LEISHMANIA in Dogs: Life Cycle, Occurrence and Zoonotic Aspects

LEISHMANIA in dogs: life cycle, occurrence and zoonotic aspects

Stig Milan Thamsborg Professor, DVM, PhD KU-SUND, [email protected] Heidi L. Enemark Seniorforsker, DVM, PhD DTU National Veterinary Institute, [email protected] Leishmaniosis One of the most important vector-borne diseases, endemic in the Mediterranean Bassin but possibly spreading in countries in Central Europe and North

The vectors arewww.onleish.org Phlebotomus spp. (“sandflies” = mosquitoes) (e.g. P. perniciosus )

- multiplies in the gut of The agent - Leishmania vectors and are trans- infantum inside a mitted by bites or faeces macrophage

www.onleish.org Hosts (vertebrates): dogs and other carnivores + homo

2 © Prof. Luis de Carvalho & Prof. Guadalupe Miró Corrales - ESCCAP Forum Lisbon 2012 Leishmaniasis in a global context

3 Leishmania in dogs, including zoonotic aspects

• Etiolgy – The parasite – Vectors • Biology and epidemiology – Life cycle – Hosts – Transmission – Prevalence • Pathogenesis and clinics • Zoonotic aspects • Diagnostics • Control – Therapy – Prevention • Re-cap and discussion

4 Etiology 1 Sub-phylum: Flagellates

Order Family Genus Diplomonadida Hexamitidae Giardia intestinalis (in organs) Trichomonadida Trichomonadidae Tritrichomonas foetus (organs) Trichomonas gallinae

Monocercomonadidae Histomonas meleagridis Trypanosomatida Trypanosomatidae Trypanosoma spp. (blood+lymphatic Leishmania spp. systems, tissues)

5 Etiology 2 Morphology ( Leishmania and Trypanosoma)

Different forms/stages:

a) Promastigot 10-15 µm (Leishmania in vector) b) Epimastigot c) Trypomastigot (typical form in blood of final host) d) Amastigot 2-3 µm (Leishmania in RES in final host)

a)+b) commonly found in vectors 6 Etiology 3 Leishmania spp. in dogs/cats in Europe

Agent Vectors Final hosts

Leishmania Phlebotomus spp. (sand Dog, fox jackal, rodents infantum flies) e.g.: cats, a.o. mammals + (s. L. chagasi i P. perniciosus , P. ariasi, homo Americas) P. perfiliewi, P. neglectus, (more genotypes) P. tobbi, P. langeroni

Leishmania P. sergenti, P. arabicus Dog+homo tropica – cutan form in dogsh (rare) (Orient sores)

The species can not be differentiated by morphology but clinics, vectors etc. and recently, PCR!! Blurry! 7 Etiology 4 Vector: Phlebotomus spp. (sandflies)

• Sandflies(Phlebotomus spp.) are mosquitoes (2-4 mm)

• Egg are laid in soil rich in organic matter, e.g., garbage • Females eat blood • Fourage during night, most activity in mornings and >18 oC; right after sunset

• During day hiding in crevices and holes, e.g. animal holes, walls, rocks, trees or sheds.

• Widespread in Mediterranean area, Africa and Middle East

• Local endemic foci in North France, South Germany and Switzerland (e.g. P. mascittii ) • http://www.ecdc.europa.eu/en/healthtopics/vectors/vector -maps/Pages/VBORNET_maps_sandflies.aspx

• Parasite development in vector: 1-2 weeks at 18 oC

8 Biology and transmission 1 Life cycle

Multiplication:

- as extracellular promastigots in the gut of sandflies

- as intracellular amastigots in dogs

9 Biology and transmission 2

Dogs comprise the main reservoir in Europe, but other reservoir host are present…

Fox (Marín-Iniesta et al., 1982) Wolf (Beck et al., 2008) Genet (Sobrino et al.,2008) Lynx (Sobrino et al.,2008) Desmer (Sobrino et al.,2008) Rat (Morillas et al., 1985) Cat (Maroli, 2007) 10 © Prof. Luis de Carvalho & Prof. Guadalupe Miró Corrales - ESCCAP Forum Lisbon 2012 Biology and transmission 3

