is implicated in human disease. S. enterica contains over 2,500 serovars, or groups of bacteria, many of which can cause disease in humans.

Salmonella serovar Enteritidis (SE) is most frequently associated with poultry and is the leading cause of human illness globally.

Most human Salmonella infections are foodborne in origin, and many animals – such as chickens – can harbor the pathogen without becoming sick themselves. The ability of SE_TAU19 to cause clinical disease in poultry interested Grayson Walker, a combined DVM and Ph.D. student in Luke Borst’s laboratory at the CVM and first author of a paper describing the research.

Dual DVM/Ph.D. student Grayson Walker. Photo by John Joyner/NC State Veterinary Medicine

“We usually think of Salmonella as harbored by chickens without harming them; however, this strain was virulent and actually made them sick,” Walker says. “We also know that Salmonella likes to stick around. This strain killed broiler chickens throughout the growing period and recurred one year later in a different flock. So, we decided to sequence the genome and see which resistance and virulence features made the strain unique.”

The team sequenced the genome of the strain and found that it included seven antimicrobial resistance genes, 120 virulence genes, and a large virulence plasmid. Plasmids are “swappable” genetic elements that can be exchanged between strains to make them more antibiotic-resistant or infectious.

“While we cannot say that it is a ‘new’ strain of Salmonella, we can say that not only is this strain deadly to poultry, antibiotic-resistant and infectious but also that it could infect humans,” Walker says. “The good news is that by sequencing the genome we now have data that could help pinpoint the origin of and contain any future outbreaks.”

The research appears in Frontiers in Veterinary Science and is supported by the United States Department of Agriculture Animal Plant Health Inspection Service’s National Bio and Agro-defense Facility Scientist Training Program. Whole-genome sequencing was completed by the FDA GenomeTrakr program funded under grant 1U18FD00678801 in the lab of co-author Sid Thakur, professor of population health and pathobiology and the director of NC State’s and the College of Veterinary Medicine’s Global Health programs. Borst, associate professor of veterinary anatomic pathology at NC State, is corresponding author.

Tracey Peake/NC State News Services

90,000 Salmonellosis in the poultry industry | biomin.net

Hatchery and control of

Salmonella

Proper management, cleaning and disinfection of the hatchery will all help to limit the incidence and spread of Salmonella . Cross-contamination can occur mainly by mixing positive and negative flocks.

If we take eggs from a positive flock, incubate and hatch them separately, in most cases they will not infect chicks from a negative flock.For typhoid fever Salmonella, chicks hatched from the parent flock will be positive. Thus, a well-managed hatchery can avoid cross-contamination but will not eliminate Salmonella from a positive herd. Probiotics, antimicrobials and ultimately vaccines can be delivered to the hatchery and help fight Salmonella.

Broiler rearing and Salmonella control

For typhoid fever caused by Salmonella, no major interventions can be taken in rearing other than antibiotic treatment.Due to the short lifespan of broilers, Salmonella gallinarum / Salmonella pullorum infestations almost always come from the breeder and not from the field.

In the case of paratyphoid salmonella, the infection can come from the breeder, but it can also occur during the rearing period. Various possible sources of infection are the previous flock, feed delivered, rodents and wildlife, backyard chickens, neighbors, other farm animals, poor cleaning and disinfection, bird disposal, and people such as staff / visitors (Vatche, 2011).

Given that the ports of entry are diverse, it is necessary to keep an eye on them to understand where the main sources are. In addition, short downtime (less than 2 weeks) and increased poultry density have a large impact on the presence and maintenance of Salmonella paratyphoid. For breeders, a good biosecurity procedure plays an important role in preventing the entry of Salmonella. If fasting before slaughter and the transport time is too long, then Salmonella also favors the reproduction.Antibiotics are not very effective at fighting Salmonella infections during growth. They can reduce the infection, but once they are removed, the infection can come back. Several other products such as probiotics, organic acids, essential oils, herbal extracts, acids, MOS and vaccines can be used to reduce / control Salmonella, but should be part of a holistic biosecurity program. Due to complex epidemiology, if used alone, the best benefit may not be achieved.

