Essential tissue destruction and endotoxaemia

Just because bacteria can invade, it does not mean they cause disease.

Definitions

Definitions of some of the words in the Workbook. Try a practice crossword puzzle.

Apoptosis: This is genetically programmed self-destruction of cells where the nucleus fragments. It occurs naturally when the cell ages or is damaged. Bacteria can induce apoptosis by activating pro-apoptotic proteins or inactivating anti-apoptotic proteins. Bacteria can thus gain entry to tissue and its nutrients this way. However, it can in some infections assist in the host’s innate defences i.e. increased sloughing of infected cells will wash away the bacteria.

Antigen: A molecule that stimulates a specific antibody response i.e. exotoxins produced by bacteria.

Autophagy: It is a recycling mechanism of cells whereby damaged cell organelles or cytoplasmic contents are encased in a double membrane and broken down by lysosomes to their base nutrients and used as raw material to produce new cell contents. Cell starvation is a major trigger.  It is also a protective mechanism of cells against intracellular bacteria. Many intracellular bacteria have developed mechanisms to delay or inhibit autophagy.

Cell lysis: Breaking down of a cell membrane to release its contents. This can be done by osmotic pressure, enzymes and pathogens.

Cytokines: Large molecules secreted by inflammatory processes that mediate and regulate immunity, inflammation and haemopoiesis.

Disseminated intravascular coagulopathy (DIC): DIC occurs when blood clots form in the capillary beds blocking the blood supply to those tissues with widespread organ failure. The clotting factors deplete, so normal blood clotting cannot occur resulting in bleeding. There are a number of causes of DIC including endotoxaemia caused by gram-negative bacteria and sepsis caused by both gram-positive and negative bacteria.

Endotoxaemia: Endotoxaemia is defined as the presence of excessive amounts of bacterial lipopolysaccharide (LPS) in the blood leading to a hyper-inflammatory disorder.

Exotoxins: Proteins produced by bacteria that cause essential tissue damage

Laminitis:   Inflammation of the hoof lamellae of hoofed animals such as horses, cattle, sheep, goats and pigs.

Limulus: Amoeba in the blood of the horseshoe crab are super sensitive to endotoxin and are an excellent test for the presence of endotoxin in intravenous products such as nutritive drips.

Lipid A: Portion of the endotoxin in gram-negative bacterial outer membranes that stimulates a hyperinflammatory response

Metabolic acidosis: Metabolic acidosis or acidic blood and tissue occurs when the body produces too many acids, cannot remove excess acids or excretes excessive amounts of bicarbonates.

Neutropaenia: Reduced numbers of neutrophils (white blood cells) in the bloodstream. Often caused by movement of neutrophils to the site of inflammation.

Respiratory acidosis: Respiratory acidosis occurs when the lungs cannot remove enough CO2.

Septicaemia (Sepsis): Septicaemia is defined as the presence of bacteria and their products, such as endotoxins or exotoxins, in the bloodstream. The layman’s term is “blood poisoning”.

Toxic neutrophils or toxic granulation of neutrophils. Blue granules and vacuoles in the cytoplasm of neutrophils. It is usually indicative of a systemic inflammatory response. Often together with Dohle bodies = large blue-grey bodies in the neutrophil cytoplasm. In chronic endotoxaemia a neutrophil left-shift may be present. This means that there are immature neutrophils in the bloodstream.

Toxoid: An antigenic virulent bacterial protein that is inactivated by formulin and used as a vaccine. It is often administered with an adjuvant.

How bacteria cause essential tissue damage or alter physiological processes

Pathogenic bacteria cause disease by either damaging essential tissues or impairing critical physiological processes within the body. They do this in one of 3 ways:

1. Direct destruction of cells. This is an unusual mechanism used by bacteria. The best example is the effect that the obligate intracellular pathogen Chlamydia has on cells where cells are destroyed by lysis and apoptosis.
2. Destruction of cells or disruption of physiological processes by bacterial exotoxins and enzymes.
3. Induce a tissue destructive inflammatory response or immune-mediated tissue destruction. Can be induced by surface antigens, endotoxins and exotoxins.

direct destruction of cells

Example: The obligate intracellular pathogen Chlamydia.

