Leptospirosis

Question 1

Describe structure of leptospires

Answer 1

• Leptospires are thin, flexible, filamentous bacteria made up of fine spirals with hook-shaped ends
• Cytoplasmic / protoplasmic cylinder wound around a straight central axial filament
• They have two non-overlapping longitudinally orientated flagella
• All contained by a layered outer envelope / membrane
• Leptospires are motile
• The peptidoglycan cell wall is closely associated with the inner membrane with lipoproteins and transmembrane proteins
• The outer membrane is composed of LPS

Question 2

Describe the aetiology of leptospirosis in dogs

Answer 2

• Leptospires were originally classified based on culture and immunological reactivity
• Now classified based on their molecular characteristics
• Over 250 serovars of L. interrogans described
• Serovars are further classified into antigenically related serogroups
  
  • Serogroups share common antigens and can cross react with antibody detection methods
• There is widespread genetic variation between the serovars from different geographical regions
• Lepto. is maintained in the environment in mammalian maintenace hosts
  
  • Different serogroups tend to inhabit a narrow range of primary reservoir hosts

Question 3

Describe the modes of transmission that are relevant for canine leptospirosis

Answer 3

• Leptispirosis can be transmitted through both direct or more commonly indirect contact

Direct:

• Bite wounds, contact with urine, venereal or transplacental transfer, ingestion of infected tissues
• Crowding increases the risk - poorly documented
• Recovered or subclinically infected animals may shed the bacteria for days to months

Indirect:

• Exposure via contaminated water, soil or food
• Spirochete may remain viable for months in the environment in moist soil
• Stagnant or slow moving warm water is ideal for survival
• Leptospires form a biofilm in aquatic environments on organic matter
• Survives best at 0-25 Celsius

Question 4

Describe the method of infection and initial replication prior to the onset of clinical disease with leptospirosis

Answer 4

• Most infection is by indirect means but the leptospires must contact and penetrate a mucosal surface - mouth, nose, eyes - or softened/abraded skin
• Upon entry, the warm body environment causes transcriptional changes that enhance pathogenicity
• Rapid multiplication occurs in the vascular space with subsequent spread to multiple tissues/organs
  
  • Liver, kidneys, spleen, CNS, eye, genital tract
• Incubation period prior to signs is ~ 7 days but varies with
  
  • Infective dose
  • Host immunity
  • Variations in the infecting strain

Question 5

Describe the initial canine host immune response to leptospirosis infection

Answer 5

• Dogs develop a specific antibody (humoral) based immune response within 7-8 days of initial infection
  
  • An early, strong antibody response is essential for clearance of the infection
• With an appropriate immune response, the infection can be cleared from most tissues
  
  • Spirochetes may persist in the kidney and be shed for days to months

Question 6

Describe the potential clinical presentations for dogs with leptospirosis

Answer 6

• Subclinical infection occurs with seroconversion but variable shedding of the organism
• Dogs with acute severe infection can present with sepsis triggered by release of endotoxin
  
  • Tissue oedema, vasculitis, haemorrhagic events
• Predominantly renal or hepatic presentations vary depending on serovars
  
  • Though the majority of these studies were based on serological diagnoses which carries a significant chance of error
• Renal colonisation - within the renal tubular epithelial cells after 2 weeks. Onset of urine shedding
  
  • Leads to interstitial nephritis

Question 7

Describe the process by which leptospirosis causes renal injury

Answer 7

• Leptospirosis infection initially occurs via mucosal penetration or otherwise
• Leptospires replicate within the vascular space and are disseminated throughout the body
• The initial leptospiremia can be associated with systemic vsculitis, SIRS and sepsis
  
  • This process can cause indirect renal injury due to microvascular disease, reduce renal perfusion, hypotension and tubular cell necrosis (high O₂ demand)
• Following leptospiremia, antibodies are generated.
  
  • Antibody/antigen complex disease can contribute to glomerular injury
• Leptospires eventually pass through the renal capillaries and enter the interstitum. By 2 weeks post-infection they have migrated into the proximal tubular cells and can be identified within the tubular lumen (shedding)
  
  • The presence of leptispires in the tubular cells can be directly toxic
  • Tubular casts caused by increased glomerular filtration of protein and immune complex disease can further cause tubular cell damage

Question 8

What is the mechanism for increased potassium loss seen in many dogs with leptospirosis?

