into three to five mice of any laboratory strain weighing ∼20 g.
C. Maintain mice in isolation.
D. Every day, check for signs of central nervous system dysfunction; if symptoms are detected, perform an autopsy on the animal.
Examination of Specimens
1. Examination of impression smears is summarized in Table 6.1.
2. Examine skin snips in teased preparations for detection of microfilariae of Onchocerca volvulus and Mansonella streptocerca as follows.
A. Tease the small bit of tissue apart in a few drops of saline to release the microfilariae.
B. Remove drops of saline to a 1- by 3-in. glass slide, cover with a no. 1 coverslip, and examine under low light for microfilariae.
C. For a permanent preparation, place 100% methanol under the coverslip to fix filariae, partially dry, remove the coverslip, and stain with Giemsa or one of the other blood stains (including the rapid stains).
3. Examine squash preparations for detection of Trichinella spp. in muscle microscopically at low power (×100) and under low light.
4. Examine scrapings of skin for Sarcoptes scabiei (scabies) microscopically at low power (×100) and under low light. Confirmation at high dry power (×400) may be necessary (Fig. 6.16). See chapter 35 on arthropods for specific/detailed skin-scraping protocol for scabies.
Figure 6.16 Sarcoptes scabiei mites from skin scrapings. (Upper) Note the two mites to the left of center. (Lower) Note the two eggs, the small nymph, and the adult mite (photographed at a higher magnification). If the light is too strong when examining the scrapings in saline, the mites will probably not be seen. doi:10.1128/9781555819002.ch6.f16
5. Inoculate cultures with ground tissue suspensions (to release organisms from the cells).
A. Place a small tissue sample in a sterile tissue grinder (Ten Broeck or Dounce) in 0.5 ml of sterile saline, and grind until tissue is broken up but not totally liquefied.
B. Add several drops of ground tissue to culture medium as follows.
a. NNN medium (see chapter 8): add drops of tissue to the bottom of the slant, where they will “pool” with condensed moisture. Incubate at room temperature (isolation of Leishmania spp.).
b. TYSGM-9 medium (see chapter 8): add drops of tissue to the liquid medium, add 3 drops of the starch suspension, and incubate at a 45 to 50° angle at 35°C for 48 h (isolation of E. histolytica).
c. Nonnutrient agar plate seeded with bacteria (see chapter 8): add drops to center of seeded agar plate, and incubate at 35 to 37°C (isolation of Acanthamoeba or Naegleria spp.).
6. Examine cultures (wear gloves at all times).
A. NNN agar for promastigotes of Leishmania spp. (see chapter 8): using a Pasteur pipette, remove a drop of fluid from interface of agar slant and culture tube, place on glass slide, cover with coverslip, and examine microscopically (×400) under low light for motile promastigotes (Fig. 6.17).
Figure 6.17 Leishmania culture sediment. Note the promastigotes from the culture sediment. The typical “rosette” formation in the right image is frequently seen. doi:10.1128/9781555819002.ch6.f17
B. TYSGM-9 medium for amebae (see chapter 8): using a sterile Pasteur pipette, remove 1 or 2 drops of material from interphase of agar and overlay, place on glass slide, cover with coverslip, and examine microscopically (×100) under low light for trophozoite motility.
C. Nonnutrient agar plate with lawn of Escherichia coli or Enterobacter sp. for detection of free-living amebae (see chapter 8): examine microscopically at low power (×100) for changes in bacterial lawn, particularly patches and tracks, indicating that the protozoan trophozoites have ingested bacteria as they move over the agar.
D. Mouse passage for detection of Toxoplasma gondii:
a. Wear canvas gloves to handle mice; sacrifice the animal(s).
b. Pin mouse to board, spray with 70% ethyl alcohol, and open peritoneal cavity with sterile scissors.
c. Using a sterile Pasteur pipette, remove fluid from the peritoneal cavity, and place in small tube or prepare smears.
d. Using one of the blood stains, prepare stained smears of peritoneal exudate, and examine microscopically at a magnification of ×1,000 for T. gondii tachyzoites.
e. Place mice and all contaminated disposable materials in bag to be autoclaved and destroyed. Place nondisposable materials (scissors) in bag to be autoclaved prior to washing.
7. Correlate all examination results (wet mount, stains, and cultures) to determine presence of organisms.
Results
The majority of the protozoa are found on the permanent stained smears (impression smears, touch or squash preparations, or teased preparations). When culture is used, permanent stained smears of the culture medium or sediment may also reveal some of the protozoa. Although infrequently used, material from animals (at autopsy) can be examined as both wet and permanent stained preparations for confirmation of protozoa. Filarial infections may be confirmed by the recovery and identification of microfilariae in skin scrapings and/or biopsy specimens.
If identifications are certain, report the organisms detected; if a presumptive identification is made, confirmation by another laboratory is suggested.
Onchocerca volvulus and Mansonella streptocerca
The use of skin snips is the method of choice for the diagnosis of human filarial infections with O. volvulus and M. streptocerca. Microfilariae of both species occur chiefly in the skin, although O. volvulus microfilariae are on rare occasions found in the blood and occasionally in the urine. For best results, the skin snip specimens should be thick enough to include the outer part of the dermal papillae. Snips may be taken in various ways. A small slice may be cut (using a razor blade) from a skin fold held between thumb and forefinger, or a slice may be taken from a small “cone” of skin pulled up by a needle. The skin snip should be so thin that significant bleeding does not occur, just a slight oozing of fluid. Corneal-scleral punches (either Holth or Walser type) have been found to be successful in taking skin snips of uniform size and depth and an average weight of 0.8 mg (range, 0.4 to 1.2 mg); this procedure is easy to perform and is painless. It has been demonstrated that in African onchocerciasis, it is preferable to take skin snips from the buttock region (above the iliac crest); in Central American onchocerciasis, the preferred skin snip sites are from the shoulders (over the scapula).
Skin snips are placed immediately in a drop of normal saline or distilled water and covered so that they will not dry; teasing the specimen with dissecting needles is not necessary but may facilitate release of the microfilariae. Microfilariae tend to emerge more rapidly in saline; however, in either fluid, the microfilariae usually emerge within 30 min to 1 h and can be examined under low-intensity light with the 10×