The example below shows conjunctivitis in a cat caused by Chlamydia felis.

(1) An environmental stable spore-like body known as the initial body will be phagocytosed by cells. (2) The acidic environment of the phago-lysosome, stimulates the initial body to change and become a larger metabolically active reticulate body. (3) Using the host ATP the reticulate body divides and at this stage will prevent the cell (epithelial cell, macrophage) from killing itself. (4) In response to host Interferon gamma the reticulate bodies go into a dormant phase where they can remain indefinitely. (5) At the end of the replicative cycle, a large number of initial bodies (non-active) will be formed. The cell will then be induced to undergo apoptosis. (6) The cell will either rupture or being dead release the infective initial bodies, allowing them to infect another cell or be excreted.

introduction to bacterial toxins

All gram-negative bacteria have lipopolysaccharide or exotoxin as a component of the outer cell membrane. This foreign antigen when released in high qualities from dying bacteria stimulates a hyper-inflammatory response in susceptible individuals that can result in a life-threatening disease known as endotoxaemia.

Pathogenic gram-positive and gram-negative bacteria are able to produce a number of protein-based exotoxins that can cause from mild to serious disease. These tend to be specific to the bacterial species and can be neutralised by specific anti-toxin antibodies produced by the adaptive immune response. Being proteins they can be inactivated using formalin and high temperatures. Vaccines against exotoxins are generally highly effective. Below is a Table that compares endotoxins and exotoxins.

Septicaemia

Septicaemia is defined as the presence of bacteria and their products, such as endotoxins or exotoxins, in the bloodstream. The layman’s term is “blood poisoning”.

The animal is said to be in septic shock when TWO (2) of the following criteria are present:

1. Hyperthermia or hypothermia (too high or too low body temperature)
2. Tachycardia (rapid heart rate)
3. Tachypnoea or hyperventilation (rapid shallow breathing)
4. Leucopaenia (too few white blood cells), leucocytosis (too many white blood cells), or >10% band (immature) neutrophils.

endotoxins and endotoxaemia

Endotoxaemia is defined as the presence of excessive amounts of bacterial lipopolysaccharide (LPS) in the blood leading to a hyper-inflammatory disorder.

LPS is a component of the gram-negative outer membrane that is primarily released when bacterial cell lyse.  It results in an exaggerated, systemic inflammatory response and is a common condition in animals, especially horses. Endotoxaemia is characterised by fever, leukocytosis, changes in vascular permeability, altered metabolic responses and activation of nonspecific host defences. It is one of the more common causes of sepsis or septicaemia.

Functions of the lipopolysaccharide (LPS) or endotoxin

1. Lipid A

Lipid A is similar in all gram-negative bacteria and since it is deeply embedded in the outer cell membrane it is only released by actively dividing and growing bacteria or when the bacterial cell lyses. Lipid A stimulates the immune system resulting in a hyper-inflammatory response that is known as endotoxaemia.

2. Core sugars

Barrier to macromolecules e.g. In the Enterobacteriales, the ompF and ompC porins exclude passage of all hydrophobic molecules and any hydrophilic molecules greater than a molecular weight of about 700 daltons. This prevents penetration of the bacteria by bile salts and other toxic molecules from the gastrointestinal tract. These sugars are genus specific and has been the target on research into more effective vaccines against gram-negative bacteria. A sugar, 2-keto-3-deoxyoctonoic acid (KDO) is unique to the core sugars and therefore its detection in blood is indicative of endotoxaemia.

