Vittaforma, Trachipleistophora, and Anncaliia spp., even when they are present in small numbers, can become established in cell cultures, thus facilitating their identification at a later time. Therefore, attempts at culturing these organisms should be made whenever possible, since many clinical laboratory personnel are familiar with cell culture methodology (11). Anncaliia algerae has been successfully grown on the rabbit kidney cell line, RH-13 (46). Cell lines from goldfish skin (GFSK-S1) and brain (GFB3C-W1) and ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth were also found to be successful. All cultures were maintained at 27°C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish, and insects (46). Expanded information on cell lines, media and supplements used for the culture of microsporidia causing human infections can be seen in Table 8.2 and Fig. 8.8 and 8.9.
Figure 8.8 (A) An E6 cell infected with E. intestinalis. Magnification, ×1,200. Note the well-defined multiple parasitophorous vacuoles (PV); N, host cell nucleus. (B) An HLF cell culture completely destroyed by E. hellem. Note the spores with everted polar tubules (at arrows), Magnification, ×600. All three species of Encephalitozoon destroy the cell culture, and often the cell cultures are completely covered by spores that either are intact or have discharged their polar tubules. Note It is very unusual to see the extruded polar tubules in routine clinical specimens such as urine or stool. (Courtesy of Govinda Visvesvara, from Visvesvara G, Clin Microbiol Rev 15:401–413, 2002). doi:10.1128/9781555819002.ch8.f8
Figure 8.9 (A) A host cell infected with Anncaliia (Brachiola) algerae. Note the arrangement of spores around the host (E6 cell) nucleus (N). A single spore is probably in the process of infecting an adjacent cell (arrowhead). Magnification, ×1,200. (B) A spore with an everted polar tubule. Magnification, ×1,200. (Courtesy of Govinda Visvesvara, from Visvesvara G, Clin Microbiol Rev 15:401–413, 2002). doi:10.1128/9781555819002.ch8.f9
Most routine clinical laboratories do not have the animal care facilities necessary to provide animal inoculation capabilities for the diagnosis of parasitic infections. Host specificity for many animal parasite species is a well-known fact which limits the types of animals available for these procedures. In certain suspect infections, animal inoculation may be requested and can be very helpful in making the diagnosis, although animal inoculation certainly does not take the place of other, more routine procedures.
The hamster is the laboratory animal of choice for the isolation of any form of Leishmania spp. A generalized infection results after intraperitoneal inoculation; spleen impression smears should be examined for the presence of organisms.
1. Aspirates or biopsy material obtained under sterile conditions from cutaneous ulcers, lymph nodes, spleen, liver, bone marrow, buffy coat cells, 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.