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Isolation and identification of feline peritoneal macrophages for in vitro studies of coronavirus-macrophage
1Cornell Feline Health Center, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401.
Abstract:
Feline peritoneal cells were collected by lavage with isotonic saline without the use of irritants or need for euthanasia of the cats. Macrophages were purified by centrifugation on Percoll followed by selective adherence. Although few macrophages could be obtained from an initial lavage, a second lavage performed on the same cat 9-11 days later yielded six times as many macrophages as the first lavage, providing sufficient numbers of cells for characterization and infection experiments. Macrophages from these subsequent lavages were not more functionally activated in phagocytosis assays than the resident macrophages from the initial lavage, and they were equally susceptible to infection with feline infectious peritonitis virus (FIPV). Infected cultures produced peak titers of 10(5.0) TCID50 per ml, and FIPV antigen was detected in a small subset (0.1-1.0%) of cells by indirect immunofluorescence. The FIPV-infected cells were identified as macrophages by their characteristic morphology and ability to phagocytize rhodamine-labeled latex beads. The successful isolation of large numbers of unactivated feline macrophages will permit in vitro studies of feline coronavirus-macrophage interactions that otherwise would not have been possible. Such studies will undoubtedly provide valuable insights into the pathogenesis of feline infectious peritonitis, an invariably fatal disease of domestic and exotic cats.
Insights
Researchers developed a method to collect large numbers of feline macrophages for studying feline infectious peritonitis virus (FIPV). This technique allows for in vitro research into FIPV-macrophage interactions and the disease
Area of Science:
- Veterinary Virology
- Immunology
- Cell Biology
Background:
- Feline infectious peritonitis (FIP) is a fatal disease in cats.
- Understanding feline coronavirus (FIPV) interactions with macrophages is crucial for FIP research.
- Previous studies were limited by the scarcity of available feline macrophages.
Purpose of the Study:
- To establish a reliable method for isolating large quantities of feline macrophages.
- To investigate the susceptibility of isolated macrophages to FIPV infection.
- To enable in vitro studies of feline coronavirus-macrophage interactions.
Main Methods:
- Feline peritoneal cells were collected via lavage.
- Macrophages were purified using Percoll centrifugation and selective adherence.
- Repeated lavages on the same cat increased macrophage yield.
- Phagocytosis assays and indirect immunofluorescence were used for characterization and infection studies.
Main Results:
- A second lavage, 9-11 days after the first, yielded six times more macrophages.
- Isolated macrophages were not more activated than resident macrophages.
- Macrophages were equally susceptible to FIPV infection.
- Infected cultures reached peak titers of 10(5.0) TCID50/ml, with FIPV antigen detected in 0.1-1.0% of cells.
Conclusions:
- A successful method for isolating abundant, unactivated feline macrophages was developed.
- This technique facilitates in vitro studies of FIPV-macrophage interactions.
- The findings will advance understanding of feline infectious peritonitis pathogenesis.