Toxoplasma gondii

Characteristics
Morphology	Toxoplasma gondii (T. gondii) belong to the phylum Apicomplexa and family Sarcocystidae. They are obligate intracellular parasitic protozoa. It is a parasite of birds and mammals. Felines are the only definitive host and the only animals that pass infective oocysts in their feces. Generally, oocysts are spherical and measure 10x12 µm, sporozoites measure 2x6 µm, tachyzoites are crescent-shaped and 2x6 µm, and tissue cysts are spheroid and have a diameter of 5 µm - 70 µm, bradyzoites measure 7x1.5 µm. Three major infectious stages and major morphological forms occur: oocyst-containing two sporocysts and four sporozoites each, quickly-multiplying tachyzoites, and slow-growing bradyzoites contained in persistent tissue cysts.
Growth Conditions	DMEM at pH 7.2 with 5% CO2 should be used to maintain and culture the tachyzoites of T. gondii. Several T. gondii strains are highly virulent and can propagate in any human tissue.

Characteristics

Morphology

Toxoplasma gondii (T. gondii) belong to the phylum Apicomplexa and family Sarcocystidae. They are obligate intracellular parasitic protozoa. It is a parasite of birds and mammals. Felines are the only definitive host and the only animals that pass infective oocysts in their feces. Generally, oocysts are spherical and measure 10x12 µm, sporozoites measure 2x6 µm, tachyzoites are crescent-shaped and 2x6 µm, and tissue cysts are spheroid and have a diameter of 5 µm - 70 µm, bradyzoites measure 7x1.5 µm. Three major infectious stages and major morphological forms occur: oocyst-containing two sporocysts and four sporozoites each, quickly-multiplying tachyzoites, and slow-growing bradyzoites contained in persistent tissue cysts.

Growth
Conditions

DMEM at pH 7.2 with 5% CO2 should be used to maintain and culture the tachyzoites of T. gondii. Several T. gondii strains are highly virulent and can propagate in any human tissue.

Health Hazards
Host Range	Cats and other felines, humans, and warm-blooded vertebrates, including most mammals and birds. Flies and cockroaches that have fed on infected cat feces can transmit oocysts to food in their own feces
Modes of Transmission	A Toxoplasma infection occurs by one of the following: Eating undercooked, contaminated meat or shellfish, drinking contaminated water, inhalation of aerosols containing oocysts, and contact with sand or soil contaminated by cat feces. Transplacental transmission is possible if the mother has an acute infection during pregnancy. Transmission is also possible through blood transfusions and organ transplants.
Signs and Symptoms	Toxoplasma gondii infection is usually non-pathogenic in immunocompetent adults. Some people who have toxoplasmosis may feel as if they have the “flu” with swollen lymph glands or muscle aches and pains that may last for a month or more. Severe toxoplasmosis, causing damage to the brain, eyes, or other organs, can develop from an acute Toxoplasma infection or one that had occurred earlier in life and is now reactivated. Severe toxoplasmosis is more likely in individuals who have weak immune systems, though occasionally, even persons with healthy immune systems may experience eye damage from toxoplasmosis.
Infectious Dose	As few as 10 sporulated oocysts in intermediate hosts
Incubation Period	If inoculated with a mixture of tachyzoites and bradyzoites, tissue cysts can form as early as 2-3 days post-infection, although a clinical presentation of toxoplasmosis may not arise.

Medical Precautions/Treatment
Prophylaxis	None readily available
Vaccines	None readily available
Treatment	In an otherwise healthy person who is not pregnant, treatment usually is not needed. If symptoms occur, they typically go away within a few weeks to months. For pregnant women or persons who have weakened immune systems, medications (Spiramycin) are available to treat toxoplasmosis.
Surveillance	Monitor for symptoms. Confirm through positive serology for IgM and IgG antibodies (i.e. by ELFA or ELISA). Identification can also be confirmed through isolation of the parasite, PCR, and ultrasound imaging.
GWU Requirements	Report all incidents to the Office of Risk Management as well as the Office of Research Safety (ORS) IMMEDIATELY FOLLOWING THE OCCURRENCE.

Containment
BSL-2/ABSL-2	Risk Group 2 classification is applied to the parasite. BSL-2 and ABSL-2 practices, including containment equipment/facilities and laboratory personal protective equipment (PPE), are recommended for activities involving infective stages of the parasites.

Spill Procedures
Small Spills (<1 liter)	If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup. T. gondii is known to be transmitted via aerosols, so caution should be taken during a spill outside of a BSC. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply an EPA registered disinfectant effective against the parasite (bleach, ethanol, glutaraldehyde), working from the perimeter towards the center. Allow 30 minutes of contact time before disposal and cleanup of spill materials.
Large Spills	Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Spill Procedures

Small Spills (<1 liter)

If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup.

T. gondii is known to be transmitted via aerosols, so caution should be taken during a spill outside of a BSC. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply an EPA registered disinfectant effective against the parasite (bleach, ethanol, glutaraldehyde), working from the perimeter towards the center. Allow 30 minutes of contact time before disposal and cleanup of spill materials.

Large Spills

Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Exposure Procedures
Mucus Membrane	Flush eyes, mouth or nose for 15 minutes at eyewash station.
Other Exposure	For an area not protected by skin, wash with soap and water for 15 minutes (open wounds, sores, etc.).
Reporting	Report ALL injuries to the PI immediately and reported to the Office of Risk Management at [email protected] IMMEDIATELY FOLLOWING THE OCCURRENCE. Exposures that involve a bloodborne-pathogen or recombinant DNA also need to be reported to the Office of Research Safety at [email protected]. If the injury requires immediate medical attention, call GWPD at 202-994-6111 or call 911.
Medical Monitoring	Seek immediate medical evaluation, treatment, and post exposure follow-up at the Employee Health Office at GWU Hospital (900 23rd St., NW, Suite G-1090, Phone: 202-715-4275). Students should go to the Students Health Office at Marvin Center. After hours treatment can be received at the GWU hospital emergency room.

Stability
Disinfection	Tachyzoites and tissue cysts are susceptible to detergents, including 10% bleach, and 70% ethanol. Oocysts are resistant to most disinfectants, but 10% formalin decreases viability significantly
Inactivation	Tissue cysts die in 6% NaCl solutions. Cysts can be killed with gamma irradiation at a dose of 1.0 kGy. High pressure processing at 300 MPa or higher inactivates tissue cysts. Heating tissue cysts to 67°C will kill them instantly. Cysts in meat can be killed by heating the meat to >60 °C or freezing it at -20 °C. Oocysts are killed if kept at a temperature of 55-60°C for 1-2 minutes
Survival Outside Host	Oocysts can survive in moist soil or water for up to 18 months. They can survive in uncovered feces for 46 days and for 334 days when covered

Personal Protective Equipment (PPE)
Minimum PPE Requirements	At minimum, personnel are required to don gloves, closed toed shoes, lab coat, and appropriate face and eye protection prior to working with T. gondii. Additional PPE may be required depending on lab specific SOPs.
Additional Precautions	Additional protection may be worn over laboratory clothing when infectious materials are directly handled, such as solid-front gowns with tight fitting wrists, gloves, and respiratory protection. Wash hands with soap and water after removing gloves.

References
CDC (https://www.cdc.gov/parasites/toxoplasmosis/) BMBL 6th Edition (https://www.cdc.gov/labs/BMBL.html) Canadian PHAC (https://www.canada.ca/en/public-health/services/laboratory-biosafety-bi…) ABSA (https://my.absa.org/riskgroups) Toxoplasma gondii: Laboratory maintenance and growth (https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpmc.26 )