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1

Generals

 Obligate intracellular Gram‐negative bacteria

 Bacteria within arthropods

Family Anaplasmataceae

 Genus Anaplasma
 Survive in erythrocytes, phagocytes and platelets

 Genus Ehrlichia
 Survive in phagocytic cells

 Genus Neorickettsia
 Survive in macrophages and mononuclear cells

Family Rickettsiaceae
 Rickettsiae (Survive in vascular epithelium)

 Genera Rickettsia, Wolbachia, Orientia

2

Bacterial reclassification- Not super important

 Reorganized by sequence comparison of 16S rRNA gene

 α‐ Proteobacteria in the genera:  Neorickettsia
 Anaplasma
 Ehrlichia

3

Genus Anaplasma

 Gram negative small coccoid – ellipsoid bacteria

 Live within cytoplasmic vacuoles of myeloid cells, neutrophils and erythrocytes- PROTECTS FROM IMMUNE SYSTEM

 Peripheral blood or tissues of mononuclear phagocyte organs

 May cause anemia, thrombocytopenia, leukopenia

 Single or in morulae (bacterial packets)

 Wright‐Giemsa (bluish purple)

 Anaplasma marginale

 Anaplasma centrale
 Anaplasma ovis
 Anaplasma bovis

Bovine anaplasmosis

 Formerly Ehrlichia bovis  Anaplasma platys

 Formerly Ehrlichia platys

 Anaplasma phagocytophilum
 Formerly Ehrlichia phagocytophilum, E. equi and E. phagocytophila

4

Anaplasma marginale

 Reservoir and transmission

 Infected ruminants

 Wild (deer) and domestic species

 REPORTABLE DISEASES

 All continents
 Biologic transmission with hard ticks

Boophilus microplus

Boophilus, Dermacentor, Ixodes and Rhipicephalus

 Mechanical transmission (less significant)

 Biting flies, contaminated fomites
 Contaminated instruments

5

Pathogenesis of Bovine Anaplasmosis

 Clinical signs and pathological changes

 Subclinical to peracutely fatal presentations

 Mortalities of ~50% in cattle >3 years of age

 Persistent infection, undulating febrile disease

 Depression, anorexia, fever, anemia and icterus

 Long term carriage occur

ERTHOCYTES-THUS ANEMIA

6

Bovine Anaplasmosis Immune Response

 Immune response
 Humoral and cell‐mediated response

 Antigenic variation present

 Msp2 gene
 Related to chronicity

 Antibody response is also directed to host antigens

 DESTRUCTION OF ERYTHROCYTES

 Immune response can be shared between other Anaplasma

 Anaplasma centrale

 Experimental vaccines are available but are not USDA approved

7

Diagnosis of Bovine Anaplasmosis

 Diagnosis
 Routine blood stains, acridine orange or IFA

 Appear purple structures near periphery of erythrocytes in GIEMSA stains

 Molecular diagnosis

 Serological methods

 Complement fixation, capillary agglutination, ELISA  Useful in detecting subclinical cases

 Treatment and control
Tetracyclines are pretty effective

 Vaccination and VECTOR CONTROL

8

Anaplasma marginale in bovine erythocytes

9

Anaplasma phagocytophilum

generals

 Human granulocytic anaplasmosis

 Tick borne fever

 Domesticated and wild ruminants

 Equine granulocytic anaplasmosis

 

dogs and horses

10

Anaplasma phagocytophilium

reservior and transmission

 Reservoir and transmission

 Rodents and wildlife (deer)

 In North America
 East is mainly the white‐footed mouse and the deer tick (Ixodes

scapularis)
 West is mainly the western black‐legged tick (Ixodes pacificus)

11

A. phagocytophilium  causes, pathogenesis

 Causes tick‐borne fever primarily in ruminants (EUROPE) and in horses, dogs and humans in North America

