Leishmaniosis in dogsSimple Steps for In Clinic Diagnosis

18 March 2020– Dr. Inbal Peled, Biogal

✓ Leishmaniasis is endemic in 88 countries on five continents—Africa, Asia, Europe, North America and South America.

✓ 12 million people are affected by leishmaniasis✓ 50 000 to 90 000 new cases of VL occur worldwide

annually✓ An estimated 600 000 to 1 million new cases of CL

occur worldwide annually.

HUMAN LEISHMANIASIS

Human Leishmaniasis - cont

• Mostly affects poor people

• Associated with:

- malnutrition,

- population displacement,

- poor housing,

- weak immune system

- environmental changes:

deforestationbuilding of dams

irrigation schemes urbanization

Human Leishmaniasis – clinical signs

MucocutaneousCutaneous (most common)

Visceral (most serious form)

partial or total destruction of mucous membranes of the nose, mouth and throat

• irregular bouts of fever• weight loss• Enlarged spleen and liver• Anemia

skin lesions, mainly ulcers, on exposed parts of the body, leaving life-long scars

Canine LeishmaniosisL. Infantum

Leishmaniosis in dogs,Simple steps for in clinic Diagnosis

✓ General aspects of the disease

✓Clinical signs

✓Diagnostics options

✓ In-clinic diagnostics

✓ Disease staging and prognosis

Topics

Leishmania Infantum Canine infection -General aspects

Canine Leishmaniosis

Cutaneous LeishmaniosisVisceral Leishmaniosis

Leishmania braziliensis*

Leishmania amazonensis

Leishmania infantum

Because the internal

organs and skin of

the dog are affected,

the canine disease

is termed

viscerocutaneous

WORLD CANINE LEISHMANIA DISTRIBUTION

http://www.cvbd.org/en/occurrence-maps/world-map/

The CVBD World

Forum is supported

by Bayer Animal

Health, a specialist in

the field of parasite

prevention.

Canine leishmaniosis is extremely common in the Mediterranean area and South America; -also found in Africa, Asia, Central America

Prevalance of L.infantum (2014)

• Spain, France, Italy and Portugal – 2.5 million

• Spreading to North Europe – Alps, Pyrenees, and north western Spain.

• South America – estimation ~ Millions • Highest in Brazil (90% of cases) and Venezuela

• Highly endemic countries• canine infection rates 70-90% • Balearic islands of Spain, Greece, Marseille area France, Naples area in Italy.

Canine leishmaniosis is extremely common in the Mediterranean area and South America;

Transmission modesSand fly transmission

Sand fly bite Dermis

Macrophages penetration and

replicationBlood

Lymph nodes, Spleen, bone

marrow, whole body

promastigotes

amastigotes

Non sand fly transmission

✓Blood products from carriers

✓Vertical

✓Venereal

Suspected yet unproven

✓ direct dog-to-dog transmission through bites or wounds

✓ other blood feeding arthropods (ticks and fleas)

Transmission modes - cont

Canine Leishmaniosis – L. Infantum

Incubation period:

3 months – 7 years

Higher Incidence

• Age at least 2 years

• Prolonged exposure to the outdoors

• Lack of topical insecticide use

• Short haircoat

• Breeds - Rottweilers, GSD and Boxers

Chronic subclinical infection40-80%

Self limited disease

Fatal disease

L.infantum infection outcome

The dog as a reservoir of L.Infantum

Long pre-patent period of infection

High concentration of protozoan amastigotes in the skin

Complete eliminationof the parasite is uncertain

Factors influencing infection outcome

OthersHost relatedParasite related

Stress

Immunosuppressive treatment

Immune status

Genetics

Susceptible breeds

Nutritional state

Co-infections

Other non-infectious debilitating diseases

Strain

VirulenceParasitic load

Proliferation in macrophages, lymphocytes, plasma cells

Lymphadenomegaly

Splenomegaly

Hyperglobulinemia

Granulomatous lesions in the skin

Over antibody production

Immune-complexes deposits in the

kidneys, joints, eye and blood vessel

walls.

Immune mediated hemolytic anemia

Co -infections

Ehrlichiosis

Hepatozoonosis

L. Infantum Pathogenesis

Canine Leishmaniosis,L.infantumDiagnosis

•History

•Clinical Findings

• Laboratory Findings

Main purposes for the diagnosis of L. infantum infection

Disease confirmation

Imported dogs

Blood donors

Breeding dogs (vertical transmission / presence in semen…)

Clinical diagnosis challenges

• Almost 50% of the affected canine population does not exhibit clinical signs

• When ill – variable clinical signs and nonspecific • Any organ can be involved• Lab findings not disease specific

(thrombocytopenia, anemia, renal disease….)• more diagnostic methods – each one has some

limitations

Typical clinical findings and history

• Skin lesions (90%)

• Weight loss

• Exercise intolerance

• Decreased appetite

• Lethargy

• Lameness

• Vomiting, and Diarrhea

• Lymphadenomegaly

local or generalized

• Splenomegaly

• Polyuria and polydipsia

• Ocular lesions

• Epistaxis

• Onychogryphosis

Variable Cutaneous lesions

• Exfoliative dermatitis with/without alopecia

• Nodular, popular or pustular dermatitis

• Erosive ulcerative dermatitis

• Nasal hyperkeratosis

• Onychogryphosis (abnormal nail growth)

Mucocutaneous ulcerationExfoliative dermatitis

Skin ulceration on the ear Exfoliative dermatitis

Visceral LeishmaniosisMucocutaneous lesion

Cutaneous signs - basic facts

• Visceral involvement

• Rarely pruritic

• Generalized or localized

Occular signs

Uveitis

• Anterior Uveitis

• Keratonconjunctivitis

• Blepharitis

Conjunctivitis and blepharitis

• Muscular atrophy

• Skin Lesions

• Weight loss

Canine LeishmaniosisLaboratory Abnormalities

CBC

• Anemia (mild to moderate non regenerative)

