-Type 2 response not protective- promotes antibody production

-Cytokines inhibitory to cell killing mechanisms

-Fungal disease and leishmania protection requires an effective type 1 response (need cells to produce IFN-gamma and TNF-alpha) then macrophages can move in to bash organism and clear infection!

-Protozoa, Trypanosomidae

-Biphasic parasite: needs 2 different hosts

-Promastigote (Flagellate), 15um long, found in vector and cultures

-Amastigote (without flagellum), 2-5um long, in vertebrate hosts, often in macrophages (within the cell)

-transmitted by sand flies (Plebotomus spp., 2-3mm, small, nocturnal, limited roaming, rural areas)

-Mediterrean region

-M>F; 2-4 years and > 7 years of age

-rarely seen in very young dogs

-long incubation time (4 months to 7 years)

-some breeds are resistant (Ibizan hound)

-GSD, Rotties, Boxers are more affected

-Immunosuppressed animals are predisposed

-seen in endemic area, 10-40% of dogs are seropositive

-30-60% of seropositive dogs are asymptomatic

-80% will develop disease

Resistant dog: temporary seropositive, no clinical signs, strong Th1 (cellular immunity), high IFNgamma, TNF, IL2, and IL12

Sensitive dog: high serum titres, clinical disease, weak Th1/high Th2 (humoral immunity), cytokines are produced IL4, IL5, IL6 and IL10

-antibodies are not protective

-formation of immune complexes which deposit in basal membranes of joints, kidneys, blood vessels, and eyes (vasculitis, glomerulonephritis, polyarthritis, and uveitis)

-opsonization of parasites may increase phagocytosis and result in infection

-impairment of cell mediated immune system

-Weakness and decreased activity

-locomotory problems, lameness

-liver, pancreas, intestinal disease

-renal insufficiency, failure

-less frequent heart disease and thrombosis

-epistaxis

-lymphadenopathy

-ocular lesions

-symmetrical alopecia and dry exfoliation (most frequent form in 60% of animals)

-starts on head and spreads to body

-ulcerative form (23% of cases) may affect bony prominences, mucocutaneous junctions and extremities (probably due to vasculitis)

-multiple nodules of variable sizes may develop around skin, eyelids and mucosae (~12% animals). Young resistant dogs are probably inoculation site

-may also develop nasal/digital hyperkeratosis, onychogryphosis, paronychia, nasal/oral depigmentation, ulcerative stomatitis, mucosal proliferations on penis, tongue, nose and mouth

-high serum proteins (usually over 8 gr/dl) unless severe loss with urine

-hyperglobulinaemia (polyclonal B-cell activation and Ig production)

-Hypoalbuminaemia (compensatory or due to protein-losing nephropathy, hepatopathy)

-Typically, "Two horns" are seen on protein electrophoresis (low albumin/globulin ratio of <1)

-CBC/kidney evaluation reveals mild, non-regenerative anaemia, thrombocytopenia and coagulation disorders

-Increased urea, later increased creatinine

-Protein/creatinine ratio in urine >0.5

-Acute-phase proteins (APP) include C-reactive protein, haptoglobin, amyloid A (may be useful in monitoring of therapy, these APPs are all increased in animals with active disease)

-100% specificity, low specificity

-Lymph nodes (30%)- afferent lymph nodes from skin lesions more positive

-Bone marrow (50-70%)- best to sample costo-chondral jxn, iliac crest, and sternum. Leishmania found in macrophages

-Spleen is most rewarding

-Sample cytology under crusts and scales

-FNAB cytology from skin nodules and plaques

-Dermatopathology H and E, Giemsa (good from scaly skin and papules), scarce in ulcerative form

-Immunohistochemistry

-IFAT, Dot-ELISA, direct agglutination

-high sensitivity and specificity (80-100%)

-in house snap tests are less reliable, OK for screening

-false positive: recent contact in the resistant dog (test in Winter--- March/April)

-false negative: prepatent phase in affected dogs (retest after 6-8 weeks)

-serology is NOT suitable for monitoring of treatment, as they remain measurable after remission

-Real time PCR of kinetoplastic DNA is highly specific and sensitive

-Requires fresh and formalin fixed tissue

-FNAB of lymph node, bone marrow and SPLEEN

-CONJUNCTIVAL SWABS detection of early positivity

-may recognize leishmanial DNA in animals which have been clinically cured for years (false positive?) and in healthy carriers

-Quantitative PCR for monitoring therapy

-Healthy- infected resistant

-Serology negative with positive PCR

-Serology low to medium

-No treatment

-Monitor serology every 3-6 months (by 2-fold increase start treatment)

-Treat clinically ill with serology high and PCR positive

-Treatment following staging

-Guidelines via LeishVet Group

-Need to perform clinical staging of leishmaniosis (4 stages)