 Pathogenesis associated with infection of neutrophils (primary) and eosinophils

 Tick pyemia (Staphylococcus infection) is commonly linked to tick‐borne fever

 Disease is mediated by the host immune response

12

Clinical and Pathological Findings for A. phagocytophilium

 Clinically
 Fever, depression, inappetance, anemia, edema, ataxia,

icterus, drop in milk yield, abortions, and leukopenia

 Immunosuppressive effects

 Pathological findings

 Hepatitis, splenomegaly, arthritis, paracortical hyperplasia in lymph nodes

13

A. phagocytophilium Vaccine and immunoaspects

 Immunological aspects

 Antigenic variability

 Major surface proteins (Msp2)

 Serological cross‐reactivity

 Ehrlichia spp, other Anaplasma spp

.  No vaccines

 Recovery with doxycycline therapy and adequate immune responses and tick control

14

A. phagocytophliium diagnosis

 Laboratory diagnosis
 In GIEMSA or Wright’s stained blood smears

 Appear like membrane‐bound morulae (1‐10 bacteria) within neutrophils of ruminants, dogs, horses and humans

 Has been propagated in tick and human leukemic cell cultures

 Serological methods  IFA, ELISA

 Molecular methods  PCR

15

A. platys

PLATLETS!

 Infectious canine cyclic thrombocytopenia

 Cycles of 1‐2 weeks interval

 Fever, lethargy, pale mucous membranes, petechial hemorrhages, epistaxis, and lymphadenopathy

 Co‐infections with Ehrlichia canis common

 Reservoir and transmission

 Rhipicephalus and Dermacentor ticks

 Laboratory diagnosis

 In GIEMSA stained blood smears
 A. platys in surface of canine platelets

 IFA, PCR

16

Rickettsiae

 Small bacteria (0.5‐1 μm)

 Gram‐negative bacteria

 Better to stain with Gimenez (red), Macchiavello (red) or Giemsa (purple) stains

 Non‐motile bacteria

 Actin hijack inside cells

 Pathogenesis includes

 Enter endothelial cells by endocytosis

 Escape from phagosome and multiply in cytoplasm and nucleus  Associated with invertebrate vectors

 In veterinary medicine

 Rickettsia rickettsii

 Rickettsia felis
 Coxiella burnetii

Rocky mountain spotted fever Typhus group
Q fever

17

Reserviors and Transmission for Rickettsia rickettsii

 Causative agent of Rocky Mountain Spotted Fever

 Reservoir and transmission

 Dogs and people in endemic areas
 Small mammals are though to be the major reservoir

 Carried naturally by ~20 species of ixodid ticks

 Dermacentor andersoni (Wood tick)

Wood tick

 D. variabilis (American dog tick)
 Transovarial and transtadial transmission- CAN GO FROM MOTHER TO EGGS

in ticks exist

American dog tick

 Mainly in Eastern North America  Seasonal incidence

18

Pathogensis of Rickettsia rickettsii

 Tick
 Replicates in epithelium
 Transferred to salivary glands and ovarian tissues

 Vertebrate
 Ticks bites injects bacteria and targets vascular endothelium

 Endocytosed, escape phagosome and multiply in cell cytoplasm and nucleus

 Damage of endothelial cell membranes

 Rickettsial phospholipases and proteases

 Necrosis, vasculitis, hemorrhages, edema, thrombosis and dyspnea

 Rarely fatal but does occur
 Nervous system disturbances (~80% cases in dog)

 Heart and kidney involvement

19

Clinical Signs of Rickettsia rickettssii

 Clinical signs
 High fever, anorexia, vomiting, diarrhea, petechiae or ecchymotic

mucous membranes, edema

  Tenderness over lymph nodes, joints and muscle

  Severe necrosis in extremities in dogs occur (severe fatal disease)

 Marked thrombocytopenia and leukopenia may be present during acute phase

20

Immunological Aspects for Rickettsia rickettssii

 Immunologic aspects
 Auto immune reactions are related to pathogenesis in

late RMSF vascular manifestations
 Humoral and cell‐mediated response occurs

 CMI most important for removal of the pathogen

 No vaccines available for RMSF

21

Lab Diagnosis of Rickettsia rickettiiss

 Culture

 Can be propagated in
 Yolk sacs of chick embryos
 Cell culture (VERO cells, endothelial cell lines)

