Rickettsia, Ehrlichia, Anaplasma and Chlamydia

Question 1

Describe the eitiology and epidemiology of Neorickettsia Helminthoeca infection, the cause of salmon poisoning disease

Answer 1

• The disease is transmitted by helminths and is endemic in the western slopes of the Cascade mountains from Washington to Colorado
• The Neorickettsial agent is a coccoid to coccobacilli to cresent shaped gram negative rickettsia
• The agent fills replicates within the cells of the mononuclear phagocyte system and often fills the cytoplasm
• The disease is carried within flukes - encysted trematode larvae
• The neorickettsial infection generally occurs when dogs eat raw salmon that harbours the fluke

Question 2

Describe the fluke lifecycle and how that might affect transmission of Neorickettsia helminthoeca.

Answer 2

• The trematode Nanophyetus salmincola harbours the neorickettsial organism throughout its lifecycle
• Lifecycle involves 3 intermediate hosts
  
  • The first intermediate host is the snail oxytrema silicula
    
    • ​The cerceriae larvae leave the snail and swim to infect the intermediate host
  • The metacercariae develop in the second intermediate host - a range of fish - salmon is most prolifically involved
    
    • The trematode can remain present throughout the salmon life including migration into salt water
  • The definitive host are numerous carnivores including dogs, cats and humans
    *

Question 3

Describe the pathogenesis of salmon poisoning disease

Answer 3

• The metacerciae infested fish is ingested
  
  • The trematode develops in the definitive host and innoculates the host with the Neorickettsial organism - likely through intestinal attachment
• The rickettsial organism initially occurs in the villus epithelial cells or the GALT/MALT
• Rickettsiae enter the blood and spread to the lymph nodes, thymus, spleen, tonsils, lungs and brain
  
  • Sepsis may result from intestinal wall damage
  • Severe GI signs may be seen - hypoalbuminemia, anaemia, haematochezia and diarrhoea

Question 4

Describe the clinical findings in dogs with Salmon Poisoning Disease

Answer 4

• Fever typically 5-7 days following ingestion of raw fish
  
  • Anorexia and depression also occur
• Marked and rapid weight loss can be seen
• Mild GIT signs that progress to severe haemorrhagic diarrhoea towards the terminal phase of the disease
  
  • May see initial severe GIT signs similar to CPV infection
• Serous nasal discharge and neurological signs may be seen
• Enlarged lymph nodes may be evident from 5 days post-infection (at the outset of clinical signs)

Question 5

How can a diagnosis of salmon poisoning disease be confirmed?

Answer 5

• Thrombocytopenia (88%), lymphopenia (77%) and eosinophilia (77%) are common
• Increases in ALKP (64%) and decreases in albumin (49%) are also common
• Trematode eggs may be seen in the faeces ~5-8 days following ingestion - does not confirm the rickettsial disease
• Organisms can be seen within macrophages aspirated from lymph nodes when appropriately stained
• PCR of lymph node aspirates or rectal smears can identify the rickettsial organism - sensitivity and specificity studies are required

Question 6

Briefly describe the therapy and treatment for dogs with salmon poisoning disease

Answer 6

• The neorickettsial organism can be effectively treated with doxycycline for 1-2 weeks
• The systemic signs should be treated supportively
  
  • Sepsis managed with appropriate broad spectrum antibiotics
  • Fluid therapy
  • Anti-emetics
  • Gastro-protection - H⁺ pump inhibitors and sucralfate
• Praziquantel to treat the trematode infestation

Question 7

Describe the pathogeneis of Wolbachia as it relates to D. Immitis infection in dogs

Answer 7

• 100% of D. Immitis harbour wolbachia with the organism being present in all life stages
• Wolbachia surface proteins (WSP) stimulate IL-8 transcription which stimulates neutrophil chemotaxis in dogs
• The presence of WSP in the pulmonary arteries and more distantly in the glomerus may explain in part the pulmonary vascular and glomerular changes that occur with D Immitis infection
• WSP also likely stimulates other inflammatory mediators

Question 8

Discuss the etiology and epidemiology of Ehrlichia Canis infection

Answer 8

• Ehrlichia Canis is the organism responsible for the disease Canine Monocytotropic Ehrlichiosis (CME)
• Ehrlichia Canis is an obligate intracellular, pleomorphic, gram negative bacterium
• Infects the monocytes and macrophages forming clusters called morulae
• Worldwide distribution
• Dogs are considered a reservoir host
• Arthropod vector is Rhipicephalus sanguineus
• Transmission is transstadial but not to the maternal eggs
• Ticks acquire larval stages by feeding on rickettsemic dogs and can transmit infection for at least 155 days
  