Dokumented but probably limited importance : • Vertical (Rosypal, 2005; Pangrazio et al. 2009; Boggiatto et al. 2011) • Blood transfusion (Owens et al., 2001, De Freitas et al., 2006; Tabar et al, 2008) • Venereal transmission (Silva et al. 2009) * Solano Gallego et al, 2011 • Other arthropods ?? (increasing evidence) – fleas, ticks (Coutinho et al. 2007; Paz et al. 2010; Dantas-Torres, 2011) 11 © Prof. Luis de Carvalho & Prof. Guadalupe Miró Corrales - ESCCAP Forum Lisbon 2012 Biology and transmission 4 Distribution in dogs in Europe

• Black line represents north limit of sandflies and endemic leishmaniosis

• Prevalence of infection in Med. Bassin: 1-40% (Ab), some places 80% (PCR) • 2.5 mio. infected dogs (Baneth, 2007)

• Most likely spreading North, e.g. in Italian Alps but also spreading within endemic areas

• Single autochtonous cases in D, F, UK, B, NL in dogs

12 Biology and transmission 5 Epidemiology

• New findings in Leishmania endemic areas : – fast and efficient spread when environmental factors are optimal, e.g., high prevalence of vectors – more widespread than expected (PCR) but only few become clinically afffected

• Cats: importance? (60% PCR+ in Sicily)

• North Europe – travelling: NL 58000/year South; risc 0.027-0.23% (Teske et al. 2002) – import of infected dogs – non-vectorborne transmission – (USA: foxhounds in NYS)

13 Pathogenesis and clinics 1 Leishmania infantum in dogs

• Pathogenesis – infects macrophages in the skin after bite – chanker formation and spread with blood/lymph to mainly RES – granulomatous inflammation with necrosis and tissue destruction when amastigotes proliferate in macrophages – multi-systemic immun-mediated pathology depending on Th1/Th2 balance – glomerulonephritis, polymyositis, haemostasis problems a.o.

• Clinics – varies with strain and host genetics – visceral form (liver,spleen, bone marrow) but SKIN LESIONS dominates commonly Top formation of goggles – typically: severe sacling with ulcers, alopecia, evt. pustules and nodules in skin initially in visceral form – poor body condition, generalised muscle atrophy, swelling of lymph nodes – also: fever, anaemia, cachexia – eye disease and kidney failure

• Incubation period: 3 mths to 7 years

• Chronic course 14 Patogenese og klinik 2 Infection versus disease

NO clinical signs Clinical signs AND/OR No laboratory Infection laboratory abnormalities abnormalities confirmed

15 © Prof. Luis de Carvalho & Prof. Guadalupe Miró Corrales - ESCCAP Forum Lisbon 2012 Patogenese og klinik 3 Tip of the iceberg

(<10%) © G. Miró

Clinical cases (always) and sero+ PCR+

Distribution of leishmaniosis in a dog population Sero+ Rarely PCR- in an endemic area (Baneth et al, 2008)

G. Miró16 Pathogenesis and clinics 3 Severe disease in some dogs!

• Rapid development with clinical disease in some dogs that do not develop protective cell-mediated immunity (CMI) despite solid humoral response • Antibody response positively correlated with severity of disease and parasite density • Other dogs remain infected for years with symptoms and develop protective CMI • Function of macrophages very important • Immune response is mixed Th1/Th2 under strong genetic regulation: • Genetically resistant: Ibizean hound • Genetically susceptible: German shepherd, rottweiler, cocker spaniel, and boxer • Single genes and SNPs identified • Dog with/without symptoms are INFECTIVE for sandflies but ill dogs much more so!!

G. Miró17 Zoonotic aspects 1

18 Zoonotic aspects 2 Global distribution in humans

L. donovani + L. infantum/chagasi (visceral) L. tropica + L. major

19 Zoonotic aspects 3 Leishmaniosis in humans

• 90% of human cases of cutan leishmaniosis in Afghanistan, Brazil, Iran, Peru, Saudi Arabia, and Syria (WHO)

• L. major : >700 cases during war in Iraq; L. tropica : visceral cases after Operation Desert Storm

• More than 90% of human cases of visceral leishmaniosis in India, Bangladesh, Nepal, Sudan, and Brazil (WHO). Continued spread to new areas due to HIV

• Co-infections of HIV and visceral leishmaniosis are reported from 34 countries in Africa, Asia, Europe, and S-America.

