Canine Leishmaniasis – a practical approach for diagnosis and treatment

Canine Leishmaniasis – a practical approach for diagnosis and treatment
CANINE LEISHMANIASIS

Canine Leishmaniasis – a practical approach for diagnosis and treatment

Canine Leishmaniasis (CanL) is a major zoonotic disease endemic in more than 80 countries in the world. It is endemic in Southern Europe, Northern Africa, the Middle East, Central Asia, parts of China, South and Central America and has emerged sporadically also in the USA. CanL is also an important concern in countries where imported disease is a veterinary and public health problem. Phlebotomine sand flies are the vectors of Leishmania infantum, the main causative agent of CanL, and also for other Leishmania species.

Dogs are considered the main animal reservoir for human visceral leishmaniasis and the disease is often fatal in people who are not treated.

Studies reporting the seroprevalence of  L. infantum in dogs from the Mediterranean area show seropositvity rates that range between 10% and 37%. Surveys using the polymerase chain reaction (PCR) for the detection of Leishmania spp. DNA in the blood or tissues of dogs, or combining PCR and serology, have revealed even higher infection rates approaching 70% in some areas. It has been estimated based on seroprevalence studies from France, Italy, Spain, and Portugal that 2.5 million dogs in these countries are infected with L. infatum. The number of infected dogs in South America is also estimated in millions with high infection rates in some areas of Brazil, Argentina and Venezuela.

Visceral leishmaniasis, caused by L. infantum, was traditionally mainly a disease of young children in the Mediterranean basin. The name, “infantum”, given to the causative agent of this disease reflects the predilection to infants. In addition to children, immoncompromised people and HIV+ patients are an important risk group for human visceral leishmaniasis worldwide.

 

Life Cycle and Transmission

Leishmania spp. are diphasic parasites whose life cycle involves infection of two types of hosts, a sand fly which harbors the flagellated extracellular promastigotes and a mammal where the intracellular amastigote stage develop. Dogs are infected by L. Infantum promastigotes that enter their skin during the bite of an infected female sand fly vector. Promastigotes invade host macrophages, transform into amastigotes and replicate in the host cell cytoplasm as intracellular amastigotes. The disease incubation period prior to the appearance of clinical signs may last months to years, during which the parasite disseminates from the skin throughout the host’s body primarily to hemolymphoid organs such as the lymph nodes, bone marrow and spleen.

Vertical in-utero transmission of L. infantum from the dam to its offspring as well as venereal transmission have been documented in dogs. In addition, transmission through blood products has been reported in humans and  dogs that received blood transfusions from infected donors. Direct dog to dog transmission via bites has been suspected but not proven.

 

Pathogenesis of Canine Leishmaniasis

Population studies in L. infantum-endemic areas have revealed that a small proportion of the dog population develops a clinical disease (10% or less of the infected dogs), another part has sub-clinical infection, and yet another part is resistant or intermittently resolves the infection without developing clinical signs. The immune responses mounted by dogs at the time of infection and afterwards are important in determining if they will develop a lasting infection and, whether and when it will progress from a sub-clinical state to clinical disease.

Dogs that are able to resist infection and either resolve it and eliminate the parasite, or restrict the infection and remain  subcliniclly infected at all times, are referred to as “clinically resistant”. Animals that are predisposed to developing clinical disease are considered “susceptible”.

Immune responses play an important part in the susceptibility of dogs to infection and the development of disease. In general, the T-helper 1 (Th1) response and its associated cytokines lead to Leishmania elimination by activated macrophages and resistance to disease development. Conversely, the T-helper 2 (Th2) response leads to parasite persistence and proliferation with increased susceptibility to disease and increased production of anti-leishmanial antibodies and hyperglobulinemia. Dogs usually develop both Th1 and Th2 responses and the balance between these responses determines the animal’s course of infection. During progressive infection, dogs become increasingly immunosupressed and may develop decreased CD4+ lymphocyte counts. Moreover, it has been demonstrated that the infectiousness of dogs with leishmaniasis to sand flies increases with the decrease in CD4+ counts.  Immune-mediated mechanisms are responsible for many of the pathological findings in CanL. Circulating immune complexes and antinuclear antibodies have been detected in animals with CanL. Glomerulonephritis associated with renal deposition of immune complexes is a hallmark of the disease. Renal pathology is present, even if not manifested clinically, in the majority of dogs with this disease.