Refinery and salmonella paratyphoid control

The reprocessing plant can play an important role in the control of salmonella . This is true for countries that permit the use of chemicals such as chlorine during processing and in the chiller. Levels of 5, 10, or 20 ppm can significantly reduce contamination. There are other effective chemicals that you can use. There are countries that allow very limited use of chemicals during processing, which is ineffective in controlling Salmonella contamination of .

In this case, focus on control before the broiler arrives for slaughter. Good hygiene, cleaning and disinfection contribute greatly to the control of Salmonella and should not be neglected. There is a link between the processing plant and the cultivation, which is a transport system, mainly chicken coops or cages. Lack of good disinfection of the cells can lead to the spread of bacteria from positive to negative flocks in the field.This system requires constant attention.

Salmonellosis

Everyone at least once in their life suffered from abdominal pain, accompanied by upset stools. If salmonella is the cause of the disease, the disease can result in severe complications.

Salmonellosis is an acute infectious disease of humans and animals caused by numerous microbes of the genus Salmonella, characterized by a variety of clinical manifestations, from asymptomatic carriage to severe forms with lesions of the gastrointestinal tract.

The causative agent of salmonellosis is a large group of bacteria belonging to the genus Salmonella. Salmonella is quite resistant to environmental factors. They remain viable for a long time at low temperatures and can even multiply in a household refrigerator. In frozen meat, they live up to 13 months, in eggs – up to 1 year, in sausages and sausages – for 6-13 days. They persist in soil, droppings and feces for several years. At the same time, boiling kills salmonella instantly, and at a temperature of 56 ° C, they die in 1-3 minutes.Therefore, regular good cooking ensures that the food is free of Salmonella.

Salmonellosis occurs with equal frequency throughout the world. Unlike other intestinal infections, it is not characterized by seasonality; outbreaks of this infection are recorded all year round. Nevertheless, a regular increase in the incidence is noted in the warm season, when conditions are created for the rapid accumulation of the pathogen in the environment and food.

Source of infection. The source of infection in salmonellosis is mainly various animals: cattle, horses, pigs, rodents, etc. Most of the diseases of salmonellosis in humans are associated with the consumption of meat from cattle, and in recent years – chickens and eggs. Meat can become infected not only during the life of the animal, but also during the process of slaughter, butchering of carcasses, during storage, transportation and during cooking. A person as a source of infection is of importance mainly in hospital (nosocomial) cases of infection

Ways of transmission of infection. The main route of transmission of infection is food, and the main factors of transmission are various food products – meat, dairy, confectionery, prepared using eggs not subjected to heat treatment. In recent years, in connection with the emergence of hospital strains of pathogens, the contact and household transmission route has become of no small importance: during hospital outbreaks, Salmonella can be found on dishes, nipples, bottles with dairy products and drinks, on the backs of beds, doorknobs, ventilation grilles, etc. dr.

Salmonellosis Clinic. From the moment of infection to the onset of symptoms, it takes from 6 hours to three days. More often the latent (incubation) period of the disease is 12 – 24 hours. The disease begins acutely: the temperature rises to 38 – 39 degrees, nausea, repeated vomiting, intense paroxysmal pain in the abdomen, chills, liquid fetid stools of brown or green color appear. With a moderate and severe course, dehydration syndrome develops: dry mucous membranes, hoarseness, a decrease in the amount of urine excreted, convulsions.

Pre-hospitalization assistance. If a product is known that has caused trouble, it should be immediately disposed of so that the rest of the family does not become infected. The patient, if there are no contraindications, should rinse the stomach as soon as possible by drinking at least 1.5 liters of water. Then you must definitely consult a doctor!

Prevention. In prevention, the main place belongs to the complex of veterinary-sanitary, medical-sanitary and anti-epidemic measures.At home, this is the correct heat treatment and storage of meat and dairy products, separate processing of raw and boiled meat and chickens, refusal of creams and dishes where eggs are used without preliminary heat treatment, adherence to cooking technology and prevention of the use of products with expired shelf life.