3. O antigen (somatic cell antigen)

• Adherence to cell surfaces.
• If long, it impedes destruction of the bacterial cells by serum components and phagocytic cells by antigenic shifts and distance complement activation. These long polysaccharide chains are specific to pathogenic bacterial strains and confer smoothness to bacterial colonies on agar. They can be detected using antibodies that are bacterial strain specific. An example is Escherichia coli  O157:H7, the zoonotic bacterial cause of haemorrhagic uraemic syndrome in children. It can be identified using serological tests –  O157 refers to the O-antigen type and H7 to the flagella antigen type.
• Confers water solubility to Lipid A  allowing it into a hydrophilic environment.

Sources of endotoxin

Any circumstance that allows gram-negative bacteria to proliferate rapidly puts animals at risk of developing endotoxaemia. Common sources are the following:

1. Gastrointestinal damage/disease. About 45% of the intestinal tract consists of gram-negative bacteria. In circumstances where there is damage to the intestine or changes in peristalses or nutrition, such as grain overload in cattle. These bacteria grow rapidly and as the nutrients wane they will also die rapidly releasing huge quantities of endotoxin. Some gram-negative bacteria penetrate the intestinal tract to cause, for example peritonitis (inflammation of the peritoneum or abdominal membranes). Most cases of endotoxaemia in horses are associated with gastrointestinal causes of colic (abdominal pain) and diarrhoea.
2. Respiratory tract. Young cattle especially develop opportunistic gram-negative bacterial infections of the lungs when they are overcrowded and stressed or suffering from viral disease.
3. Peracute gram-negative mastitis (inflammation of the udder) in dairy cows.
4. Other serious gram-negative infections. Female animals suffering from pyometra (pus in the uterus) and post-parturient uterus infections. Neonates with umbilical infections. Gram-negative septicaemia.

pathogenesis of endotoxaemia

Initiation of endotoxaemia

Once in the bloodstream, Lipid A binds with circulating LPS-binding protein (LBP). LBP is synthesised by the liver. The LBP-LPS complex binds to the CD14 molecules on monocytic cells. The resulting complex activates pattern receptor molecules on cells such as toll-like receptor 4 (TLR4). This affects the transfer of LPS to myeloid differentiation protein-2 (MD2) which stimulates monocytes, dendritic cells, macrophages and B-cells to upregulate the production of proinflammatory cytokines, nitrous oxide (NO) and isosanoids (prostaglandins and leukotriens). Furthermore, LPS may also be recognised by pattern recognition receptors on cells, further stimulating the transcription of several proinflammatory cytokines.

Proinflammatory cytokines such as TNFα, IL1 and IL6 induce release of acute phase proteins from the liver. These include fibrinogen, C-reactive protein, mannose-binding lectin, complement, serum amyloid A, haptoglobin, cerruloplasmin and vasoactive amines.

This results in haemodynamic derangement, abnormal body temperature, progressive hypoperfusion of the vasculature, hypoxic injury to susceptible cells, quantitative adjustments in the leukocytes and platelets, DIC and multiple organ failure.

Note that small amounts of Lipid A will stimulate a protective inflammatory response, whereas large quantities of Lipid A can lead to an excessive inflammatory response which is auto-destructive.

Consequences in brief:

The initial production of cytokines by the monocytes and macrophages leads to:

1. The production of a variety of cytokines including prostaglandins; leukotrienes, interleukins and tumour necrosis factor.
2. Stimulation of the coagulation cascade
3. Stimulation of the alternative complement pathway

The pathophysiology of peracute and acute endotoxaemia

1. Fever

An early clinical sign in endotoxaemia is fever. IL-1 stimulates the production of Prostaglandin E2 in the hypothalamus, which stimulates the production of cAMP increasing the temperature set point of the thermoregulatory centre. This leads to mammals shivering to increase heat production and vasoconstriction of blood vessels in the skin to reduce heat loss.

2. Effect on the vascular system

Chemical mediators such as prostaglandins (PGs) and nitrous oxide (NO) mediate capillary vasodilation which increases flow to and slows down the blood flow in the tissues. Prostaglandins and their effects on vasoactive amines like histamine and bradykinin increase capillary leakage – 1) by increasing the spaces between endothelial cells and allowing protein rich-fluid and even red blood cells to move into the interstitium; and 2) increasing blood pressure within the capillaries; resulting in oedema and haemorrhaging. The loss of fluid also results in haemoconcentration and embolisms of the capillary bed which decreases the volume of blood returning the heart. Thus, the heart rate will increase, but the cardiac output will decrease. Later on, cardiac depressant factor will result in a decrease in heart rate.