Susceptibility and resistance to CanL are influenced by the dog’s genetic makeup. The presence of severe CanL among Ibizian hounds in the Balearic islands of Spain is rare and significantly lower than among other breeds. It has been shown that this breed produces a predominantly cellular response against L. infantum. Other breeds that originate from areas that are not enzootic for leishmaniosis such as the Boxer, Rottweiler and German shepherd are overrepresented in CanL surveys.

 

Clinical findings in canine leishmaniasis and clinical staging

The history reported by owners of dogs with CanL typically includes the appearance of skin lesions, ocular abnormalities, or epistaxis.

These are commonly accompanied by weight loss, exercise intolerance and lethargy. However, dogs with renal disease or some other internal organ involvement and no dermal lesions should also be considered as potentially suffering from the disease. On physical examination, the main clinical signs associated with CanL are dermal lesions, lymphadenomegaly, splenomegaly, abnormal nails growth (onychogryposis) and poor body condition. Additional findings include: nose bleed (epistaxis), renal disease with possible polyuria and polydipsia, vomiting, gastrointestinal disease, melena, rhinitis and lameness.

Sixteen to 80% of the dogs with clinical disease due to leishmaniasis have ocular or pericolar lesions including keratoconjunctivitis and uveitis. The dermal lesions associated with CanL include exfoliative dermatitis which can be generalized or localized over the face, ears and limbs. Nodular dermatitis has been reported mainly in Boxers, and cutaneous ulceration is frequently found with bleeding from local ulceration sites.  A mild form of papular dermatitis has also been described.

The most common serum biochemistry findings in dogs with CanL are hyperproteinemia with hyperglobulinemia and occasional hypoalbuminemia resulting in a decreased albumin/globulin ratio. Markedly elevated activities of liver enzymes or azotemia are found in some dogs with CanL. Epistaxis, ocular abnormalities or real disease may be the only presenting clinical findings in CanL therefore Leishmaniasis should be considered among the differential diagnoses when such clinical signs are present. Dogs with marked hyperglobulinemia with no apparent cause should also be evaluated for CanL.

Dogs with marked hyperglobulinemia with no apparent cause should also be evaluated for CanL.

 A clinical staging system for CanL provided by the Leishvet group can be found at https://www.leishvet.org/fact-sheet/clinical-staging/. It divides the disease into 4 clinical stages based on clinical signs, clinicopathological abnormalities and level of anti-leishmanial antibodies. This system is useful for deciding on the best therapy for each dog and for considering prognosis. If the dog’s condition worsens or improves, the clinical stage may change.

 

Diagnosis

The diagnosis of CanL is performed for different indications (Table 1).

Purpose of the

diagnosis of CanL

  1. Confirmation of disease in dog with compatible clinical signs
  2. Evaluation of blood donor
  3. Exportation / Importation
  4. Epidemiological survey
  5. Monitoring of “health status”
  6. Monitoring of treatment

1. Cytology and histopathology

Leishmania amastigotes can be demonstrated by cytology from the skin, lymph nodes, spleen. bone marrow, abdominal fluid, joint fluid or other fluids, tissues and organs. This is possible when preparations are stained with Romanowsky type stains such as Giemsa or quick commercial stains.

However, detection of amastigotes by cytology is frequently unsuccesful due to a low number of detectable parasites present even in animals with a full blown clinical disease. Amastigotes may also be viewed in histophathologic formalin-fixed, parafifin-embedded biopsy sections of the skin and other organs. An immunohistochemical staining method can be employed to detect and verify the presence of Leishmania organisms in tissues.

