9 Salmonella Serotypes, Taxonomy, Epidemiology

Salmonella Serotypes and Taxonomy

• There are approximately 2,500 different serotypes of Salmonella.
• The Kauffman-White scheme standardizes Salmonella taxonomy using O (somatic) and H (flagellar) antigens.
• Serovar names are based on the geographic location where Salmonella was first identified.
• The Salmonella genus includes two species: S. enterica and S. bongori.
• There are six subspecies of S. enterica.
• 60% of all Salmonella serotypes are grouped under S. enterica subsp. Entererica.
• Within this group, O-antigens A, B, C1, C2, D, and E account for 99% of warm-blooded animal infections.
• Group B O-antigens are the most common type isolated from horses.
• Salmonella subspecies are further classified into serotypes based on biochemical differences.
• Reporting laboratories might abbreviate Salmonella enterica subsp. Enterica serotype Typhimurium as Salmonella Typhimurium.

Salmonella Epidemiology and Prevalence

• S. Typhimurium is the most common serotype of Salmonella found in horses with diarrhea.
• There are no host-adaptive strains in North America.
• S. Abortusequi is a host-adaptive strain causing abortion in pregnant mares in Asia and Africa.
• Agona, Anatum, and Typhimurium var. Cophenhagen, Newport, Infantis, Java, Javina, Saintpaul, London, Senftenberg, Krefled, and Miami are other common serotypes reported in horses with diarrhea.
• Salmonella is highly contagious, persistent in the environment, and causes nosocomial infections.
• Fecal shedding prevalence varies with geographic location, the horse's health status, and the detection method used.
• Normal horses on farms have a fecal shedding rate of less than 1%.
• Horses in a hospital setting have a much higher fecal shedding rate (4-6%).
• Fecal shedding is more prevalent in sick horses in the ICU, especially those with colic or diarrhea.
• Horses can harbor Salmonella in their mesenteric lymph nodes for an extended period, even without a recognized "true carrier" status.

Salmonella Risk Factors and Pathogenesis

• Risk factors include the serotype, inoculation dose, and the host's immunity.
• Specific risk factors for horses include colic, transport, antibiotic use, general anesthesia, feed changes, and concurrent illnesses, especially those involving the gastrointestinal system with impactions.
• The primary transmission route is fecal-oral.
• Salmonella invades the gastrointestinal mucosa and is phagocytosed by macrophages and dendritic cells.
• Salmonella proliferates within macrophages, leading to the dissemination of the organism (intracellular organism makes it challenging to treat with antibiotics).
• In adult horses, this dissemination is limited to the intestine and mesenteric lymph nodes.
• In foals, the organism can spread more widely, potentially resulting in septicemia.

Endotoxins and Their Characteristics

• Endotoxemia is the presence of endotoxin in the bloodstream.
• Toxemia is the presence of bacterial toxins in the bloodstream; endotoxin is one example.
• Sepsis is a systemic inflammatory response syndrome caused by an infection.
• Septicemia is an infection in the bloodstream that can lead to sepsis.
• Endotoxin is a lipopolysaccharide found in the outer cell envelope of gram-negative bacteria.
• LPS consists of a hydrophilic portion (inner core, outer core, and O-Specific side chain) and a hydrophobic portion (Lipid A).
• O-specific side chains vary among bacteria and are responsible for the "smooth" appearance of gram-negative bacterial colonies; without them, colonies appear "rough”.
• Lipid A anchors the LPS molecule in the cell wall and is the toxic component of gram-negative bacteria.

Sources and Conditions Associated with Endotoxemia

• The source of endotoxin is the cell wall of gram-negative bacteria and is released upon bacterial death or rapid multiplication.
• Sources in the body include damaged gastrointestinal mucosa (due to colitis, strangulating lesions, or ischemic bowel), gram-negative bacteremia (common in foals), gram-negative pneumonia, metritis, or wounds.
• Endotoxemia is common in horses with GIT disease with mucosal compromise, septic neonatal foals, post-foaling metritis/retained placenta, and gram-negative pneumonia/pleuritis.
• The GIT is a large source of free endotoxin.
• In healthy horses, a small amount of endotoxin crosses the intact mucosal barrier, reaches the portal circulation, and is processed by the liver.

