for the culture of Plasmodium falciparum were described in 1976 (42) and have been improved and modified since that time (43, 44). Life cycle stages of the five Plasmodium spp. that infect humans have been established in vitro. Of these five, P. falciparum and P. knowlesi are the only species for which all stages have been cultured in vitro. The life cycle includes the exoerythrocytic stage (within liver cells), the erythrocytic stage (within erythrocytes or precursor reticulocytes), and the sporogonic stage (within the vector). Culture media generally consist of a basic tissue culture medium to which serum and erythrocytes are added. Most of the culture methods have been directed toward the stage found in the erythrocyte. This stage has been cultivated in petri dishes or other containers in a candle jar to generate elevated CO2 levels or in a more controlled CO2 atmosphere. Later developments employed continuous-flow systems to reduce the labor-intensive requirement for replenishing the system with fresh media. The exoerythrocytic and sporogonic life cycle stages have also been cultivated in vitro. Although cultivation is of great help in understanding the biology of Plasmodium, it does not lend itself to use for routine diagnostic purposes (7).
The availability of the microaerophilous stationary phase (MASP) culture technique, in which the parasites proliferate in a settled layer of blood cells, has provided an opportunity to study Babesia, a formerly obscure disease agent regarded as within the purview of veterinary parasitology, in the laboratory. A number of Babesia spp. have been established in continuous culture using the MASP technique. It is possible to study the basic biology of the organism—as well as host-microbe interactions, immune factors triggered by the parasite, factors involved in innate resistance of young animals to infection, and antimicrobial susceptibility—to a degree not possible before the availability of cultures. These culture systems can produce quantities of parasite nucleic acid needed for defining phylogenetic relationships, developing diagnostic methods for parasite detection in asymptomatic individuals, and producing parasite antigens and attenuated strains of Babesia that could be used for immunization (5).
The in vitro cultivation of Cryptosporidium has improved significantly in recent years. These obligate intracellular parasites colonize the epithelium of the digestive and respiratory tracts, are often difficult to obtain in significant numbers, produce durable oocysts that defy conventional chemical disinfection methods, and are persistently infectious when stored at refrigerator temperatures (4 to 8°C). While continuous culture and oocyst production have not yet been achieved in vitro, routine methods for parasite preparation and cell culture infection and assays for parasite life cycle development have been established. Parasite yields tend to be limited, but in vitro growth is sufficient to support a variety of research studies, including assessing potential drug therapies, evaluating oocyst disinfection methods, and characterizing life cycle stage development and differentiation (1). Recent studies indicate that primary human intestinal epithelial cells (PECs) support Cryptosporidium better than other existing cell lines (45).
Although various microsporidia that infect humans can be identified from clinical specimens by serologic and/or molecular methods, none of these methods are commercially available. Unfortunately, in some cases microscopic examination of biopsy specimens does not yield conclusive results. It is also possible that microsporidial organisms may be present in very small numbers, which can be easily missed during routine histologic examinations. Some microsporidia such as Encephalitozoon, Enterocytozoon, 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,
