Leishmania challenge model
View Patent ↗The present invention provides a method for effectively and reproducibly infecting canines with Leishmania infantum using sand flies to vector the parasite. The inventive method comprises several steps, including ensuring canines are naïve to Leishmania , infecting the canines using bites of Leishmania -infected sand fly bites, and evaluating successful transmission of the Leishmania parasites.
1. A method for infecting canine animals with a Leishmania parasite comprising the steps of:
a. testing the canine animals to ensure they are free of Leishmania and not previously exposed to sand fly salivary proteins (naïve);
b. transmitting the Leishmania parasite to the canines by means of Leishmania -infected sand fly bites; and
c. quantifying the parasite load/fly and the % metacycles/fly (scoring the sand flies) post transmission to verify success of transmission, thereby infecting the canine animals.
2. The method of claim 1 further characterized in that the transmitting to the canines is performed using 10 to 50 bites of the infected sand flies.
3. The method of claim 1 or 2 wherein the infected sand flies harbor greater than or equal to 30,000 Leishmania parasites, and wherein greater than or equal to 50% of the parasites are metacycles.
4. The method of claim 3 further characterized in that the Leishmania parasite belongs to species infantum .
5. The method of claim 1 or 2 further characterized in that the testing of the canines includes testing for previous exposure to sand fly salivary peptides or proteins, or testing for the presence in the canine animals of Leishmania -specific components, including antigens, nucleic acids, proteins, peptides, or membrane components, or testing for the presence of host-produced components, including antibodies against said Leishmania components.
6. The method of claim 1 or 2 wherein the Leishmania parasite is a virulent strain isolated from a sick canine animal that is infected with the Leishmania parasite.
7. The method of claim 1 or 2 wherein the transmitting is done using multiple delivery or bite sites on the canine animals, to mimic what occurs under field conditions wherein canines are infected with Leishmania parasites via the biting action of Leishmania -infected sand flies.
8. The method of claim 1 , which models naturally occurring Leishmania infection in canines, comprising the steps of:
a. selecting canines six months or younger for infection;
b. testing the canines for previous exposure to sand fly salivary proteins;
c. infecting vector sand flies with a virulent isolate of L. infantum or other Leishmania spp., obtained from a canine infected with the L. infantum or other Leishmania spp., wherein only passages 5 or 4 or 3 or earlier of the L. infantum or other Leishmania spp. are used to carry out the infection, and wherein the infecting is accomplished by supplying the sand flies with blood containing L. infantum or other Leishmania spp.;
d. monitoring the sand fly infection to follow the progression of the infection along the sand fly midgut;
e. assessing the state of bacterial colonization in the sand fly;
f. assessing an incremental increase in total parasite number with time and an incremental increase in appearance of metacyclics with time;
g. scoring sand flies prior to every transmission event;
h. using blood from Leishmania -infected canine(s) to infect groups of flies 1 or 2 or 3 times a week;
i. carrying out on multiple sites over multiple transmissions of Leishmania spp. to dogs via sand fly bites; and
j. scoring of sand flies used for transmission to assess feeding success and infection status, thereby infecting the canines, and modeling naturally occurring Leishmania infection in canines.