  • The organism can “over-winter” in the tick awaiting the spring season to transmit the organism

Question 9

Describe the pathogenesis of Ehrlichia Canis with regards to initial infection and replication

Answer 9

• E. Canis contains a single circular chromosome
  
  • Many genes are encoded
  • Minimal metabolic pathway enzyme genes - uses host cytoplasmic enzymes for metabolism
• Has numerous pores or channels in the outer membrane regulated by the genome
  
  • These allow uptake and regulation of nutrient uptake during intracellular development
• Incubation period is 8-20 days
• Replication by binary fission occurs within the macrophages/monocytes within membrane bound vacuoles
  
  • Direct cell-cell spread may occur through cytoplasmic projections
  • Late stage morulae may lead to host cell rupture and spread into the surrounding environment
    *

Question 10

What mechanisms allow E canis to avoid detection by the immune system and minimise immune stimulation

Answer 10

• E Canis lack lipopolysaccharide and peptidoglycan in the cell wall
  
  • Poor wall strength
  • Less activation of TLR that rapidly respond to LPS and peptidoglycan
• Multiplication in sub-membrane vacuoles helps the organism evade the immune system and cellular lysosymes
• E Canis can induce down-regulation of MHC II
  
  • This interferes with antigen recognition and presentation, cell-cell adhesion, cytokine production, humoral reactions
• Inhibition of phagosome / lysosome function
  
  • This is one of the targets of oxytetracycline
    
    • Inhibits synthesis of a fusion hindering protein

Question 11

Describe the three stages of infection with Ehrlichia Canis

Answer 11

1. Acute
   
   • Lasts 1-4 weeks
   • Infection can be cleared at this point with appropriate management
2. Subclinical
   
   • Occurs with inadequate management of the acute disease
   • Dogs may remain in this stage for months to years
   • Clinically healthy carrier status
   • Thrombocytopenia persists during this phase
   • Likely that the spleen harbours the infection primarily
3. Chronic
   
   • Not all dogs develop chronic severe disease with spontaneous recovery reported
   • Specific conditions leading to development of the chronic disease are unclear
   • Severe pancytopenia due to bone marrow hypoplasia
   • Haematological abnormalities due to both chronic inflammatory and immune mediated mechanisms

Question 12

Describe the clinical findings and pathophysiology for the chronic severe form of Ehrlichia canis infection

Answer 12

• Severe pancytopenia caused by a combination of:
  
  • Bone marrow hypoplasia
  • Chronic inflammation
  • Immune mediate processes
• Thrombocytopenia is the most common abnormality
  
  • Increased platelet consumption
  • Decreased platelet half-life
    
    • Sequestration and immune mediate destruction within the spleen
  • Platelet auto-antibodies have been identified
  • Platelet migration-inhibition factor
    
    • Produced by lymphocytes once exposed to infected monocytes
• Platelet dysfunction / thrombocytopathy
  
  • Exacerbates the clinical signs associated with thrombocytopenia
• Gammaglobulinemia
  
  • Antibodies produced against E canis are ineffectual
  • These antibodies may contribute to hyperviscosity
  • The gammaglobulinaemia is usually polyclonal and not only due to Ab directed against E canis
• Extensive plasma cell infiltration of bone marrow and parenchymal organs
  
  • Hyperimmune mechanisms

Question 13

Describe the pathophysiological immune response / mechanisms involved in the clinical course of chronic severe E Canis infection

Answer 13

• Hyperimmune mechanisms are likely present
  
  • Extensive plasma cell infiltration
  • Ab titres do not correlate with the high gammaglobulinemia
• Immune complex disease plays a part
  
  • Coombs test positive
  • Auto-agglutination positive
• IgG2 is the principle antibody produced and appears around day 15 following initial infection
  
  • IgG 2 has been shown to be more efficient at inducing an auto-antibody response in a mouse model
  • Class switching helps promote a Th1 response and production of INF-g and TNF-a
• Cell mediated immunity plays a major role - hence GSD are more susceptible to the disease than others

Question 14

Describe the potential clinical signs in dogs with chronic Ehrlichia canis infection

Answer 14

1. Multisystemic signs
   
   • Bleeding diathesis - petechiae or eccymoses
   • Epistaxis
   • Lymphadenomegly
   • Splenomegaly
2. Ocular signs
   
   • Hyphaemia
   • Retinal haemorrhage
   • Retinal detachment
   • Chorioretinitis
   • Anterior uveitis
3. Neuromuscular signs
   
