Diseases transmitted by sand flies

Leishmaniasis

sandfly image

Leishmaniasis is caused by the protozoan parasite Leishmania infantum, which is transmitted by sand flies of the Phlebotomus species. Dogs are the major reservoir for this infection. This infection causes disease in humans, particularly immunosuppressed adults and children. It is extremely common in countries surrounding the Mediterranean and in South America. Most recently it has spread across the USA and Canada, particularly in the fox hound breed, despite the fact that there are no competent sand fly vectors in these areas. Online maps are available to help determine if a country a dog has travelled to/from has leishmania infection there. Both direct dog-to-dog transmission and vertical transmission have been suggested, and spread through contaminated blood products has been reported.

In endemic areas the infection prevalence in dogs may approach 90% but most dogs are asymptomatic as their immune response plays a large part in determining the outcome of infection; an ineffective cell mediated immune response with a pronounced humoral response is associated with clinical disease.  Leishmaniasis is a chronic disease with a long incubation period - it can be 3 months to 7 years after infection that clinical signs develop.

 

 

 

What are the clinical signs?

Leishmania skin lesions

Skin lesions (scaling is common, also alopecia, ulceration) especially head and pressure points

Lymphadenopathy

Ocular signs (e.g. uveitis, conjunctivitis, keratoconjunctivitis sicca)

Splenomegaly

Weight loss & poor body condition

Fever

 

What other clinical signs may be seen with clinical leishmaniasis?

typical signs of leishmaniasis, with scaling skin and alopecia around the  nose and eyes.

Onychogryphosis (abnormal nails)

Epistaxis

Renal disease-associated signs e.g. polyuria & polydipsia

Joint disease signs - polyarthritis

Anaemia

 

 

 

 

 

 

 

What clinicopathological changes can occur with clinical leishmaniasis?

The following non-specific changes are not infrequently seen with clinical leishmaniasis:

Routine haematology: May see non-regenerative anaemia or possibly regenerative anaemia due to immune-mediated haemolysis (less common). Occasionally see thrombocytopenia.

Serum biochemistry: Hyperproteinaemia (due to hyperglobulinaemia) and hypoalbuminaemia are common. Azotaemia can occur. Raised liver parameters may be present.

Urine analysis: May show poor urine concentrating ability (SG < 1.030) and/or proteinuria (as evidenced by an elevated urine protein to creatinine ratio: UPC (≥ 0.5).

 

How do I diagnose whether a dog has leishmania infection and/or disease?

1. Demonstration of Leishmania infantum organisms in cytology or histopathology samples

Finding leishmania organisms on stained smears or sections prepared from fine needle aspirates, impression smears or tissue biopsies from affected tissues is useful to confirm infection, although the examiner needs to be experienced in identifying organisms.

Organisms can be scarse and only present in certain organs, reducing the sensitivity of this technique. Samples should only be obtained in dogs in which clinical leishmaniosis is suspected due to the presence of consistent clinical signs and/or clinicopathological changes.

Suitable samples include bone marrow, lymph node, skin or joint fluid. Samples showing pathology consistent with leishmania infection e.g. (pyo)granulomatous or lymphoplasmacytic inflammation or lymph node reactive hyperplasia, as well as organisms, are more likely to represent clinically significant leishmania infection.

2. Serology - antibody testing

The detection of leishmania-specific serum IgG antibodies in the serum of dogs using quantitative techniques is very useful in the diagnosis of leishmania infection. Antibodies are typically detected by either ELISA or immunofluroescence (IFAT) and both are regarded as being good tests although some subjectivity occurs in the interpretation of IFAT titres. Although it can take a few months for dogs to seroconvert (up to 22 months, although median is 5 months) the long incubation period with leishmania means that most sick dogs are likely to be antibody positive.

High antibody levels are consistent with clinical leishmaniasis.

Low antibody levels are not usually indicative of disease, although infection is implied, and in these cases further montoring (repeat serology every 3-6 months) and /or additional diagnostic tests such as PCR are indicated to help confirm or exclude clinical leishmania disease. Serology results can also help determine when treatment courses can be completed.

3. Polymerase chain reaction (PCR)

PCR is a sensitive and specific technique that can amplify DNA from Leishmania infantum in various samples. PCRs based on amplifying leishmania kinetoplast DNA (kDNA), such as the PCR offered by Acarus, Langford Veterinary Services, are particularly sensitive due to the many thousands of kDNA sequences present in each Leishmania organism. Real-time PCR, as offered by Acarus, additionally allows quantification of leishmania DNA in the sample; thus the level of any leishmania infection present can be determined (higher levels consistent with severe disease), and additionally response to treatment can be monitored (as leishmania organism numbers should fall quite quickly with effective treatment). PCR results can also help determine when treatment courses can be completed (negative PCR results should be obtained before treatment is stopped).