 

2. Serology

Various serological methods for the detection of anti-Leishmania antibodies have been developed. These include the indirect immunofluoresence assay (IFA), enzyme-linked immunosorbent assays (ELISA), and direct agglutination assays (DAT). Purified whole antigens and recombinant antigens are frequently used in commercial serology kits for the detection of leishmaniasis antibodies in dogs. In general, good sensitivities and specificities are achieved with most of these assays for the diagnosis of the majority of CanL clinical cases. Most kits provide a qualitative result of positive or negative, and it is recommended to follow a positive qualitative result with a quantitative test which will provide a titer that can be followed during treatment of the dog.

Successful treatment is frequently associated with a marked decrease of antibody levels over time, whereas unsuccessful treatment or relapse to clinical disease after disease remission is associated with increased antibody levels. Sub-clinically infected dogs are often seronegative or have low anti-leishmanial antibody levels, which may be missed by qualitative serology as well as in some cases also by quantitative IFAT or laboratory ELISA. Overall, serology is recommended for the diagnosis of dogs with suspicion of clinical disease, and if convenient, it can be initially performed using a rapid kit produced by a reputable company such as Biogal.

 

3. PCR

Detection of parasite DNA in the blood or tissues of infected dogs by PCR allows specific diagnosis. Several different assays with various target sequences using genomic or kinetoplast DNA (kDNA) have been developed for CanL. PCR can be performed on DNA extracted from tissues, blood or even from histopathologic specimens. Assays based on kDNA are the most sensitive target for PCR detection in infected issues.

The preferred sites for sampling dogs for PCR are the bone marrow, lymph node, spleen and non-invasively by swabs from the conjunctiva. PCR on blood and other body fluids is considered less sensitive and dogs with overt disease may be negative. On the other hand, hemolymphoid tissue PCR can be used for detection of infection of sub-clinically seronegative infected dogs.

Treatment of canine leishmaniasis

The main drugs used for treatment of CanL include pentavalent antimony meglumine antimoniate (Glucantime®), milrefosine (Milteforan®), and allopurinol (Alloril®, Zylol®).

Allopurinol, which is administered orally for long term, is often combined with either megluimine antimoniate which is injected daily for 4-6 weeks, or with miltefosine which is administered orally daily for 4 weeks. Treatment with allopurinol is rarely stopped before 12 months of treatment, and is sometimes continued for the dog’s lifetime depending on its clinical response and laboratory test results. Allopurinol is the only drug recommended for treatment of dogs by the World Health Organization (WHO) Because this drug is not used to treat human leishmaniasis, there is less concern about the development of drug-resistant strains that are harmful to humans. It has been shown to provide clinical improvement to treated dogs also as monotherapy. Anti-leishmanial treatment is often successful in clinical cure of dogs with mild to moderate disease, however, it may only achieve temporary clinical improvement in some cases it is frequently not associated with total elimination of the parasite. Treated dogs should be monitored over a long period of time and will often remain carriers of the disease and may experience clinical disease relapse. Carriers could be infectious to sand flies and treatment of CanL can be stopped when the al of the following three conditions are met: (1) reversal of clinical signs; (2) normalization of the hematology, blood biochemistry profile and urinalysis; and (3) negative serology with titers that   reach a level below the cut-off titer of quantitative serological assays.

Ancilliary treatment of CanL includes treatment with domepridone (Leishguard®) which is a dopamine D2 receptor antagonist reported to have immunostimulant properties via the stimulation of prolactin secretion which acts as a pro-inflammatory cytokine. An additional ancilliary treatment includes a dietary supplement of nucleotides and active hexose as an adjunctive therapy.

 

Prevention of canine leishmaniasis

Clinically cured dogs often remain parasitologically positive and infectious to the sandfly vector.

The use of topical insecticides against CanL in collars or spot-on formulation containing pyrethroids has been shown to be effective in reducing disease transmission. Delthamethrin-impregnated collars and permethrin with imidacloprid spot on drops have been reported to significantly reduce the number of sand fly bites to dogs under experimental transmission and in field conditions, and thus to decrease the risk of infection. Commercial vaccines against CanL are marketed in Europe and Brazil, and have been proven to decrease the occurrence of clinical disease due to L.infantum.