   • Meningitis / meningeal haemorrhage
   • CNS or PNS signs depend on the area and severity of the nervous system that is affected
4. Concurrent infection
   
   • Especially other tick borne diseases
5. Cardiac disease
   
   • Myocardial injury could cocur

Question 15

List and briefly describe the available diagnostic tests for canine Ehrlichia Canis

Answer 15

• Routine haematology
  
  • Thrombocytopenia +/- pancytopenia
  • May see a well differentiated granular lymphocytosis
• Routine biochemistry
  
  • Elevated globulin (usually polyclonal)
  • Decreased albumin
  • Elevated liver enzymes
• Coagulation studies
  
  • Prolonged bleeding times
• Cytology
  
  • Can identify morulae in monocytes
    
    • Blood smears / buffy coat smears
    • Tissue aspirates - lymph node, spleen
  • May see plasmacytosis
• Urine
  
  • Increased UP:Cr
• Fluorescent antibody testing
  
  • Serological gold standard, but not perfect
  • IgG tested - does not appear until ~15 days post infection
  • Positive titre may represent current acute illness, recovered illness or persistent subclinical infection
  • Needs to be interpreted with history and clinical signs as antibodies can persist for ~1 year
• ELISA (point of care)
  
  • Multiple antigens assessed
• PCR
  
  • Blood PCR may be insensitive
  • Splenic or tissue aspirates
  • Real-time PCR most useful
  • Ideal to assess for clearance of an infection when FA positive antibodies are high following treatment

Question 16

Describe the treatment options for the various phases of canine monocytotropic ehrlichiosis caused by Ehrlichia Canis

Answer 16

• Acute phase
  
  • Tetracyclines and chloramphenicol are effective
  • Doxycycline at 10 mg PO q 24 hours for ~ 4 weeks is recommended
  • The earlier the treatment is initiated, the better the chance of clinical resolution
  • Clinical improvement typically starts from 24-48 hours post initiation of antibiotic therapy
  • Supportive care is also necessary
    
    • Blood products incl platelet rich plasma
    • Fluid therapy
    • Desmopressin acetate to stabilise platelet function. Water deprivation to avoid overhydration
  • Short course of glucocorticoids - 1-2 mg/kg q 24
• Subclinical phase
  
  • Monitor platelet counts
    
    • Generally returns to normal in 10-14 days
  • Proteins and antibody titres can remain elevated for up to 12 months
• Chronic phase
  
  • Treatment can be attempted but is generally unrewarding.
  • Multisystemic inflammatory disease with bone marrow hypoplasia contributes to death

Question 17

Describe the important aspects of and options for prevention of Ehrlichia Canis infection in dogs

Answer 17

• No vaccination is available for E canis
• The primary method of prevention is adequate tick control
• In endemic areas or for kenneled dogs, daily doxycycline at 6.6 mg/kg PO may prophylactically prevent initial infection
  
  • 100 mg per dog has been shown effective in a study from the Middle East and Africa
• With additions to a kennel, tick treatment and E Canis serology should be performed with isolation until results are at hand
  
  • If seropositive in a dog without clinical signs, then PCR from a splenic aspirate can be used to confirm current infection
  • Alternatively, if positive, treatment could be administered in conjunction with isolation
• Care with transfusions. Testing of blood donors in endemic areas is recommended.

Question 18

Briefly contrast Ehrlichia ewingii with Ehrlichia Canis infection

Answer 18

• E. Ewingii is spread primarily by the Lone Star tick - A. americanum
  
  • ​R. sanguineus may also spread this disease as for E canis
• E. Ewingii is the cause of canine granulocytotropic ehrlichia
  
  • Infects granulocytes compared with monocytes
• The distribution of the disease varies due to the tick location - primarily in the south east of North America, compared with a worldwide distribution
• White-tailed deer are the primary reservoir species
  
  • Numerous potential reservoir mammals have been identified for E canis
• The clinical disease is acute rather than chronic and therefore occurs seasonally in line with the tick lifecycle
• The organism can delay neutrophil apoptosis extending the neutrophil life to allow for replication
• As for E canis, immunosuppression predisposes to infection

Question 19

Briefly document the aetiology of Anaplasma phagocytophilum infection

Answer 19

• A phagocytophilum is a gram-negative, coccoid to ellipsoid bacteria that lack a cell wall. They are obligate aerobes and lack a glycolytic pathway - thus obligate intracellular parasites
• Similar to Ehrlichia, they have no LPS or peptidoglycan within the outer cell membrane
• They inhabit cell membrane derived vacuoles in immature or mature haematopoietic cells of mammalian hosts
• A phagocytophilum preferentially infects neutrophils and replicates via binary fision

