or CSF may be used for inoculation.
2. Material (0.25 to 1 ml) obtained from these sources should be inoculated under sterile conditions by the intraperitoneal route. Material from patients with mucocutaneous leishmaniasis should be inoculated by the intranasal route or into the feet.
3. Young (either sex) hamsters (2 to 4 months old) should be used for this procedure.
4. If the animal dies several days after inoculation, splenic aspirates should be examined for the presence of organisms. The material should be prepared as thick blood films and stained with Giemsa or other blood stains.
Note The infection develops slowly in hamsters; several months may be required to produce a detectable infection. For this reason, culture procedures are usually selected as more rapid means of parasite recovery. Intranasal lesions or those in the feet may develop very slowly; experimental animals should be kept for 9 to 12 months before a negative report is sent out.
Several laboratory animals can be used for the recovery of trypanosomes. Organisms are usually found in the blood of the animals within the first week after inoculation; however, if no organisms are found, the animals should be checked at the end of 2 and 4 weeks before results are reported as negative. Trypanosomes are infectious, so extreme care should be used when examining any blood or tissue suspected to contain these organisms.
1. Blood, lymph node aspirates, tissue, or CSF obtained under sterile conditions may be used for inoculation.
2. Material (up to 2 ml) should be inoculated intraperitoneally into guinea pigs or white rats for Trypanosoma gambiense and T. rhodesiense and into white mice for T. cruzi.
3. The number of organisms in the blood may vary; therefore, smears should be prepared frequently (every few days) over a 4-week period after inoculation.
Note Rats should be checked for the presence of T. lewisi (common parasite in rats) before inoculation to prevent a possible false-positive result.
All common laboratory animals can be infected by T. gondii. White rats and mice are generally used. White rats develop a chronic infection that can be a useful means of maintaining a strain of organisms. Mice that have been inoculated by the intraperitoneal route develop a fulminating infection that leads to death within a few days. Tremendous numbers of organisms can be recovered from the ascitic fluid. These specimens should be handled carefully to avoid an accidental laboratory infection.
General Procedure
1. The Toxoplasma organisms are found throughout the body after dissemination via the bloodstream. Any body tissue or fluid can be used for animal inoculation; the most common specimens are blood, lymph node fluid, and CSF.
2. The material used for inoculation (the amount may be very small [less than 0.25 ml]) should be obtained under sterile conditions and should be inoculated via the intraperitoneal route.
3. White mice of either sex and any age can be used. The animals should be checked daily for symptoms of illness.
4. After several days, the organisms can be recovered at necropsy from the peritoneal fluid of the mouse. The material should be prepared as thick blood films and stained with Giemsa or other blood stains.
5. Blind passage of peritoneal fluid to additional mice is recommended if the original mice appear to be negative.
6. If mice survive for up to 6 months, serum can be tested for the presence of antibody.
Procedure for Tissue
1. Grind the tissue in sterile 0.85% NaCl.
2. Prepare a 10% suspension (10% tissue, 90% NaCl).
3. Inoculate six mice via the intraperitoneal route. Each mouse should receive 1 ml.
4. The mice can be reinoculated the following day with an additional 1-ml dose.
5. Check the mice daily for symptoms.
6. If the animals are not sick at the end of 6 days, examine 1 drop of peritoneal fluid from each mouse directly and then stain it with Giemsa stain.
Xenodiagnosis is a technique that uses the arthropod host as an indicator of infection. Uninfected reduviid bugs are allowed to feed on the blood of a patient who is suspected of having Chagas’ disease (T. cruzi infection) (47) (Fig. 8.10). After 30 to 60 days, feces from the bugs are examined over a 3-month time frame for the presence of developmental stages of the parasite, which are found in the hindgut of the vector. This type of procedure is used primarily in South America for field work, and the appropriate bugs are raised in various laboratories specifically for this purpose. The term “xenodiagnosis” has also been applied to the diagnosis of trichinosis (Trichinella spiralis). Muscle tissue from a patient suspected of having the disease is fed to uninfected rats; the rats are then checked after the appropriate time for the presence of T. spiralis larvae, particularly in the diaphragm. This procedure is rarely requested and is not available in most clinical laboratories.
Figure 8.10 Illustration of the process of xenodiagnosis used for the diagnosis of Chagas’ disease. (Illustration by Sharon Belkin.)doi:10.1128/9781555819002.ch8.f10
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