Samples suitable for PCR are tissues in which lesions are believed to be present based on clinical examination or clinicopathological results; otherwise the sensitivity of detection by PCR, in descending order, is bone marrow or lymph node aspirates > skin aspirates > conjunctival swabs > buffy coat > whole blood. So although blood is often positive by PCR in clinical leishmania disease, a negative result cannot be used to rule out disease, and in these cases concurrent serology testing is important to determine if clinical leishmania disease is likely in cases with consistent clinical signs and/or clinicopathological changes. Additionally PCR can sometimes generate positive results in dogs that are leshmania infected but which do not have clinical disease, so again testing for antibodies is helpful to determine the stage of Leishmania infection and whether disease is likely to occur.  Repeat testing of asymptomatic dogs is always recommended to assess for any progression although repeat serology in this context may be more useful than PCR.

Conclusion:

Together with clinical signs and clinicopathological changes, combined Leishmania PCR and antibody testing gives the maximum information for helping you determine whether a dog is leishmania infected and/or has leishmania disease.

 

When is treatment for leishmaniasis indicated?

Treatment is indicated if a dog has active leishmania infection with associated clinical signs. The only time asymptomatic dogs may need treatment is if leishmania infection has been demonstrated (e.g. by cytology or PCR) in association with an antibody titre (could be low or negative on initial testing) which is found to rise on repeat testing 2-3 months later.

 

What treatment options are there?

Commonly used protocols (see table below for drug doses):

Protocol 1. Meglumine antimonate and allopurinol for 28 days, followed by allopurinol alone for at least 6-12 months.

Protocol 2. Miltefosine and allopurinol for 28 days, followed by allopurinol alone for at least 6-12 months.

Supportive care may also be required e.g. antibiotics if 2o bacterial pyoderma, treatment for malassezia if present, intravenous fluid therapy if renal disease.  Monitoring is also important (see below) to assess response and decide when allopurinol treatment can be stopped.

Meglumine antimonate and miltefosine are licensed for the treatment of canine leishmaniosis in several European countries but they require importation and authorisation in non-endemic countries such as the UK (see below).

 
  Drug Dose Possible Side Effects
Protocol 1 Meglumine antimonate
(e.g. Glucantime)
100 mg/kg SC q24h for
28 days (but some have used 50 mg/kg BID)
Pain, swelling at injection site – try and use different injection sites daily and massage well afterwards (sometimes anti-inflammatory prednisolone is required for 3-5 days if severe reactions).  Fever, loss of appetite and diarrhoea are occasionally reported.  Transient increase in liver enzymes sometimes seen.  Renotoxicity is very rare.
Protocol 1 plus Allopurinol 10 mg/kg PO BID for 6-12 months Xanthine uroliths – can monitor using ultrasound but although xanthine crystalluria is common, urolithiasis seems to be relatively uncommon.  If occurs then need dietary management and/or reduce dose of allopurinol to 5 mg/kg BID or 2.5 mg/kg BID, ensuring control of Leishmania remains.
Protocol 2 Miltefosine (e.g. Milteforan) 2mg/kg PO q24h with food for
28 days
Vomiting, diarrhoea (usually occurs within 5-7 days of starting treatment and is self-limiting over 1-2 days, so no treatment usually required)
Protocol 2 plus Allopurinol 10 mg/kg PO BID for 6-12 months Xanthine uroliths – can monitor using ultrasound but although xanthine crystalluria is common, urolithiasis seems to be relatively uncommon. If occurs then need dietary management and/or reduce dose of allopurinol to 5 mg/kg BID or 2.5 mg/kg BID, ensuring control of Leishmania remains.

Other therapeutic protocols for leishmaniosis less favoured include amphoteracin B, metronidazole in combination with spiramycin, marbofloxacin and domperidone.  Domperidone is a dopamine D2 receptor antagonist with prokinetic and anti-emetic effects; the DA antagonist effects result in an increase in serotonin which stimulates prolactin release which is a proinflammatory lymphocyte-derived cytokine – this may be involved with its anti-leishmania activity which has yet to be fully elucidated. Very little published evidence on its efficacy exists but an increasing number of vets are usuing it abroad, so clients sometimes return from periods abroad with this drug.  A recent study found that 1 mg/kg PO BID domperidone for a month was associated with either controlling or reducing clinical signs and antibody titres in infected dogs, however it was those dogs with very mild or earlier infections that responded best.  It may be that domperidone may be more effective when combined with other drugs such as miltefosine, but further studies are required.

 

How do I import these drugs?

