Ref. CDVD011
32 tests/kit
CANINE LEISHMANIA
Dog
Canine Leishmania
Canine Leishmania is an enzyme immunoassay (EIA) for the detection and semi-quantitative measuring of specific antibodies against canine Leishmania antigen in dog sera. To perform the test following the recommended procedure requires approximately 30 minutes.
General Information
Canine leishmaniasis is a parasitic zoonosis, endemic, caused by a
flagellated protozoan included in the Leishmania genus.
Symptomatically, the disease may manifest itself in three different forms :
Cutaneous : It’s characterized by wounds on the skin with appearance
of little nodules in the ears, elbows, tarsus, neck, between fingers, and
around eyes. These little nodules open up, appearing dry or wet ulcers. It
may also appear alopecia, furfuraceous desquamation, dermatosclerosis,
etc.
b) Visceral : It’s characterized by anaemia, adynamia, hyperthermia,
muscular fibrillation, nasal hemorrhages, hepatomegaly, splenomegaly,
adenopahies, progressive slimming without loss of appetite, purulent
conjunctivitis which are resistant to antibiotic therapy, keratitis with
cornea pigmentation, corneal ulcers, and, in the last phases, back third
paralysis that may spread to the front third and neck.
Cutaneous visceral : It’s characterized by a combination of cutaneous
and visceral symptoms.
Once the animal is infested, The infection may take different courses,
depending basically on the immunity condition and other factors of the
individual :
Latent or unapparent infection : The parasite is localized into the
cells of the reticular endothelial system, where it reproduces.
Acute infection : When the infection turns acute, the animal begins to
manifest symptoms of the disease. In this moment the Leishmanias begin to
be phagocyted by macrophages of the organism, where they reproduce until
they destroy them, thus remaining free in the bloodstream, where they are
again phagocyted invading different tissues and organs. In this phase, if
the animal is bitten by a transmitter phlebotomus, the Leishmania go into
the intestine of the animal, where they develop to the flagellate phase (leptomone)
and go to the vector’s pharynx, from which they will move to another
animal when the phlebotomus bites it.
Chronic infection : After acute phase, the disease develops up to the
chronic form, which culminates in the animal’s death.
Biological Cycle
In natural conditions the Leishmania has an evolutionary cycle, in which
an amastigote phase, of intracellular growth in the vertebrate host,
alternates with a promastigote phase, extracellular in the vector insect.
When the Leishmanias are localized into the vertebrate host, they parasite the cells of the phagocitary mononuclear system. In the interior of the macrophage, the Leishmania is found in amastigote form. This has a round or ovoid corpuscle form, immobile, from 2 to 4 µm of diameter, with a round nucleus, a kinetonucleus, and it lacks flagellum. It multiplies by simple binary fission, until the host’s cell is destroyed and the released amastigots are phagocyted by new macrophages. When the vector, haematophagous dipterous of Phlebotomus genus, feeds on infested animals, ingests cells parasited by amastigots. When blood arrives at the insect’s stomach, the protozoa are released by the breaking of the macrophages transforming themselves into promatigotes of 15 to 20µm long, spindle-shaped and with external flagellum. These promastigotes multiply by longitudinal division, and they are first located in the medium intestine and later in the pharynx and the buccal cativity.
When the insect bites a healthy host, he inoculates the promastigotes. These promastigotes are phagocyted by the macrophages, transforming themselves into amastigotes, so re-starting the cycle again.
Therefore the role played by hosts and vectors is of vital importance in the epidemiological chain.
The ecological System in which the population of parasites remains indefinitely, may be regarded as a reservoir of the Leishmania specie.
The system basically consists of reservoirs and vectors.
Reservoirs : Canines and gnawing are the reservoirs of the Leishmania
donovani complex.
Vectors : Basically Phlebotomus, although might be involved fleas, ticks
and flies.
Leishmaniasis in men :
In our case the visceral leishmaniasis caused by L. donovani, acquires an
special importance, since excluding India and Central Kenya where man is
the reservoir, the phlebotomus and mammalian reservoirs are the natural
hosts, and man is the accidental host. In China, Mediterranean basin and
Brazil the reservoirs of the L. donovani complex are the canines. The
vectors are : Phlebotomus chinesis(China), P. ariasi, P. perniciosus and
other species(Mediterranean basin) and Lutzonya longipalpis(Brazil)
There have been occasional findings of L. donovani in wild carnivores : fox, jackal, wolves and rats. Thus, they can constitute the primary reservoir from which has been formed the secondary synantropic canine reservoir.
Incidence:
Even though there aren’t too many reports about it, it’s known that
there are endemic areas in which there is a positive correlation between
the casuistry of cases in animals and cases in men, which leads the WHO to
include it into the diseases list of obligatory declaration. It isn’t
too risky to affirm that the incidence in endemic areas must be at present
more than 30%, with a tendency to increase.
Diagnosis:
Given the increase of disease’s prevalence, now the clinical veterinary
ought to have available the appropriate means to carry out a quick and
reliable diagnosis, even those who exercise their activity in free areas
or with low prevalence, for only an animal that has traveled to an
enzootic area is enough to catch the disease.
Leishmaniasis is a disease with specific treatment, so it requires an
accurate diagnosis
Principle of the test
The Cypress Diagnostic Canine Leishmania test is based on the principle of an enzyme Immunoassay (EIA). The assay system utilizes an inactivated specific antigen. This specific antigen is coated on the microtiter well.
First, the test sample or controls are allowed to react with the solid phase specific antigen. If there are specific antibodies, they will bound to the antigen. After 10 minutes incubation at Room Temperature, the wells are washed with the washing solution to remove unbound material.
In a second step, a Protein A/HRPO conjugate is added to the wells resulting in the specific antibody being sandwiched between the solid phase antigen and enzyme conjugate Protein A. After 5 minutes incubation at Room Temperature, the wells are washed with the washing solution to remove unbound material. A substrate /chromogen solution is added and incubated 10 minutes , resulting in the development of a blue color in the wells where a complex Antigen/Anti-Leishmania antibody/Protein A - HRPO conjugate was formed previously.
The color development is stopped with the addition of the stop reaction
solution and the color turns yellow. The yellow color is measured
spectrophotometrically at 450 nm.
The concentration of dog specific IgG antibody against canine Leishmania
is proportional to the color intensity of the test sample.
Rev.09.95