-laboratory tests to be done (CBC/general panel including renal values, blood protein electrophoresis and TP; urine analysis with sediment and P/C)

-No treatment

-Allopurinol alone (inhibits xanthine oxidase)

-Antimonials alone (pentavalent antimonials bind to polypeptides, inhibit DNA topoisomerase glycolytic enzymes and fatty acid beta oxidation)

-Miltefosine alone (exerts activity by inhibiting cytochrome c oxidase and causing apoptosis-like cell death. This may affect membrane integrity and mitochondrial function of the parasite)

-Miltefosine + allopurinol

-Antimonials +allopurinol

-Meglumine antimoniate

-used to treat leishmaniasis (classical therapy)

-given by injection into a muscle

-side effects include loss of appetite, nausea, abdominal pain, cough, feeling tired, muscle pain, irregular heartbeat, and kidney problems

-belongs to a group of medications known as the pentavalent antimonials

-Pentavalent antimonials bind to polypeptides, inhibit DNA topoisomerase glycolytic enzymes and fatty acid beta-oxidation. Electron microscopy studies show antimonial-induced changes in parasite cell membranes.

-a dopamine D2 receptor antagonist which stimulates the release of serotonin----> production of prolactin occurs---> stimulation of cell-mediated immunity

-Leishguard (Esteve) and on the market in Spain and Italy

-Used as an immunostimulator

-Both in prevention in healthy dogs and as therapy in diseased animals

-Inexpensive and safe!

-Protein electrophoresis on day 30, 90 and every 6 months thereafter

-Any other abnormal value (e.g. kidneys)

-Serum testing every 6-12 months (decreases very slowly). For negative serum titre (good immunological recovery and need to perform PCR for parasitological cure)

-For a 2 fold increase (evidence of relapse)

-RT-PCR used when patient is serology negative

-Acute phase protein monitoring (high in Leishmania infected)---- C-reactive protein, haptoglobin and amyloid A

-75% of dogs survive at least 4 years if they are followed with current protocols (including a new treatment course at every relapse)

-Long term remission with long term allopurinol administration

-Worst prognosis if severe renal damage

-Dogs in endemic areas should not spend the night outside

-Fine mesh net should be applied to windows

-Repellents on animals in the environment (DEET, piperonylbutoxide)

-Pyrethroid collars (e.g. Scalibor, Seresto/Foresto), permethrin spot-on have repellent activity when given every 3-4 weeks

Helpful Reminder:

-MOA of Pyrethroids (axonic excitotoxins)- toxic effects are mediated through preventing the closure of voltage-gated sodium channels in axonal membranes

-MOA of Permethrin is to act on the nerve cell membrane to disrupt the sodium channel current

-Canileish brand

-excreted secreted proteins, parasite surface antigen and saponin: stimulates the cell-mediated immune system and production of IFN-gamma

-Only in seronegative dogs

-3 injections the first year then annual thereafter

-It causes seroconversion (positive IFI)

-In order to differentiate vaccinated from diseased animals, a special test is needed

• Spleen is the most rewarding
• Lymph nodes (30%)- afferent lnn from skin lesions more positive
• Bone marrow (50-70%)- Leishmania found in macrophages
• Sample under crusts and scales
• FNAB cytology from skin nodules/plaques

• High serum proteins (usually over 8 gr/dl) unless severe loss in urine
• hyperglobulinemia (polyclonal B cell activation and Ig production)
• hypoalbuminemia (compensatory or due to protein losing nephropathy, hepatopathy)
• typical "two horns" protein electrophoresis
  • low albumin/globulin ratio (<1)

• Measurement of circulating antibody via IFAT, Dot-ELISA, direct agglutination
• high sensitivity and specificity (80-100%)
• in house snap tests are less reliable (ok to screen)
• False positives due to recent contact with resistant dog (test in winter March/April)
• False negatives due to prepatent phase in affected dogs (retest after 6-8 weeks)
• not suitable for monitoring of treatment-remain measurable after remission

• highly specific and sensitive
• use fresh and formalin fixed tissue
• FNAB of lymph node, bone marrow and spleen
• Conjunctival swab for detection of early positive
• may recognize leishmanial DNA in animals which have been clinically cured (false positive?) and in healthy carriers
• PCR for monitoring therapy

• Healthy infected and sensitive
  • serology high and PCR positive
  • will develop the disease
• Clinically ill
  • serology high and PCR positive

• Excreted secreted proteins, parasite surface antigen and saponin: stimulate the cell mediated immune system and produce IFNγ
• Only use in seronegative dogs
• 3 injections in the first year then yearly thereafter
• Causes seroconversion
• In order to differentiate vaccinated from diseased- need a special test