Authorisation and importing Glucantime and Miltefosine in the UK

Apply for a SIC:

Veterinary surgeons wishing to import these drugs need to apply to the Veterinary Medicines Directorate (VMD) for a Special Import Certificate (SIC). The country of origin of the drug being imported is also required; meglumine antimonate can be imported from France and Spain, and miltefosine from Spain. An online web-based service for SICs is available for veterinary surgeons at the VMD’s Special Import site.

Select the 'Apply for Special Import Certificate (To import a veterinary medicinal product authorised within the EU)' option, which will then require you to enter your RVS membership number to continue.  The first time you do this, you will need to register yourself and your practice. This on-line application is free.

IImport the Drug:

Either identify a UK-based importing drug wholesaler to import the drug on your behalf from another EU country

OR

arrange to import it yourself directly from a wholesale drug supplier in an EU country where the product is licensed.

If using a UK-based importing drug wholesaler, you will require copy of a valid SIC as part of the ordering process. If the drug is being imported direct by you as the veterinary surgeon from an EU wholesale drug supplier, the SIC may also be required or at least confirmation that the importer is a registered veterinary surgeon.

 

How do I monitor response to treatment?

What response should I expect?

Response to therapy is good to poor depending on the degree of organ dysfunction. The majority of dogs improve clinically during the 1st month of treatment, usually seeing an improvement starting after a week or so. Dogs with renal dysfunction have a poorer response but can still do well. Improvements in blood qPCR results are usually seen quite quickly if the dog was initially positive on blood qPCR; the UPC and globulin levels may also improve quite quickly, but this is variable. Antibody titres remain elevated for longer periods of time and usually only start to decline a few months (4 - 6 months) after starting treatment – thus there is usually no point in looking for a decline in antibody levels within the 1st 4 months of treatment.
Monitoring after starting treatment:

Monitoring of dogs is recommended at the following approximate time points after starting treatment: 1 month, at 4 months, at 6 months and then every 6-12 monthly thereafter as required depending on progress.

Monitoring at 1 and 4 month checks should include evaluation of any organ dysfunction, serum albumin and globulin levels, urine protein: creatinine ratio and blood qPCR. In successful treatment, these will often normalise during this period. The 1 month testing is useful to evaluate the efficacy of the meglumine antimonate or miltefosine given and a marked improvement in test results suggests that the treatment can be stopped after a month (allopurinol, however, should be continued for at least 6 months).  Rarely the improvement is not as good as expected and in these cases consideration can be made to giving meglumine antimonate or miltefosine for another month, but please ask for advice before doing this.  For the subsequent checks, antibody levels should be evaluated as well. 

Finishing Treatment:

After one year of successful treatment, consideration may be given to discontinuing allopurinol, particularly in a non-endemic region. All clinicopathological parameters should be within reference range and the antibody titres borderline or negative, and blood qPCR negative, before allopurinol treatment is stopped. If a bone marrow sample is available at this time, a negative PCR provides further support for discontinuation. Discontinuation of therapy is ultimately dependent on recovery of the dog’s immunological ability to control the infection. Some dogs never regain this ability and will require continual therapy (allopurinol plus intermittent meglumine or miltefosine as required) to keep the infection under control.

 

What is the prognosis?

Although clinical response is good in the majority of dogs, it is guarded to poor in dogs with evidence of disseminated immune-mediated disease, and those with severe renal disease or failure.

Treatment does not clear infection; but many dogs with low infection loads (low serology, qPCR usually negative) remain asymptomatic for long periods – monitoring is important as described above.

 

What about vaccination?

A promising vaccine (CaniLeish by Virbac) has recently been launched in UK; it does not prevent infection but reduces risk of developing clinical sign by promoting cell-mediated immunity.

The vaccine is made up of Leishmania infantum excreted secretory proteins (~50-100) with adjuvant (local vaccine reactions are relatively common).  It is recommended for healthy seronegative dogs only - so you need to do serology before vaccination.

Vaccination induces antibody production that may cause seropositive results on conventional ELISA and IFAT Leishmania antibody testing, so vaccination will interfere with screening for natural infection using serology. However vaccine-induced antibodies are probably quite transient but vaccination status must be known when assessing dogs for natural infection with serology. Vaccination will not induce PCR positivity so PCR can still be used for diagnosis of leishmania infection in a vaccinated dog.

Despite the existence of vaccination, the importance of vector (sand fly) control in the prevention of leishmaniosis cannot be overemphasised.  All owners must be advised to do this, even in vaccinated dogs.  The use of synthetic pyrethroids that repel sandflies, and keeping dogs indoors between 7pm and 7am is recommended when sandflies are active (May to October for Europe).