Question 20

List and briefly describe the vectors, hosts and geographic range for Anaplasma phagocytophilum

Answer 20

• The primary vector for A phagocytophilum are Ixodes ticks
• A large number of small mammals (including mice, voles, woodrat, squirrel and chipmunk) together with deer have been implicated as natural reservoir hosts
  
  • Birds may help to distribute ticks geographically
• The infection is primarily seen in the regions of ixodes tick habitation - certain segments of North America and eurasia (Northern Hemishpere)
• Co-infections with other tick transmitted diseases are common, especially Borrelia burgdorferi

Question 21

Briefly describe the pathogenesis of A**naplasma phagocytophilum

Answer 21

• Spread by ticks within 24-48 hours of initiation of tick attachment.
  
  • As the organism resides in the salivary glands infection may occur sooner
• Incubation period is 1-2 weeks
• Early replication, cellular adhesion and internalisation, secretory transport, endothelial adhesion, inhibition of apoptosis, evasion of the immune system, multiplication and release
• The clinical disease is associated with mild to moderate thrombocytopenia, lymphopenia and mild anaemia
  
  • Specific mechanisms of causation not completely known
• Many alterations in cytokine profiles are describe including over production of IL-8
  
  • Alterations in blood cell migration / chemotaxis and bone marrow suppressive

Question 22

Briefly detail the etiology and epidemiology of the cause for Rocky Mountain Spotted Fever

Answer 22

• RMSF is caused by rickettsia rickettsii, a tick born obligate intracellular bacteria
• The bacteria is primarily located and the disease prevalent in regions aligned with the tick population
  
  • Dermacentor andersoni and Dermacentor variabilis are the natural tick vectors for R rickettsii
• The disease is seen in most states of the USA, southern Canada, Mexico, Central America and northern South America
• Transfer between ticks can occur horizontally when they feed on infected mammals during acute infection and rickettsemia
• Ticks can also be infected transstadially or transovarially through generations
• Delayed transmission between the infected tick and new host is incompletely understood - 5-20 hours after attachment

Question 23

Describe the basic pathophysiological mechanism of disease caused by Rickettsia rickettsii in Rocky Mountain Spotted Fever

Answer 23

• The rickettsial organism is transmitted by the bite of an infected Dermacentor tick
• The bacteria is disseminated in the blood and replicates within the endothelial cells of the small arteries and venules
  
  • Replication triggers endothelial cell inflammation, activation of platelets and the coagulation cascade together with complement
  • Widespread coagulation cause reductions in plasminogen, antithrombin III and increased FDPs
  • Coagulation consumption is not tyically severe enough to cause DIC
• Elevated platelet associated Ig levels - immune trigger for platelet destruction
• Progressive necrotizing vasculitis can lead to thrombocytopenia and thrombosis
• Increased aldosterone and ADH levels have been described in people
  
  • Hyponatremia
  • Increased ECV and fluid overload
  • Central oedema and increased intracranial pressure
• Peripheral vascular collapse may occur in severe infection

Question 24

List the major clinical signs associated with Rocky Mountain Spotted Fever caused by Rickettsia rickettsii

Answer 24

• The disease occurs seasonally in line with tick activity
• Subclinical infection with seropositivty common in dogs from endemic areas
• Fever (within 2-3 days of tick attachment)
• Early cutaneous lesions
  
  • hyperemia of the lips, penile sheath, pinna, extremities, ventral abdomen (rare)
• Scrotal / epididymal swelling
• Stiff gait and joint/muscle pain/swelling
• Pettechial and ecchymotic haemorrhages
  
  • Usually mucosal with retinal lesions most common
• Neurological signs - meningitis / encephalomyelitis are seen in severe cases or where treatment is delayed
• Necrosis of the extremities and permanent organ damage can occur from thrombosis and advanced vascular disease
• Shock, cardiovascular collapse and oliguria are common in the terminal phase of the disease

Question 25

List the common clinical pathological abnormalities seen with acute Rocky Mountain Spotted Fever in dogs

Answer 25

• Mild to moderate thrombocytopenia
• Mild coagulopathy - prolonged ACT
  
  • Can see coagulation profile consistent with early DIC
• Hyperglycemia
• Increased liver enzymes - ALT, ALP, AST
• Hypoalbuminemia
  
  • Due to vascular inflammation and leakage
• Hypercholesterolemia
• Hyponatremia, hypochloremia, metabolic acidosis
• Proteinuria +/- bilirubinuria

Question 26

What tests can be utilised to confirm a suspected diagnosis of Rickettsia rickettsii infection?