In order to prevent hospital salmonellosis, a whole range of measures has been developed, the knowledge and implementation of which is mandatory for employees of relevant institutions.

Doctor-bacteriologist of the educational establishment “MKDTs” Smal A.P.

90,000 The US Biotechnology Center reported an outbreak of Salmonella strain associated with the consumption of fish and seafood

As part of the ongoing risk analysis of the supply of unsafe fish products, the Rosselkhoznadzor subordinate FSBI “NCBRP” informs that, according to the National Center for Biotechnological Information, 155 cases of Thompson salmonella caused by the consumption of fish and seafood manufactured by Northeast Seafood.The vast majority of patients lived in the Denver area where the company is located.
As reported by Food Safety News https://www.foodsafetynews.com/2021/11/biotechnology-center-finds-outbreak-strain-of-salmonella-in-155-cases-linked-to-seafood/
US Administration The Food and Drug Administration (FDA) has reported positive testing of product samples from Northeast Seafood Products Inc. on the Thompson Salmonella strain, which caused an outbreak of disease, after which the company was temporarily closed and withdrew a wide range of fish and seafood produced by it from restaurants, as well as from a number of American retail chains.Symptoms of a Salmonella infection can include diarrhea, abdominal cramps, and fever within 12 to 72 hours after eating contaminated food. Otherwise healthy adults usually get sick for four to seven days. However, in some cases, the diarrhea can be so severe that patients need to be hospitalized. However, some people may be infected with salmonella bacteria and not get sick, show no symptoms, but they still have the ability to spread the infection to other people.Currently, US health officials continue to monitor the situation as it usually takes an extended period of time to determine if a person with Salmonella is part of a specified outbreak.
Experts also express concern that consumers may store portions of recalled products in their home freezers and continue to consume them. They note the insidious nature of the disease: food contaminated with Salmonella bacteria usually does not look spoiled, does not have any specific odor or unpleasant taste, and anyone can get Salmonella infection, but infants, children, the elderly and people with weakened immune systems are more prone to high risk of infection.Most recover without medical attention and are not tested for Salmonella, so the true number of affected consumers is likely to far outnumber those reported, and the outbreak may not be limited to states where the disease has been identified.
We would like to remind that information on the problems in connection with the spread of infections caused by the consumption of fish and seafood in the world is available on the website of the FSBI “NCBRP” in the section “Analysis of veterinary risks” / “Analytical articles” http: // fishquality.ru / analiz-veterinarnykh-riskov / analiticheskie-stati /

90,000 HIV-related epidemic of deadly salmonella in Africa – scientists

Dugan and his colleagues have sequenced the genomes of over 120 strains of Salmonella, compared them and produced a “family tree” describing the relationship between different cultures of bacteria. To do this, scientists have identified 10.6 thousand single nucleotide polymorphisms – differences in one “letter” -nucleotide in DNA – and compared their sets in the genomes of Salmonella.

It turned out that all strains of atypical salmonella are divided into two groups, which scientists have designated as “line 1” and “line 2”. The genome of these bacteria differs markedly from the DNA structure of “ordinary” Salmonella strains from the Typhimurium serovar, which they are part of. For example, the genomes of common and atypical Salmonella differ from each other by 700 mutations, while the maximum number of differences between bacteria from different lines does not exceed 450.

Scientists superimposed the resulting family tree on a map of Africa and determined the time of origin and the location of the source of the “parent” strains of iNTS.It turned out that “line 1” originated about 50 years ago in Malawi, in areas that are associated with the beginning of the first AIDS epidemics in Africa. The iNTS group then infiltrated Kenya, the Democratic Republic of the Congo, Uganda and Mozambique.

The second line appeared much later – in 1977 in the Democratic Republic of the Congo. It spread along the same countries of tropical Africa along approximately the same routes as “Line 1”, but managed to penetrate further – to Nigeria, Mali and some other northern countries.Beginning in 2003, this line began to rapidly displace bacteria from the first group, since the bacilli from “line 1” were not resistant to chloramphenicol, the antibiotic with which doctors tried to save victims of Salmonella.