 33‐35°C with a generation time of ~9h

 Need to have glutamate as nutrient

 Serological methods
 Immunofluorescence and ELISA commonly used

 Mainly detect circulating IgG
 Negative or low titers can be present early in the diseases

 Laboratory diagnosis

 Molecular diagnosis- Have to bx hemorrage sites because you can't just pull blood
 PCR is now widely used and accepted  Very sensitive and specific
 Limitations of PCR

22

Treatment and Control of Ricketsia Rickettsii

 Treatment and Control
 Susceptible to chloramphenicol, fluoroquinolones and

tetracyclines

 Dogs need aggressive supportive therapy and possibly steroids

 NEED TICK CONTROL

23

Coxiella burnetii

Reservoir

 Worldwide

 Survives in environment
 Different from other related rickettsiae

 Have a endospore‐like growth phase

 Can be disseminated by airborne route - DOES NOT NEED A VECTOR

 Natural hosts and vectors include

 ~125 mammalian species
 Arthropods

 Ticks, mites, fleas, lice and flies

 Environmental persistence

24

Transmission of Coxiella burnetti

 Transmission
 Human cases of Q‐fever can be traced to

 Sheep, cattle and goats
 Direct or via unpasteurized milk products, feces, sperm and

reproductive discharges
 Wind can carry “spore like stages”

 BIO‐WEAPON, REPORTABLE

 In humans

 Subclinical influenza like occupational diseases

 Farmers, abattoir workers, veterinarians

 Endocarditis in chronic presentations

25

Pathogenesis of C. burnetti

 Mildly affected animals can have latent infection

 Persist particularly in lactating mammary gland and pregnant uterus

 Shedding of bacteria occurs during pregnancy

 Activated during parturition

 Sporadic aborts occur

 Pathological changes

 Severe vasculitis, placentitis, splenomegaly, mild hepatitis, fever

ENDOTHELIUM and RESPIRATORY

26

Immunologic Aspects of C. burnetti

 Vaccine is available in some countries for at risk people

 Uses the avirulent phase II
 Has altered LPS and are killed by macrophages

 Whole cell killed vaccine to researchers

 Coxevac (France) vaccine for small ruminants

 Phase I vaccine containing inactivated C. burnetii strain Nine Mile

 Two antigenic phases, Phase 1 and Phase 2

 Phase 2 antibodies are indicative of acute infection Antibodies to Phase 1 indicative of chronic infection

27

Lab Diagnosis of C. burnetti

 Tissue stains
 Gimenez, Giemsa, modified Ziehl Neelsen

 Doesn’t differentiate between Coxiella and Chlamydophila psittaci

 Isolation in cell culture or embryonated eggs  Serological methods

 IFA, ELISA and others

 Molecular diagnosis  PCR

28

Treatment and Control of C. burnetti

Treatment and Control

 Major challenge

 Antimicrobials are not effective in the acidic phagosome where C. burnetii resides

 Need to add alkalinizing agents

 Chloroquine + Tetracycline

 Chloramphenicol, clarithromycin, enrofloxacin and trimethoprim‐sulfa

29

Ehrlichiae

 White blood cell obligate intracellular bacteria

 Multiply within membrane‐lined intracytoplasmic vesicles

30

Ehrlichiae canis

Reservoir and transmission
 Agent of CANINE MONOCYTIC EHRLICHIOSIS

 Only few cases of human monocytic ehrlichiosis due to E. canis  Brown dog tick (Rhipicephalus sanguineus)

 Obtain the bacteria from infected dogs only during acute diseases (~2 weeks)

 Dog can remain infected for years, despite therapy

 Puppies and particular breeds are more susceptible

 All continents (except Australia, low to no cases)