Mechanism of Action of LPS

• Excessive endotoxin levels can overwhelm the mononuclear phagocytic system's ability to clear it.
• LPS gains access to circulation and forms aggregates in plasma due to its hydrophobic nature.
• LPS aggregates interact with lipopolysaccharide-binding protein, which disperses the endotoxin molecule into monomers.
• LPS-binding protein "shuttles" LPS to inflammatory cells (monocytes) and is synthesized by the liver.
• LPS binding protein levels increase during inflammation (surgery, colitis, etc.) and is an acute phase protein.
• LPS binding protein transfers endotoxin to the cell surface receptor complex CD14-MD2, primarily on mononuclear cells.
• Soluble CD14 exists, and endotoxin-soluble CD14 complexes interact with cells lacking membrane-bound CD14.
• The CD14 receptor lacks the ability to transmit the endotoxin signal to the interior of the cell.
• TLR-4 is the missing link between CD14 and the cytosolic environment.
• CD14 and endotoxin interact with TLR4, transmitting a stimulatory signal to the interior of the cell.
• The intracellular domain of TLR4 exposes binding sites for intracellular proteins involved in the cell signaling process.
• Intracellular signaling results in cell activation.
• Activation of transcription factors (Nuclear factor κB (NFκB) and mitogen-activated protein kinase) promotes gene transcription of inflammatory mediators.
• Cytokines are primary mediators of LPS effects.
• Cytokines are produced by different cells with a wide range of effects.
• The primary groups of cytokines studied include TNF, interleukins (IL-1, IL6), eicosanoids (arachidonic acid metabolites), and tissue factor.
• Inflammatory mediators cause endothelial cell dysfunction and damage due to increased vascular permeability and the inability of endothelial cells to respond to vasoconstrictive amines.
• LPS results in neutrophil activation and margination of neutrophils into tissues (often the lungs).
• Margination results from adhesion molecules on endothelial cells and neutrophils interacting, causing neutrophils to become "sticky."
• Neutropenia is a consistent feature of LPS.
• LPS can cause abnormalities of coagulation (DIC).
• can cause Shock and organ failure are caused by vascular dysfunction and can include multiple organ failure (including laminitis).
• Endotoxemia plays a significant role in gastrointestinal diseases.
• LPS is one of several Pathogen-Associated Molecular Patterns (PAMP) recognized by TLR-4.
• Cell walls from gram-positive bacteria (e.g., Lipoteichoic Acid) are other PAMPs that bind to TLR-2.
• Not all cases of sepsis result from endotoxin in the bloodstream.
• Physical examination findings alone may not differentiate a foal with septicemia from a gram-positive infection or a horse with clostridial myositis.

Clinical Signs of Endotoxemia

• Initial signs (15-45 minutes): Anorexia, yawning, sweating, depression, colic, muscle fasciculation, recumbency, increased HR and RR, and fever.
• Mucous membranes show hyperemia.
• Later signs (continued exposure): Mucous membranes darken (toxic-purple), body temperature decreases, blood flow decreases, severe depression, poor arterial pulse ("shock").
• potential for Organ Failure: Renal failure, laminitis, liver disease, and abortion in pregnant mares are possible.
• Hemogram/Biochemistry Profile: Leukopenia/neutropenia 2° to margination of neutrophils.
• PCV or TP may be elevated; PCV may be increased, and TP is normal or low due to GIT loss.
• Electrolyte abnormalities are common- glucose is frequently elevated in adults (insulin resistance), neonate foals can be low, and Azotemia (prerenal to renal).
• Hemostatic abnormalities include signs of disseminated intravascular coagulation such as spontaneous venous thrombosis or prolonged bleeding after venipuncture.
• Abnormalities of platelet count, fibrinogen, fibrin degradation products (FDP), coagulation times (PT and APTT), and anti-thrombin concentrations are laboratory findings associated with DIC.

Diagnosis and Clinical Presentations of Salmonella

• Diagnosis is based on clinical signs, blood work showing low neutrophil count, and underlying disease.
• Endotoxin quantification is possible but mainly done for research.
• Salmonella can present with acute diarrhea; depression/fever/leukopenia without diarrhea; inapparent infections; septicemia (especially in foals); chronic diarrhea.
• Acute diarrhea: Three hallmarks- Diarrhea, Fever, Neutropenia; other signs may include Colic, signs of endotoxemia, edema, and depression.
• Additionally there may be fever and neutropenia without diarrhea or with only a mild diarrhea.
• Prerenal to renal azotemia, electrolyte abnormalities, and hypoproteinemia can be present on blood work.
• Chronic diarrhea: ranges between periods of normal and not, weight loss, and intermittent fever.
• Possible complications: Laminitis, Renal Failure, DIC, Chronic Diarrhea, GI infarction/necrosis.
• Fecal culture: Use an enrichment medium and collect multiple samples
• PCR is more sensitive than culture but does not provide serotyping or sensitivity information.