The origins of these bacteria and their trajectory overlap in many ways with the spread of the HIV epidemic in Africa. This allowed scientists to assume that this subspecies of Salmonella developed in the population of HIV carriers in the countries of central and eastern Africa.

“It is believed that the HIV epidemic in tropical Africa began in the central regions of the continent and spread eastward, in much the same way as iNTS. Our results indicate that the development of this form of salmonellosis and its spread may have been intensified by the large population of people with compromised immunity, “added co-author Robert Kinsley of the Sanger Institute.

It is not yet clear how these bacteria spread and how they infect healthy people.Scientists plan to find the answer to these questions by continuing to study the genome of Salmonella.

Seva and Biotecon Diagnostics presented a new real-time diagnostic kit / News / News and publications / Ceva Russia

Libourne, 10 September 2019 A new diagnostic kit was presented at the SafePork Science Symposium in Berlin to help pig farms fight Salmonella infection in real time.

It was developed and manufactured by Biotecon Diagnostics in collaboration with the international veterinary biopharmaceutical company Seva. The highly sensitive DNA test will accurately identify and differentiate between vaccine strains and field strains of Salmonella Typhimurium (STM), the most common enzootic Salmonella. It will complement the Salmoporc vaccine, which is widely used in many European countries.

If Salmonella is found on the farm by any of the established methods, differentiation can be made on any sample without the need for lengthy culture tests.Results can be obtained overnight or within 24 hours, compared to five days for existing tests. The test is safe and reliable.

Vaccination is the only way to sustainably reduce Salmonella Typhimurium and its spread along the food chain. If STM is found on the farm where the vaccine is used, it must be determined whether it is a vaccine strain, which should not be of concern, or a field strain. strain, which is a serious cause for concern “, commented Dr. Rieke Schmelz, veterinarian and salmonella specialist, Seva.

To check for Salmonella, the veterinarian takes samples from pigs and the environment at several locations. Salmonella is found on 80-100 percent of pig farms in most countries.

Reducing the prevalence of Salmonella on the farm through vaccination will also reduce the need for antibiotics. Dr. Schmelz noted. “ Seva is committed to caring for the future of antibiotics by providing pig farms with practical tools and services to maintain a high level of preventive health.Vaccination will help prevent a further increase in MDR Salmonella.

Further information on vaccinations and the new diagnostic method can be found at www.stop-salmonella.com

At the presentation of the diagnostic kit: (left to right) Heiko Rüdiger, Veterinary Director of Ceva Santé Animale; Dr. Rike Schmelz , Ceva Santé Animal and Dr. Christoph Kunas, Product Manager, Biotecon Diagnostics

A new diagnostic kit developed by Biotecon Diagnostics in collaboration with Ceva.

90,000 2.4.5. Definition of Salmonella / Consultant Plus

2.4.5. Definition of Salmonella.

Pathogenic microorganisms, including salmonella, should not be found in the mass of the product, which is indicated in the table. 1, 2, 3, 4.

2.4.5.1. The method is based on the use of enrichment media to increase the growth of Salmonella, their isolation on special agar media, followed by serological reactions.

2.4.5.2.Analysis.

When examining sterilized liquid mixtures and products, the volume (mass) of the investigated product should be (100 +/- 1.0) cubic meters. cm; for fermented milk liquid products (before neutralization) – (50 +/- 0.5) cubic meters. cm, except for “Baldyrgan” and starters, where the volume is (100 +/- 1.0) cubic meters. cm; pasty products, ready-made cereals, drinks are investigated in mass (volume) (50 +/- 0.5) cubic meters. cm (g).