• 70% of human cases among adults i S-E associated with HIV (WHO)

• Estimate: 700 new human cases per year in S-E

• Leishmaniosis is common among travellers/tourist of S-E

• Human diagnostics in DK in 2011 (Overlæge Jørgen Kurtzhals): – 6 microscopy (4719 malaria; 6145 cryptosporidiose) – 104 antibodies (531 malaria-species; 1097 toxoplasma- schistosomes-echinococcosis)

20 Diagnostics 1 Diagnostics, dogs

• Tentative diagnosis: – History and clinics (obs. demodicose) – Lab test: haemogram, biochemistry and urine analysis

• Detection of parasites – Smear from marrow or lymph node/Giemsa staining ( DTU-Vet ) – Aspirate lymph node, marrow, blood, conjunctiva or skin biopsy : PCR or qPCR (most sensitive + species)( Idexx ) – Histology on skin biopsy (sensitivity increased by immuno-histochemistry) – (in vitro culture to promastigote stage) • Indirecte detection - serology – At earliest 8-12 weeks after infection – IFAT, ELISA ( Laboklin )(semi-quantiative test to follow treatement success) – Snap test in clinics (first detection)

21 Diagnostics 2 Clinical pathology in dogs

• Blood: – normochrom, normycytic anaemia – evt. thrombocytopenia – leukopenia – hyperglobulinaemia – hypoalbumimaemia

• Biochemistry: – Varying degrees of uraemia – Increase protein/creatinin

• Urin analysis – Proteinuri – Haematuri

22 Control 1 Chemotherapy of leishmaniosis in dogs

Drug Type Dosage Adm. Side effects Meglumine pentavalent 75-100 mg/kg Sc Lokcal swellings antimoniate antimoniate daily in 4-8 Resistance in vitro (enzyme weeks inhibition) Allopurinol purin analog 10-20 mg/kg (2- Po Cases of nephrolithiasis (inhibit RNA) 3 x day) in 6-18 (xanthin) mths. (min. 20 Often medication rest of mg/kg daily) life! Miltefosine alkylophospho- 2 mg/kg daily in Po GI problems if not fed lipid 4 weeks with other diet (damage to membranes) Combinations: meglumin+allopurinol miltefosine+allopurinol

+ additional symptomatic treatments and correct diet

23 Control 2 Aspects of control in dogs

• Treat when clinical disease (+clinical pathology) and/or detection of parasite

• Euthanasia considered if organ damage or severe cachexia

• Early treatment is imperative

• Relapse is not uncommon (monitored by increased titres)

• Use single drug in non-endemic countries but drug combinations may work faster and with less relapse

• Follow up: during treatment and every half year (biochemistry and Ab)

• Inform the owner: – Treatment does not remove the infection! – Expensive and LONG TERM

• Amphotericin B: also effect but for humans only

24 Control 3 Prevention in dogs

• Inside during mosquito risk period (early and late) April-November (SE)

• Environmental cleaning: remove breeding spots for sandflies (organic material) + nets on houses

• Repellantia (e.g. collars, spot-on, spray)

• Collars with pyrethroids can have effect for 6 mths.

• Treatment/medical prevention often inefficacious but a treated dogs using repellent is less infective to others

• Kennels and other places with high dog density: increased vector control

• Vaccine for dogs on market now (NB: only uninfected dogs) – Reduction of clinical disease and potentially less transmission

In relation to control in humans: • Notifiable in some SE countries • Euthanasia of infected dogs in some countries outside EU. Does not permanently reduce the incidence in humans 25 Re-cap and discussion Leishmania in dogs/humans

• Grave, invalidating disease with continuous expenses for treatment

• Many dogs infect but only very few get ill (healthy carriers)

• Best control through environmental clean-up, topical insecticies and vaccination

• Avoid imported cases – well-defined risk area + PCR-test

• Diseased dogs need treatment or euthanasia; treated dogs still infective

• In DK transmission from infected dogs unlikely

• Vets have a responsibility to minimize the risk of transmission to humans !

• We need better disease data in humans! (notifiable)