Salmonella is one of the leading causes of bacterial gastroenteritis in people in the United States. Salmonella is not considered part of the normal gut microbiome in humans, who become infected with this bacterium via ingestion of contaminated food (eggs, poultry, pork, beef, and vegetables). Beef products have been linked to a relatively small proportion of salmonellosis cases in humans compared to poultry-related products. However, an increasing interest exists in reducing salmonellosis cases in humans from animal sources, including beef. Salmonella is an example of evolutionary success, as the genomic backbone of Salmonella has evolved to adapt to a broad range of hosts, environments, and nutritional stressors. The virulence of Salmonella strains is determined by a wide array of genes involved in facilitating each step of the invasion process. The first step of the host invasion is the attachment to the epithelial cells lining the gut using molecules called adhesins. Then, Salmonella uses needle-like molecular superstructures called type III secretory system 1 and 2 to introduce approximately 40 effector molecules into the epithelial cells, subverting the internal cellular processes in the latter and inducing the endocytosis of the bacteria, giving rise to Salmonella-containing vacuoles (SCV). Subsequently, Salmonella thrives within the SVCs and can be transported to neighboring epithelial cells or deeper in the intestinal mucosa. The most comprehensive way of elucidating the virulence genes in Salmonella is sequencing the whole genome (whole genome sequencing, WGS). However, only a few studies have been published that describe the genomic features associated with virulence in Salmonella circulating in cattle in the United States. The overall objective of this project was to characterize the virulence genes present in Salmonella isolates from cattle. Specifically, this study[MC1] detected virulence-associated genes in Salmonella isolates from cattle, compared the genomic features between the Salmonella isolated from beef and dairy cattle, and reconstructed their phylogeny.
[MC1]reword to remove all "we" in every summary.
"this project", "this study", etc.
This study completed the sequencing of the whole genome of 300 Salmonella isolates. These isolates were obtained through active surveillance of all beef and dairy cattle throughout their hospitalization at Colorado State University’s James L. Voss Veterinary Teaching Hospital (JLVVTH) from 2002-2012, regardless of the type and severity of health problems (a total of 476 bovine patients). The whole genome sequencing was performed using long reads (Nanopore platform), which were assembled using Flye. Genomes were polished with Medaka. ABRicate was employed to search for virulence genes with the virulence factor database (VFDB). The genes detected by ABRicate were evaluated against the available metadata. The variables used for analysis were the year of sampling, sex, breed, state, and clinical history in the past 48 hours: systemic signs, diarrhea, fever, previous surgery, affected body system (musculoskeletal, gastrointestinal, respiratory, renal, hepatic, or reproductive), and type of hospital housing (isolation units or food animal pens). The metadata that was used for statistical analysis was dichotomized. Scoary was utilized to detect associations between individual virulence genes and each variable in the metadata. The phylogenetic relationship between isolates was visualized via interactive tree of life (iTOL) using the output from the pangenome analysis conducted with the Prokka-annotated assemblies in Roary.
The Salmonella isolates (n=300) were previously characterized as belonging to 32 different serotypes. The most prevalent serotypes were Montevideo (n=43, 14.33%), Muenster (n=40, 13.33%), and Newport (n=36, 12%). The isolates originated from cattle in 12 farms (3 farms accounted for 81.34% of the Salmonella isolates). Most isolates were collected from 2002 through 2007 and originated from Colorado dairy cows and heifers exhibiting systemic signs, including gastrointestinal signs. Missing data was present in at least 37 isolates but could involve more isolates in a few variables (age, fever, and leukopenia). On average, Salmonella isolates harbored 148.3 virulence genes (minimum: 133; median: 149; maximum: 168). Most virulence genes were present in at least 80% of the isolates (n=137). Nine virulence genes were present in 60-80% of the isolates, seven in 40-60% of the isolates, six in 20-40% of the isolates, and 26 virulence genes were present in less than 20% of the isolates. These rare virulence genes encode for nutritional and metabolic factors, immune modulators, effector delivery systems and toxins, and proteins involved in adherence and stress survival. Significant associations were detected between virulence genes in Salmonella in cattle and the year of collection, cattle age at admission, the presence of diarrhea or fever in the past 48 hours, and hospital housing. In some cases, the same Salmonella virulence genes were associated with cattle age at admission, the presence of diarrhea or fever in the past 48 hours, and hospital housing, showing the same direction in the association (either positive or negative) suggesting a set of virulence genes may increase the fitness of Salmonella to induce diarrhea and fever in older cattle to a degree requiring isolation of the animals. Another set of genes seems to be more prevalent in milder cases in younger cattle and encode for exotoxins. Further investigation of these findings is warranted using molecular bacteriology tools, which were outside the scope of this study. Interestingly, three virulence genes were positively associated with cases of diarrhea and fever in older cattle to a degree requiring isolation of the animals were also more prevalent in Salmonella isolates collected 2002-2007 (that is, less prevalent in 2008-2012 Salmonella isolates with an OR < 1). This finding may indicate a temporal trend. Additionally, even though no strong clustering of the phylogenetic tree existed related to the metadata variables available for the Salmonella isolates, some of the isolates appear within the same clade in the phylogenetic tree.
This study demonstrated some genes are more prevalent in Salmonella from older cattle showing gastrointestinal signs (such as diarrhea) and fever. This could suggest that Salmonella may need different sets of genes for virulence depending on the age of the bovine host.