2.4.5.3. Under sterile conditions in a glass with a capacity of 200 cubic meters. cm measure (100.0 +/- 1.0) cubic meters.cm; (50 +/- 0.5) cc cm or weighed (50 +/- 0.5) g of the test product and aseptically transferred into flasks with a capacity of 1000.0 cubic meters, respectively. cm, 400.0 cc cm or 250.0 cubic meters. cm, containing (400.0 +/- 1.0) cubic meters. cm, (200 +/- 1.0) cc. cm of liquid enrichment medium, observing the ratio of 1: 5 product and culture medium. Mix everything thoroughly. In case of poor dissolution of the product, the samples are subjected to mechanical shaking on an apparatus for shaking liquids (shooter-apparatus). Magnesium medium, Müller, Kaufmann medium, selenite medium (p.4.2.4.1, 4.2.4.4, 4.2.4.5, 4.2.4.6).

Sowing of liquid products in different volumes of enrichment media of double concentration (1: 1) is allowed.

Sour milk products are neutralized before being introduced into the enrichment medium (p. 2.3.4).

Crops are kept in a thermostat at (37 +/- 1) degrees. C within 18 – 24 hours.

2.4.5.4. The next day, sowing is carried out from the enrichment media onto the surface of well-dried dishes with differential diagnostic media of Ploskirev and bismuth-sulfite agar (p.4.2.4.7). To obtain individual colonies, a minimum amount of inoculum is taken with a loop and inoculated with a streak. Plates with crops are placed in a thermostat at (37 +/- 1) deg. C for 24 – 48 hours. The crops are checked twice: after (24 +/- 1) and (48 +/- 3) hours after incubation.

2.4.5.5. Processing of results.

2.4.5.5.1. On Ploskirev’s medium, Salmonella colonies are colorless, dense; on bismuth-sulfite agar, they are black, with a characteristic metallic luster, while a black staining of the medium under the colony is observed.

In the absence of typical Salmonella colonies on each of the media, the final test result is recorded as negative, i.e. there are no salmonella in the mass (volume) of the product under study.

If there are typical or suspicious colonies for Salmonella on any of the nutrient media on Petri dishes, further study them.

2.4.5.6. A well-isolated colony is selected from each medium on Petri dishes containing a suspected colony of Salmonella and plated by streak and prick on three-sugar agar with urea (p.4.2.4.8) or Kligler’s environment (p. 4.2.4.10). Test tubes with crops are kept in a thermostat at (37 +/- 1) deg. C within 24 hours. Identification of cultures seeded on Kligler’s medium or three-sugar agar with urea is carried out by fermentation of lactose, glucose, sucrose and urea splitting:

– reddening or yellowing of the beveled part of the column of the medium indicates the formation of acid as a result of fermentation of lactose, sucrose or both sugars;

– redness of the bar itself indicates glucose decoupling;

– restoration of the color of the medium to the original (pale pink) indicates the cleavage of urea;

– blackening of the medium in the column indicates the formation of hydrogen sulfide.

The mechanism of action of Kligler’s medium is identical to that of trisugar agar (except for urea, which is absent in this medium).

2.4.5.7. Interpretation of the results.

If cultures ferment lactose to gas and break down urea, they do not belong to Salmonella bacteria.

Cultures that do not ferment lactose and do not break down urea, but which ferment glucose (with or without gas formation) are undergoing further study (cultures fermenting glucose without gas formation are suspicious of typhoid or dysentery; cultures fermenting glucose to gas producing in the environment, bubbles and producing hydrogen sulfide may belong to bacteria of the genus Salmonella).

——————————–

<*> Among bacteria of the genus Salmonella, up to 15% of strains that do not produce hydrogen sulfide are found.

If not a single reaction characteristic of Salmonella was found in any of the test tubes, then the result is considered negative and a conclusion is made about the absence of bacteria of the genus Salmonella in the studied mass (volume) of the product.

2.4.5.8. If both tubes show the typical color of the media for Salmonella, a serological test is performed.For this purpose, a small amount of culture is taken with a loop from tubes with three-sugar agar or Kligler’s medium, emulsified in a drop of saline on a glass slide. Add a drop of polyvalent Salmonella O-serum (4.2.4.11) to the solution and gently shake the slide to mix the liquids.

A positive reaction to salmonella (agglutination) is observed within 30 – 60 seconds. Negative control reaction (culture + saline solution) is mandatory.