 Tropical and subtropical latitudes

31

Ehrlichiae canis Pathogenesis

monocytes live longer, so it can be CHRONIC

subclinical infections usually

32

Acute and Chronic Diseases of E. canis

 Acute diseases
 Fever, malaise, depression, inappetance, weight loss, pale

mucous membranes, lymphadenopathy, epistaxis

 Thrombocytopenia, leukopenia and anemia (pancytopenia)

 Chronic diseases
 Dyspnea, enlargement of spleen, lymph nodes and liver

 Polyarthritis, CNS disturbances and pulmonary infiltration

 Secondary infections

33

E. canis Immunological aspects

 Immunological aspects

 Progression to the latent and chronic occurs mainly in dogs with genetic predisposition and possibly impaired cell‐ mediated immunity

 Latent infections may reside in spleen, liver, bone marrow  Not known at this time

 Cellular and humoral immune‐mediated response to infected mononuclear cells and platelets contribute to:

 Blood cell destruction
 Bone marrow depression  Polyarthritis
 Uveitis

 Immune complex deposition possible

34

Lab Diagnosis of E. canis

 Laboratory diagnosis
 Giemsa‐stained smears of buffy coat

 Colonies (morulae) less than 4 μm in diameter are demonstrable

 Scarce and found mainly during acute stage

 Canine cell lines can be used for culture and isolation

 Serology

 IFA

 Molecular  PCR

35

E. chaffeenis

E. chaffeensis
 Human monocytic ehrlichiosis
 Closely related to E. canis
 Its vector is Dermacentor variabilis (American dog tick)  Mammalian reservoir is the deer

36

E. ewingii

E. ewingii
 Agent of canine granulocytic ehrlichiosis
 Amblyoma americanum (Lone star tick)
 Mainly in central North America
 Similar disease to A. phagocytophilum infection

37

Treatment and Control of E. canis, ewingii and chaffeenis

 Treatment and Control
 Tetracyclines and imidocarb dipropionate

 Mainly in acute cases
 Less so in advanced cases

 Doxycycline and steroids
 Late‐stage monocytic ehrlichiosis

 POOR PROGNOSIS

PREVENTION  Tick control

38

African heartwater

Ehrlichia ruminantium causative agent

 REPORTABLE DISEASE

 Mainly in ruminants in Africa and parts of the Caribbean

 Passed only by parenteral introduction to the blood

 Tick vector is Amblyomma spp.

 Replicates in reticuloendothelial cells
 Macrophages, endothelial cells, and neutrophils

39

Pathogenesis of African heartwater

CS; edema, and likes to go to pericardium can see NS signs

40

Acute, Subclinical, and Percute CS of African Heartwater

 Peracute form
 Fever of several hours
 Collapse and death under convulsion

 Acute form- Very common
 Fever followed by neurological signs

 Hyperexitability, muscle tremors, ataxia, deficit in conscious proprioception, head pressing, coma and seizures

 Death within 2‐10 days

 Mortality 6‐80%

 Subclinical form is also described
 Hydropericardium, hydrothorax, congestion

 Splenomegaly and extensive hemorrhages

41

Pathological Findings of African heartwater

Widespread vasculitis with effusion
 Epithelial and endothelial hemorrhages
Pericardial effusion and encephalitis can occur

 Enlarged spleen, liver and lymph nodes
 Bone marrow depression

42

What is one of the findings on necropsy from african heartwater?

hemorraghic lesions in muliple organs

43

Immunological and Lab diagnosis of  E. ruminantium

 Immunologic aspects
 Some cattle breeds and newborns appear to be resistant

 In surviving cattle, cell‐mediated immunity persists for up to 5 years (unless a different isolate)