Answer 26

1. Serology
   
   • Increased IgM or a four-fold increase in IgG
   • Micro-IF - indirectefluorescent antibody test or ELISA
     
     • These kits can idetify and quantify IgG or IgM
   • IgG does not increase until 2-3 weeks post infection
   • IgG titres generally decrease in 3-5 months after active infection
2. Direct immunodetection
   
   • Immunohistochemistry of biopsy tissue containing significant vasculitis lesion.
   • Can detected ~ 70% of infections - moderate sensitivity. Very good specificity
   • Biopsy from mucosal haemorrhages or vesicles is most useful
   • Can diagnose the disease from days 3-4 of the disease
3. Genetic detection - PCR
   
   • Variable sensitivities depending on the assay and primer that is used
   • Low sensitivity on serum tests may be attributed to low numbers of organisms circulating in the blood
4. Rickettsial isolation
   
   • For laboratory use

Question 27

Briefly describe the etiology of a typical chlamydial infection

Answer 27

• Chlamydia are obligate intracellular bacteria that exist in two main forms
• Elementary bodies are infectious and contain a rigid cell wall
  
  • Transient extracellular migration to infect new cells
• Once the cell is entered they transform into a larger vegetative form - reticulate body
• The reticulate body lacks a cell wall and are non-infectious
• Replication occurs via budding and fission within the vesicle
• The RB’s then transform / develop into a large membrane bound population of elementary bodies within ~ 2 days
• Cell lysis allows the new EBs to infect adjacent cells
• Spread is generally via mucosal contact or via aerosols
• Immunity involves cellular and humoral mechanism

Question 28

Discuss the pathogenesis of Chlamydophyla felis infection in cats

Answer 28

• Cp felis is primarily a pathogen of the upper respiratory tract and conjunctiva
  
  • Signs therefore include conjunctivits and nasal signs
• The organism can spread systemically and infect many organs
• The initial febrile response is due to chlamydemia and release of Il-1, IL-6, TNF and interferons
• The infection can become chronic and insidious
  
  • Chronic disease may be due to carrier status or repeat exposure
• Assymptomatic carrier status occurs
• Atypical reticulate bodies have been identified in people with arthritis…
• Shedding from the reproductive tract occurs within 1 week of conjunctival innoculation if > 4-6 months of age
• Reproductive shedding may increase in late pregnancy

Question 29

Describe the common clinical features of Chlamydophila felis infection in cats

Answer 29

• Conjunctivitis is the most common clinical sign - blepharospasm, conjunctival hyperemia, chemosis ocular discharge
  
  • Acute, chronic and recurrent
  • Corneal disease is rare
  • Concurrent viral diseases are common
• Most cats remain clinically well
• Subclinical pneumonia may occur
• Nasal signs may be present, but are rarely present alone without conjunctivitis
• Systemic signs are possible but rare and include a polyarthritis like syndrome
• Reproductive signs and shedding can occur
  
  • Reports of reproductive signs have generally been limited to experimental studies

Question 30

Discuss the available laboratory methods for confirming a clinical diagnosis of Chlamydophila felis

Answer 30

• Organism cultivation
  
  • Generally reserved for research
  • Requires vigorous swabbing to obtain epithelial cells containing the reticulate bodies
• Microscopy and Enzyme-linked immunosorbent assay detection
  
  • Direct FA techniques using MABs are more specific that attempting cytological diagnosis
  • ELISA - variable to low sensitivity and specificity
• Nucleic Acid detection - PCR
  
  • Rapid and sensitive
  • Allow speciation
  • 87.5% sensitive in the first month of infection
  • 72.9% sensitive in the second month
  • Special transport media not required as non-viable organisms can be detected
• Serological testing
  
  • Not particularly helpful due to highly variable results
  • High titres likely to reflect active infection
  • High titres can be seen with persistent infections
  • Seronegative animals are unlikely to be carriers

Question 31

Briefly discuss the usefulness and potential complications of the chlamydia vaccination in cats.

When is vaccination indicated?

Answer 31

• MLV provides better protection than the inactivated vaccines
• Colonisation of the mucosae is not completely prevented
• Replication is minimized following vaccination, therefore clinical signs are reduced
• Transmission to other cats can still occur
• Post-vaccine fever, anorexia, lethargy and limb soreness has been seen 7-21 days post-vaccine with the combined vaccines
• These vaccines are only indicated for cats in a high risk / high prevlaence environment
• Even then, vaccination needs to be accompanied by an appropriate treatment regime of all in contact cats
• Hygiene and isolation with treatment work well to minimise the risk in cattery situations