I haven't worked with Theileria, but I assume that cryopreservation of blood stages should be similar to other hemoparasites such as Plasmodium. If you wish I can send you a protocol for cryopreservation of Plasmodium, but it only applies to 1 ml specimens. I would think that you will need to use cryopreserved specimens to infect a cattle / sheep by IV inoculation, and then have the ticks feed upon the infected animal.

Dear Dr. McAllister,

Thanks for your answer. I would be grateful if you send me the protocol for cryopreservation of Plasmodium.

Actually, I want to infect ticks in an artificial blood feeding system and since the piroplasms are not available throughout the year, therefore I have to cryopreserve Theileria blood stage so that I could use them any time I desire...

Regards,

Shahin

CRYOPRESERVATION METHOD FOR APICOMPLEXAN HEMOPARASITES

(method we use for Plasmodium gallinaceum, in chickens and Aedes aegypti mosquitoes)

When practical, perform procedures using aseptic practices (preferably in a laminar flow hood or biosafety cabinet). However, there may be few if any consequences if aseptic procedures cannot be strictly followed.

1. Prepare sterile Alsever's solution and place on ice.

2. Slowly add 1 ml DMSO to 9 ml pre-chilled Alsever's solution, mixing constantly and keeping on ice to avoid heating of the mixture.

3. Identify animal with a high parasitemia.

4. Label sterile cryovials (1.5 - 2 ml capacity) in anticipation.

5. Collect blood into a 10 ml vacuum heparin blood tube (fill the tube completely), or into a sterile syringe with heparin.

6. Place blood tube on ice and take to lab for immediate processing.

7. Transfer the blood into a larger sterile tube and place on ice.

8. Slowly (over the course of 2 or 3 minutes) add an equal volume of Alsever's/DMSO mixture to the blood, steadily mixing the entire time while on ice (either by gentle shaking and tapping of tube as solution is dripped into it, or by slowly pipetting Alsever's/DMSO mixture in-and-out of the blood with gradually increasing volumes until all is mixed). Too rapid mixing can cause osmotic shock to cells or generation of heat during dilution of DMSO.

9. Transfer 1 - 1.5 ml of mix into each cryovial

10. Place cryovials into pre-chilled (4C) container made for the purpose and place into -80 freezer overnight. For example, use a "Freezer Buddy" containing isopropanol, or a solid block container. A less controlled method is to wrap cryovials in thick cotton. The goal is to achieve a slow steady freeze at -1C / minute.

11. Transfer to liquid nitrogen for long term storage.

RECOVERY AND PASSAGE OF CRYOPRESERVED HEMOPARASITES:

1. Rapidly thaw cryovial in 37C water bath.

2. Inoculate IV into susceptible animal.

3. Monitor parasitemia daily. Onset may be delayed and parasitemia may be low.

4. Transfer infection from parasitemic animal to a another susceptible animal by IV inoculation of blood.

5. Monitor parasitemia daily. Parasitemia and symptoms may become severe.

6. Infect vector arthropods by feeding on fresh heparinized blood placed into a blood feeder apparatus at body temperature, or by feeding arthropods directly upon the animal.

7. After waiting an appropriate pre-patent period, confirm patent infection of arthropods by cytological inspection of salivary glands, and infect susceptible animals by exposure to infected arthropod vectors or by subcutanous inoculation of dissected sporozoites. This maintains sexual competency of the parasite strain.

Composition of Alsever's solution (g):

NaCl (4.2)

Citric Acid•3Na•2H2O (8.0)

Citric Acid•H2O (0.55)

D-Glucose (20.5)

Water (1000)

Cryopreservation of infected blood with heamoparasite  in liquid nitrogen then RECOVERY AND PASSAGE into splenoectomized calf in order to increase parasitemia .

what is a best preservation protocol for Theileria equi ...??