If no agglutination is detected during serological testing, the final result is recorded as negative. Any agglutination that appears on the glass indicates the likelihood of Salmonella being present.

2.4.5.9. When isolating cultures of gram-negative rods fermenting glucose with or without the formation of gas, not fermenting lactose and sucrose, producing or not producing hydrogen sulfide and having a clear serological characteristic, it is believed that bacteria of the genus Salmonella are present in the studied mass (volume) of the product.

(PDF) IMPROVEMENT OF ALLOCATION AND IDENTIFICATION OF SALMONELLA ENTERICA BACTERIA OF ARIZONAE SUBSPECIES

RJOAS, 2 (50), February 2016

15

Salmonella Arizonae isolates were first obtained in 1939 by Mary E. Caldwell6 Ryerson by staff members of the Department of Bacteriology and Zoology at the University of Arizona

. The source of this pathogen was Heloderma suspectrum –

Arizona Gila monster. Later, references to the synonyms

of this pathogen Paracolobactrum arizonae [1] and Arizona hinshawii [2] appear in the literature, and only in 2002

it was assigned to a separate subspecies.In the 9th edition of the Kaufmann-White scheme

[3], published in 2007 by the World Health Organization,

mentioned the existence of 99 serovariants of this subspecies. However, unlike Salmonella

of other Salmonella Enterica subspecies, Salmonella serovars of

Arizonae and Diarizonae subspecies do not have names, but are designated by the antigenic formula

structure. Moreover, the issues of the epizootic significance of serovariants and their

identification within the subspecies Arizonae have been insufficiently studied.

The source and natural reservoir of Salmonella of these serovars

are reptiles (lizards, snakes, turtles). It should be mentioned that

, in addition to reptiles, are susceptible to this pathogen are turkeys,

piglets, sheep [4], dogs, cats, monkeys, goats [5].

In literary sources, you can often find a description of

clinical cases of human diseases caused by the causative agent Salmonella

Arizonae with a fundamentally different form of manifestation of the disease, including

fatal diseases.Indicates the forms of manifestation of the disease

arizonous etiology such as meningitis [6], including in newborns [7],

gastroenteritis in children with microcephaly [8], otitis media, osteomyelitis [9], pleurisy,

sinusitis, peritonitis , bacteremia [10]. There is a description of a case of

septic arthritis of the hip joint in a 10-month-old child [11].

Also note the possibility of this disease without concomitant

for salmonella infection of gastroenteritis [12].

Sporadic outbreaks of arizoonosis in humans are most common

in the southwestern United States, where the majority of the population

are Hispanics, among whom the common consumption of snake meat

, as well as the use of drugs based on snake

offal [13]. In addition to the countries of Latin America and the United States, this problem

covers the countries of South Asia and the United Kingdom [14].

The most susceptible are newborns and small children.From the

large number of clinical cases, it can be concluded that

the infectious process intensifies and takes on an acute form due to the low

immune status. It is believed that the disease in humans is associated with increased

permeability of the blood-brain barrier, congenital injuries,

lack of formed cellular immunity, and also caused by

vertical infection from the mother. Patients who have had arizoonosis often

subsequently acquire neurological complications, hearing loss,

hydrocephalus [15], etc.

As antibiotic therapy for Salmonella arizona infection

broad-spectrum antibiotics are used – ampicillin, gentamicin,

fluoroquinolones and third-generation cephalosporins [16].

In animal husbandry and poultry farming, the problem of Arizona-infection is the most pressing

in turkey breeding. Turkey farming in our country is intensively developing

. An increase in the production and consumption of turkey products requires

to improve the methods of veterinary and sanitary control and veterinary

sanitary examination, taking into account the specificity of the product and the characteristics of the pathogen

[17].In accordance with Council Directives 2009/158 / EC of 30 November 2009 on

on the veterinary and sanitary conditions governing intra-Community trade

and imports from third countries of poultry and hatching eggs, the hatching egg

turkey is subject to mandatory control for the presence of the causative agent

salmonellosis subspecies Arizonae [18].