Further reading on canine leishmaniasis
Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. (2008). Canine leishmaniosis – new concepts and insights on an expanding zoonosis: part one. Trends Parasitol 24:324-30.
Mirό G, Cardoso L, Pennisi MG, Oliva G, and Baneth G. (2008). Canine leishmaniosis- New Concepts and Insights on an Expanding Zoonosis – part two. Trends Parasitol. 24: 371-377.
Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P, Oliva G, and Baneth G. (2009). Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet. Parasitol. 165: 1-18.
Solano-Gallego L, Miró G, Koutinas A, Cardoso L, Pennisi MG, Ferrer L,
Bourdeau P, Oliva G, Baneth G, The LeishVet Group. (2011). LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vectors. 2011;4:86.
Miró G, Petersen C, Cardoso L, Bourdeau P, Baneth G, Solano-Gallego L, Pennisi MG, Ferrer L, Oliva G. (2017). Novel Areas for Prevention and Control of Canine Leishmaniosis. Trends Parasitol 33:718-730.
Solano-Gallego L, Cardoso L, Pennisi MG, Petersen C, Bourdeau P, Oliva G, Miró G, Ferrer L, Baneth G. (2017). Diagnostic Challenges in the Era of Canine Leishmania infantum Vaccines. Trends Parasitol. 2017 33:706-717.
Dantas-Torres F, Miró G, Baneth G, Bourdeau P, Breitschwerdt E, Capelli G, Cardoso L, Day MJ, Dobler G, Ferrer L, Irwin P, Jongejan F, Kempf VAJ, Kohn B, Lappin M, Little S, Madder M, Maggi R, Maia C, Marcondes M, Naucke T, Oliva G, Pennisi MG, Penzhorn BL, Peregrine A, Pfeffer M, Roura X, Sainz A, Shin S, Solano-Gallego L, Straubinger RK, Tasker S, Traub R, Wright I, Bowman DD, Gradoni L, Otranto D. (2019). Canine Leishmaniasis Control in the Context of One Health. Emerg Infect Dis. 2019: 25(12):1-4.
Dr. Baneth is a professor of Veterinary Parasitology and Infectious Diseases at the Hebrew University of Jerusalem in Israel. Dr. Baneth graduated from the Hebrew University Koret School of Veterinary Medicine in Israel in 1990. He did a Small Animal internship and residency at the Hebrew University until 1994 followed by a fellowship in Internal Medicine and Infectious Diseases Research at the College of Veterinary Medicine, North Carolina State University during 1994 and 1995. He received a PhD in veterinary parasitology from the Hebrew University in 2000. Prof. Baneth served as the head of the Small Animal Internal Medicine Department at the Hebrew University Veterinary Teaching Hospital. He is a diplomate of the European College of Veterinary Clinical Pathology (ECVCP), an associate member of the European Veterinary Parasitology College (EVPC) and an editorial advisory board member for the Journal Veterinary Parasitology since 2006. He is the vice president of the LeishVet group for standardization of the diagnosis, treatment and prevention of canine leishmaniasis, a member of Board of Directors, Israel Society for Parasitology, Protozoology and Tropical Diseases, and a founding member of the Tropical Council for Companion Animal Parasites (TroCCAP). Dr. Baneth was the chairman of the World Small Animal Veterinary Association (WSAVA) Scientific Advisory Committee (SAC) during 2014-2019 and was an advisor to the European Food Safety Authority (EFSA) on leishmaniosis.
Dr. Baneth heads a laboratory on infectious diseases and One Health. His research interests focus on the pathogenesis, diagnosis and treatment of veterinary and zoonotic vector-borne infectious diseases including leishmaniosis, relapsing fever borreliosis, canine ehrlichiosis, babesiosis, hepatozoonosis, trypanosomiasis and dirofilariasis.  Dr. Baneth is involved in the study of zoonotic and veterinary diseases in the Mediterranean Basin, Uzbekistan, Ethiopia, Southern Europe and South America. He is the author of more than 300 scientific publications and book chapters.

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