Simultaneous with leukocyte activation, endotoxin binds to complement proteins to activate the lectin-dependent and alternative complement pathways, which further promotes inflammation through the production of peptide products.

3. Effect on coagulation

Intravascular coagulation is initiated primarily through tissue factor activation and proinflammatory mediators i.e. fibrinogen release. The coagulation pathway is also stimulated when there is an increase in endothelial leakage. The release and activation of large quantities of clotting factors results microemboli (blood clots) within the microcapillary beds and eventually clotting factor depletion. This condition is known as   disseminated intravascular coagulopathy (DIC). Animals with DIC will bleed – this is evidenced by the presence of petechiae (pin-point bleeding sites), ecchymoses and suggillations (very large bleeding sites) on mucosae and will also have multi-organ failure due to ischaemic necrosis.

Poor tissue perfusion results in metabolic acidosis.

4. Effect on leucocytes

Complement and IL8 induce neutrophil chemotaxis and diapedesis into the tissues. This initially causes depletes the neutrophils in the bloodstream (neutropaenia). This is a diagnostic characteristic of acute endotoxaemia in horses. Often toxic neutrophils are present. Later on immature (band) neutrophils will be released from the bone marrow creating a left-shift (from mature neutrophils to immature neutrophils) in the bloodstream.

In endotoxaemia there is an initial drop in lymphocytes (lymphopaenia) and later on a lymphocytosis.

5. Acute respiratory distress syndrome (ARDS, shock lung)

There are a number of causes of ARDS, including endotoxaemia and sepsis. The high production of cytokines by activated macrophages (cytokine storm) results in hyper inflammation in the lungs with an initial high blood flow to the lungs (hypertension) which later is decreased (hypotension) as a result of a decrease in cardiac output (see effect on the vascular system). Marginated neutrophils release their cytoplasmic granules containing proteolytic enzymes into the tissue causing injury to the pneumocytes and endothelium. This will allow marked protein leakage, haemorrhage and movement of phagocytes into the interstitium and alveoli. The extravascularised proteins and surfactant will form hyaline membranes which line the alveoli.  All these factors interact to result in a marked decrease in gaseous exchange within the lung leading to cyanosis (blue discolouration) and a respiratory acidosis which compounds the metabolic acidosis already present.

The severe systemic endothelial leakage, coagulopathy, decrease gaseous exchange in the lungs and decreased cardiac output leads to poor tissue perfusion and therefore multiple organ dysfunction as a result of hypoxia. Organs most susceptible to ischaemic damage are the heart, lungs, liver, brain, kidney and intestinal tract. There is also a species-associated organ susceptibility. For example, dogs are more susceptible to lung dysfunction whereas horses tend to develop more GIT dysfunction.

6. Effect on the gastrointestinal tract

Thomboxane A₂ reduces intestinal motility resulting in ileus and delays stomach emptying.

The pathophysiology of chronic endotoxaemia

1. Laminitis in horses

Laminitis is inflammation of the hoof lamellae and is diagnosed mainly in horses and cattle, but any hoofed animal is susceptible. About 10% of lameness in horses is due to laminitis. One of the causes of laminitis is endotoxaemia which often plagues animals that have recovered from endotoxaemia or those who were less severely affected.

Endotoxaemia can lead to increased inflammation of the hoof due to increased capillary pooling. The increase in inflammatory cytokines can result in the production of matrix metalloproteases which breakdown the basement membrane of the lamellae. This effect is likely to be minor. More likely is that the decreased nutrient and oxygen supply to the lamellae will result in ischaemic necrosis of the lamellae.

