Factors influencing tissue cyst yield in a murine model of chronic toxoplasmosis

Abstract

Recent advances into the unique biology of Toxoplasma tissue cysts and the bradyzoites they house necessitates optimization of tissue cyst recovery from infected mouse brains. Here, we present data from 68 tissue cyst purifications of Type II ME49 tissue cysts in CBA/J mice performed over a period of two years. The effects if infecting with both tissue culture tachyzoites as well as ex vivo tissue cysts were assessed. Significant mortality was restricted to tachyzoite infections with female mice being more susceptible. Infection with tissue cysts was associated with both lower overall symptomology and mortality exhibiting no sex bias. Cumulatively, host sex did not impact overall tissue cyst yields, although, tachyzoite initiated infections generated significantly higher yields compared to tissue cyst-initiated infections. Notably, serial passage of tissue cysts was accompanied with a decreasing trend for subsequent cyst recovery. The time of tissue cyst harvest, a potential reflection of bradyzoite physiological state, had no significant impact on subsequent cyst yield at the selected time points. In aggregate, the data reveal the considerable heterogeneity associated with tissue cyst yield making the design of adequately powered experiments critical. This is particularly the case for drug studies where overall tissue cyst burden currently serves as the primary and often sole metric of efficacy, as the data presented here demonstrate that cyst recovery between preparations of untreated animals can mirror the reported effects of drug treatment.

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides inToxoplasma gondiitachyzoites and bradyzoites

“…To determine if the loss of TgLaforin impacted the overall viability/infectivity of in vivo tissue cysts, we examined the disease progression in WT, ΔTgLaf, and COMP infected animals following injection of 20 tissue cysts i.p. Consistent with prior data, infection with tissue cysts results in markedly lower pathology and consequent mortality during the acute phase for WT as well as both the ΔTgLaf and COMP lines ( Figure 7A, B ) (68). The death rate from cyst infection did not differ statistically among the three lines.…”

“…The capacity of ΔTgLaf cysts to initiate new infections was assessed in vivo . Consistent with other studies (7, 68), infection with tissue cysts is associated with limited symptomology and low overall mortality. Similar to what was observed with tachyzoite-initiated infections, the ΔTgLaf parasites consistently presented with lower cyst burdens relative to WT cyst infections.…”

“…To test this hypothesis, equal numbers of male and female CBA/J mice were infected with 100 tachyzoites intraperitoneally (i.p.) and monitored daily using a previously developed five-stage body index score to track the severity of symptoms associated with a tachyzoite infection over the course of 28 days (68).…”

“…Type II ME49∆HXGPRT ("WT"-the parental line utilized to generate all other lines in this study): This line was generated in a previous study using CRISPR/Cas9 targeting of TgHXGPRT and selection with 6-Tx (68).…”

“…Mice were then monitored and assigned a body index score daily. Monitoring frequency increased to twice a day once symptomatic throughout the course of infection as previously described (68). When symptomatic, mice were administered a gel diet and wet chow on the cage floor and given 0.25-0.5 mL saline solution subcutaneously as needed.…”