• Thrombocytopenia

Serum Biochemistry

• Hyperproteinemia

• Hyperglobulinemia

• Hypoalbuminemia

• Renal azotemia

• Elevated liver enzymes

Urinalysis

• Proteinuria

• Isosthenuria (depending on renal

stage)

Laboratory Abnormalities

Canine Leishmaniosis – Specific Diagnostic methods

• Parasitological: cytology/histology(requires experience in slide examination)

✓ Skin lesions✓ Bone marrow ✓ Spleen ✓ Lymph Node✓ Liver ✓ Rare- WBC

• Serological

• Molecular: PCR

• IFAT: Indirect Fluorescent Antibody Test• Cross reactivity with other Trypanosoma spp

• ELISA• Higher specificity • Technically easier to perform

• Rapid lateral flow

.Qualitative

.Quick, simple, yes/no result

.point of care test

Serology

Canine Leishmaniosis diagnostics

• Negative serologic test results may reflect delayed seroconversion in some animals, which can take several months to occur

• Affected by vaccination• Performed in a referral Lab

SEROLOGIC TESTSLimitations

✓Goal: Detection of Antibodies in the blood

✓Antibodies indicate exposure to pathogen (past and present) but not necessarily disease

✓Antibodies develop within 3 weeks after infection (following establishment of disease)

✓Antibodies can persist for months to years post exposure without presence of disease

✓Antibodies can persist regardless of effective treatment

MOLECULAR TESTSPolymerase Chain Reaction (PCR)

✓ Traditionally restricted to high complexity laboratories with dedicated space and equipment.

✓ New technology is now available for use in regular clinical laboratory environments.

✓ Detects and amplifies a specific sequence of of the

pathogen’s DNA

✓ Allows for sensitive and specific diagnosis of infection

✓ Monitoring treatment efficacy

Biogal’s PCRundiagnostic devices

Molecular detection- PCR

✓First choice samples: bone marrow, lymph node, skin and conjunctival swabs.

✓ Less sensitive samples: blood, buffy coat and urine.

✓Most sensitive technique: real-time PCR.

✓Results are not affected by vaccinations (DNA not present in the vaccine)

✓ PCRun: in-clinic diagnosis

Source: LeishVet guidelines for the practical management of canine leishmaniosis

Clinical stages Serology PCR Clinical signs Laboratory findings Prognosis

Stage I Mild disease

Negative to low positive antibody

levels+

mild clinical Lymphadenomegaly/papular

dermatitis

Usually normalcreatinine < 1.4 mg/dl;

non-proteinuric: UPC < 0.5Good

Stage II Moderate

disease

Low to high positive antibody

levels+

stage I + Cutaneous lesions

anorexia, weight loss,

fever, epistaxis

• Mild non-regenerative anemia

• High Glob

• Low Alb

• serum hyperviscositysyndrome

Normal renal profileUPC < 0.5

Good to guardedCreatinine <1.4 mg/dl;

UPC = 0.5-1

Stage III Severe disease

Medium to high positive antibody

levels+

stages I and II, +immune-complex related

lesions: vasculitis, arthritis, uveitis

glomerulonephritis.

Creatinine < 1.4 mg/dl; UPC > 1 Guarded

to poorCKD stage II(Creatinine 1.4 -2 mg/dl)

Stage IV Very severe

disease

Medium to high positive antibody

levels+

stage III + Pulmonary thromboembolism,

nephrotic syndrome end stage renal disease

CKD stage III (creatinine 2-5 mg/dl)

PoorCKD stage IV (creatinine > 5

mg/dl)

Nephrotic syndrome: marked proteinuria UPC > 5

Leishmania infantum canine infection

Meglumine Antimoniate, Allopurinol, Miltefosine, Domperidone

Prevention:

• Vector control- Parasiticides with repellent activity

• Vaccines

Prognosis: Good to poor depending on disease stage

• Treatment is rarely, if ever, curative and dogs usually remain infected for life

• With no treatment - death within 2-3 years from onset of clinical signs due to renal failure.

Treatment

PARAMETERS

➢ Clinical history and physical examination

➢ CBC, Biochemistry

➢ Complete urinalyisis & UPC

➢ Quantitative serology*

➢ PCR

FREQUENCY

Sick treated dogsClinically healthy

Infected dogs

After the first month of treatment and then every 3-4 months during the first year.

Later on, every 6-12 month in dogs fully recovered clinically with treatment.

Every 3-6 months

Not before 6 months after initial treatment and every 6-12 months

Every 3-6 months

At the same time as serology Every 3-6 months

*Some dogs have a significant decrease in antibody levels (i.e. more than three two-fold dilution difference between monitoring samples) associated with clinical improvement within 6-12 months of therapy. A marked increase in antibody levels (i.e. more than three two-fold dilution difference between monitoring samples) should be interpreted as a marker of disease relapse, especially in dogs following the discontinuation of treatment.

Source: LeishVet guidelines for the practical management of canine leishmaniosis

Take home messages

• Serology alone DOES NOT provide a definitive diagnosis

• Full comprehensive diagnosis and monitoring:

✓ Physical examination

✓ CBC, Biochemistry, UA

✓ Serology & PCR

• First choice samples for PCR

• Bone marrow / Lymph nodes

• Skin

• Conjunctival swabs

• Buffy coat

• Peripheral blood

Thank you

inbalp@biogal.com