Answer 8

• Unsaturated fatty acids in the glycolipid fraction from leptospires specifically inhibits the Na⁺K⁺ ATPase pump
• This leads to increased intracellular sodium levels
• Reduced sodium uptake from the tubular fluid at the proximal convoluted tubule leads to increased delivery of sodium to the MD and enhanced glomerular blood flow
  
  • Further promotes increased tubular fluid flow
• Increased tubular fluid flow increases the potassium delivery to the distal tubule and collecting ducts
• In the principal cells of the collecting duct, sodium is resorbed in exchange for potassium
  
  • Increased potassium excretion and increased urine output

Question 9

Describe the potential liver changes that commonly accompany leptospirosis in dogs

Answer 9

• Liver can be damaged by the generalised vasculitis process during initial leptospiremia
• Certain leptospires prodice toxins that can cause major hepatic dysfunction due to subcellular damage
  
  • Icterus in canine leptospirosis is typically correlated with the degree of liver damage as haemolysis does not occur
• Serovar Grippotyphosa has been associated with the development of chronic active hepatitis
• Likely that initial cellular injury +/- persistence of the organism in the liver contribute the the damage
  
  • Altered circulation
  • Hepatitis and fibrosis
  • Immunological disturbances
    
    • All lead to chronic inflammation and this can lead to severe fibrosis and cirrhosis

Question 10

Describe the potential severe clinical signs at presentation for dogs with acute leptospirosis prior to the onset of renal / hepatic injury

Answer 10

• Fever, shivering and muscle pain are earliest clinical signs
• Vomiting, dehydration and vasculitis / SIRS can occur
  
  • Tachypnoea, rapid / irregular pulse / poor CRT
• Coagulation deficits can become apparent
  
  • haematemesis, haematochezia, melena, epistaxis, petechiae

Death can occur by this point due to sepsis - prior to the development of hepatic or renal injury

Question 11

Describe the systemic clinical signs that can be seen with acute canine leptospiral infection following the initial SIRS/sepsis response

Answer 11

• If a dog survives the initial leptosiremia and sepsis event, then widespread parenchymal injury can follow
• Renal injury
  
  • Renomegaly, pain and signs of AKI
• Hepatic injury
  
  • Icterus and intrahepatic cholestasis
  • With severity and chronicity, chronic hepatitis, hepatic fibrosis and liver failure
• Respiratory signs
  
  • Coughing and dyspnoea
  • Interstitial pneumonia and pulmonary haemorrhage
• Gastrointestinal signs
  
  • Vomiting / melena / haematochezia
  • Intussusception
• Cardiac manifestations
  
  • Ventricular arrhythmia and myocardial damage

Question 12

Describe the standard serological method for diagnosing leptospirosis infection in dogs

Answer 12

• Microscopic agglutination test
  
  • Requires growth and cultivation of various serogroup spirochetes in lab
  • Requires dark-field microscopy
• The sprichetes are grown in liquid media
• The patient’s serum is then exposed to the spirochetes at serial dilutions
  
  • When antibodies are present, they will cause microscopic agglutination of the spirochetes - this can be visualised using dark field microscopy
• Initial screening at 1:100
  
  • For those that are positive at this dilution, further two-fold dilutions are performed
• The highest reported titre is ofter 1:3200 - and suggests a high portion of antibody is present directed towards that particular serogroup.
• The test is serogroup specific and cannot differentiate serovars

Question 13

Describe the interpretation of and sensitivity and specificity findings for the MAT in diagnosing leptospirosis.

How might the sensitivity of the MAT test be improved?

Answer 13

• The MAT identifies antibodies to specific leptospiral antigen
  
  • False negative results can be seen if the infective serogroup is not included in the test panel
  • False negative results may occur early in the course of disease prior to the development of Ab (< 7-8 days)
• A single test with a cut-off of 800 (1:800) has been reported to have a sensitivity of 22-67% when compared to identification of a rising titre on repeated testing (3-week interval)
• Identification of a four-fold decrease in titre following antimicrobial therapy is also supportive of an active infection
• The titre rise may be brief or may not occur with rapid and appropriate treatment.

Question 14

Describe the limitations of dark-field microscopy and culture in the diagnosis of leptospirosis in dogs

Answer 14

• Leptospires require special transport media and can be killed with in appropriate handling
• Innoculation of an appropriate culture bottle patient side is required.
• Culture can take 3-6 months for results to be returned (or to have a negative result returned)
• Culturing pathogenic organisms is hazardous
• Following culture, serological or PCR testing is still required to accurately identify the serovar for epidemiological purposes
• Dark-filed microscopy requires large numbers of intact live motile organisms
  
  • Fibrins strands, cellular fibrils and other bacteria can be mistaken for leptospires
  • Not recommended as a singular test