 Laboratory diagnosis
 Demonstration of agent in Giemsa‐stained smears

 Molecular and serological diagnosis

44

Treatment and Prevention of E. ruminantium

Treatment
 Tetracycline

Effective if early during diseases presentations

 Prevention

Tick control and vaccination are important

 Young calves and lambs are vaccinated with virulent bacteria

 Older cattle infected & immediately treated

45

Generals for Neorickettsia

 Small, non‐motile, coccoid, intracytoplasmic
 Found within vacuoles of monocytes, macrophages and

enterocytes

 Gram‐negative bacteria
 Stain readily with Macchiavello and Giemsa stain

 Cannot be cultivated in cell‐free media or chicken embryos

 Fluke serve as vectors
 All stages within the life cycle of fluke are infectious

46

Salmon posioning disease

 Caused by Neorickettsia helminthoeca in Pacific Northwest coast

 Range of the snail intermediate

 Elokomin fluke fever is a mild form of salmon poisoning disease

 Infects lymphoreticular tissues of canids (LN, Peyers patches)

 Target cell are the canine mononuclear cells
 Multiplies in cytoplasm, frequently forming multiple morulae and

filling the entire cell

 Infects dogs after ingestion of fluke (Nanophyetus salmincola)

 Neorickettsia helminthoeca is maintained by transovarial passage in the helminth

 Found throughout the life cycle of the fluke  INCLUDING FREE‐SWIMMING CERCARIA

encysted in salmonid fish

47

Host of salmon posioning and pathogenesis

 Generally only members of family Canidae  Reported in captive polar bears and raccoons

48

Acute diseases - salmon poisioning

 Acute diseases

 Fever, depression, dehydration, anorexia, vomiting, weight loss, hemorrhagic diarrhea, and lymphadenopathy

 From proliferation of reticuloendothelial components

 Case fatality high in untreated dogs
 Recovered animals are immune to re‐infection

49

Diagnosis and Prevention of Salmon Poisioning

 Diagnosis
 Detection of fluke eggs in feces, history, clinical signs,

LOCATION

 Organism in lymph node aspirates

 Has been propagated in cell culture media

 Serology/molecular
 IFA, can be unreliable, PCR best

 Differential Diagnosis
 Canine Parvovirus 2 and canine distemper

 Prevention
 Not allow dogs to eat raw fish

 Sick animals
 Supportive care

 Control vomiting and diarrhea  Maintain acid‐base balance

 Tetracycline, penicillin G, chloramphenicol and sulfonamides  No vaccine

50

Potomac Horse Fever

 Also known as:
 Equine monocytic ehrlichiosis

 Equine scours
 Acute diarrheic illness of equids
 Caused by Neorickettsia (Ehrlichia) risticii

 Affinity for blood monocytes, tissue macrophages, and intestinal epithelial cells

51

Epidemelogy of Potomac Horse Fever

  Very little is known

  US, Canada, South America

 Serological Europe, Asia and Australia

 Proximity to bodies of water

 Seasonal pattern  Mainly summer

  Very little is known

  US, Canada, South America

 Serological Europe, Asia and Australia

 Proximity to bodies of water

 Seasonal pattern  Mainly summer

 Infectious life cycle involves an intermediate snail reservoir and trematode cercaria

 Aquatic insects also suspected, mayflies

52

Potomac Horse Fever Clinical Signs

 Primary clinical sign is acute, watery diarrhea
 Fever, anorexia, mild colic, depression, dehydration,

laminitis and leukopenia

 Case fatality is 5‐30%

 Enterocyte infection with loss of microvilli and malabsorption of sodium and chloride ions and lack of water resorption

 DIARRHEA

53

Dx and Tx of Potomac Horse Fever

 Diagnosis  History

 Wright‐stained blood smears  Not reliable

 Serology ‐ unreliable  IFA, ELISA

 Molecular detection  PCR

 Treatment

 Available vaccine is questionable  Heterogeneity among isolates
 Deficiency in antibody response

 IV administration of tetracycline or oxytetracycline early in the course of diseases

54

Aeyptianosis

 Diseases caused by Aegyptianella pullorum

 Affects poultry and wild birds

 Vector are tick of genus Argus

 Present with ruffled feathers, anorexia, diarrhea, anemia and hyperthermia

 Lesions include hepatosplenomegaly and punctiform hemorrhages of serosal surfaces

  Treatment with tetracyclines

  Control of ticks is important

55

Salmoning poisiong and potmac horse fever cause....

GI SIGNS