 2. Effect of endotoxins on the reproductive tract.

Endotoxins have a direct effect on the reproductive tract in that the production of prostaglandin F2 alpha and TNF increase uterine muscle contraction. They are also believed to decrease embryogenesis at the blastocyst stage. There is also a reduction in progesterone and an increase in cortisol. This leads to either embryonic absorption or abortion dependent on the stage of gestation.

Diagnosis of endotoxaemia

The diagnosis of endotoxaemia is dependent on the following:

1. Clinical examination: Fever early in disease, later on, animals may have a sub-normal temperature. Rapid breathing and heart rate, pulse may be rapid and weak. Mucosal bleedings; discolouration of mucosae (purple, deep red, pale); toxic gum line (red-blue line on the edge of the gums; decreased intestinal sounds; and affected organs that may have a gram-negative infection.  Diarrhoea may be present.
2. Haematology and blood chemistry: Leukopaenia, neutropaenia in early disease later on a left shift. Metabolic acidosis and lowered levels of oxygen in the blood (Blood-gas analyser). Increased bleeding time and other coagulopathies. A profound neutropaenia (which starts within an hour), with toxic neutrophils and a left shift is highly indicative of endotoxaemia.
3. ​​​​​Post-mortem examination: Endotoxin is rapidly cleared from the body, so evidence of a gram-negative infection with generalised congestion and haemorrhages.

If necessary – Measures of endotoxin: (these are often not practical to use)

1. Endotoxin activity assay = chemi-luminescence test. This is a test where anti-LPS IgM antibodies bind to Lipid A in a blood sample. This LPS-antiLPs antibody complex stimulates neutrophils to release oxygen radicals (oxidative burst). These radicals oxidise luminol that emits measurable light.
2. Lipopolysaccharide binding protein (LBP) is detected in a sandwich or competitive ELISA format. An electroluminescence marker measures the presence of LBP. Most tests are species specific. However, the competitive ELISA format allows multi-species testing.
3. sCD14 measurement in plasma uses a cytometric assay.
4. Limulus amoebocyte lysate assay. It is rarely done as the endotoxin is rapidly cleared from the bloodstream. However, it is used by manufacturers of intravenous infusions and vaccines to ensure that the levels of endotoxin are low in those products. Animals injected with products containing endotoxins can develop a fever response. This test exposes the amoeba naturally in the blood of the horseshoe crab to the test substance. If the amoeba die then endotoxin is present.

Treatment of endotoxaemia

(You will learn about this in BVSc4, so details are not essential to learn at this stage.)

1. Control source of endotoxin. Treat the colic, mastitis, pneumonia etc. Antibiotic therapy. Surgical removal of dead tissue.
2. Hydration and maintenance of oxygen perfusion of tissues. Increase the blood volume to ensure that the cardiac output is maintained. Check that the kidney is functional. Will also correct electrolyte balances that occur. Plasma can also be administered.
3. Hyperimmune plasma. Effect is controversial, more likely to protect animals at risk before clinical signs of endotoxaemia are present.
4. Polymixin B. This is an antibiotic that binds Lipid A allowing the Lipid A to be cleared from the body. It is usually administered intravenously to horses at low doses in the early stages of endotoxaemia. (It is highly toxic at bacteriocidal doses)
5. Non-steroidal anti-inflammatories. The cyclooxygenase inhibitor, Flunixin reduces the production of vasoactive amines in a hydrated animals. A specific anti COX2 firocoxib has a similar effect.  DMSO an antioxidant may have a mild positive effect.
6. Steroidal antiinflammatories. Studies on the cortisones are controversial, whilst there is some improvement in haemodynamics, the long-term survival of animals is not improved. Not advised for horses.
7. Treatment of laminitis in horses – ice packs decrease neutrophil activity and the build-up of inflammatory cytokines. Inhibitors of neutrophil adhesion to endothelial cells – experimental.
8. There are a number of antiinflammatory agonists entering the market. Some may be beneficial such as ethyl pyruvate, beta-agonists and ketamine that can be administered as adjunct therapies.