Potomac Horse Fever (PHF)

• PHF is also known as Equine Monocytic Ehrlichiosis and is caused by Neorickettsia risticii, an obligate intracellular parasite.
• The organism resides in monocytes, macrophages, and GI cells within the cytoplasm of infected cells.
• transmission is not ticks, but through a fresh water snail- (Juga sp.)!
• The snail is parasitized by a trematode that is infected with N. risticii.
• DNA of N. risticii has also been found in aquatic insects (stoneflies, caddisflies, and mayflies) that serve as trematode vectors.
• Horses are likely infected by ingesting infected insect vectors.
• Fecal-oral transmission is unlikely.
• Risk factors include time of year (summer/fall), geographic location, and proximity to water.
• Clinical signs/syndromes: Signs include diarrhea, laminitis, and abortion. Infections are are likely subclinical or missed.
• Laminitis occurs more commonly with PHF than other types of colitis.
• Initial bloodwork shows hemoconcentration and decreased WBC, followed by possible leukocytosis, pre-renal to renal azotemia, and electrolyte abnormalities
• Diagnosis is made based on season & geographic location, clinical signs, and response to treatment.
• Feces and blood can be tested by PCR at UCDavis and Texas A&M.
• PHF is treated with oxytetracycline with response expected in 24-48 hours, but be careful because it can worsen diarrhea or chelate calcium-cause cardiovascular collapse if not PHF
• Supportive treatments include fluid therapy and supportive care for diarrhea.

Other Causes of Diarrhea in Adult Horses

• C. perfringens (A & C) and C. difficile can cause antibiotic-associated diarrhea.
• C. difficile has a new name – Clostridiodes difficile.
• Both organisms are potentially zoonotic.
• Clinical signs are similar to other causes, including hemorrhagic diarrhea.
• Specific treatment: Metronidazole
• Coronavirus is a new emerging disease that is species-specific but very infectious.
• Signs include fever, anorexia, colic, and low white count with only 20% having diarrhea.
• Most horses recover well; neurologic signs and hyperammonemia are rare.
• Large outbreaks have been reported with short clinical signs (1-4 days) but outbreaks can last weeks.
• Diagnosis: PCR for organism in feces.

Treatment of Salmonella and PHF

• Supportive treatment includes fluids (crystalloids, colloids), correction of acid/base imbalances, and electrolyte imbalances.
• Treatment of endotoxin should also be considered.
• Removing the source of endotoxin and inhibiting its release into circulation may include Plasma or Endoserum.
• Neutralization of endotoxin before it interacts with inflammatory cells should also happen.
• Plasma or Endoserum (a plasma product) contains antibodies against the core and lipid A regions of LPS.
• Polymyxin B is a cationic polypeptide that binds to the anionic portion of lipid A at doses of 1000-6000 IU/kg IV bid. It can cause nephrotoxicity, especially in dehydrated and azotemic horses.
• Biosponge® (Di-tri-octahedral smectite) binds endotoxins in the lumen of the GIT when given orally.
• Prevention of the synthesis/release/activation of inflammatory mediators in necessary.
• NSAIDs inhibit prostaglandin production. Flunixin meglumine is preferred to other NSAIDs at 0.25 mg/kg TID to QID is the "anti-endotoxic" dose.
• Pentoxifylline is a methylxanthine derivative and phosphodiesterase inhibitor that inhibits the production of various cytokines and can increases RBC deformability.
• DMSO (Dimethyl sulfoxide) is an anti-inflammatory- free radical scavenger, doses range from 1-0.1 mg/kg
• Antibiotics are indicated if septicemia occurs, foals less than 3 months old, or severe leukopenia/neutropenia occurs.
• Non-steroidal anti-inflammatory drugs (NSAIDs) have analgesic, anti-inflammatory, and anti-endotoxic effects.
• Bismuth subsalicylates (Pepto-Bismol) or Kaolin/pectin may be more effective in moderate/severe cases as oral protectants.
• Biosponge® is an aluminomagnesium silicate that has been shown to absorption of endotoxins and demonstrate C. difficile and perfringens exotoxin absorption in vitro.
• The use of probiotics can reduce the growth of pathogenic bacteria.
• Saccharomyces boulardii has been shown to be beneficial in humans and horses with colitis.

Prevention

• Isolating all horses with diarrhea is recommended, especially Salmonella suspects!
• Preventing spread relies on proper hygiene and cleaning contaminated areas with chlorox or phenols.
• Prevention/Control PHF: a Killed vaccine is available but only decreases clinical signs and does not prevent transmission.