Mice Infected with Low-virulence Strains of Toxoplasma gondii Lose their Innate Aversion to Cat Urine, Even after Extensive Parasite Clearance

Toxoplasma gondii chronic infection in rodent secondary hosts has been reported to lead to a loss of innate, hard-wired fear toward cats, its primary host. However the generality of this response across T. gondii strains and the underlying mechanism for this pathogen mediated behavioral change remain unknown. To begin exploring these questions, we evaluated the effects of infection with two previously uninvestigated isolates from the three major North American clonal lineages of T. gondii, Type III and an attenuated strain of Type I. Using an hour-long open field activity assay optimized for this purpose, we measured mouse aversion toward predator and non-predator urines. We show that loss of innate aversion of cat urine is a general trait caused by infection with any of the three major clonal lineages of parasite. Surprisingly, we found that infection with the attenuated Type I parasite results in sustained loss of aversion at times post infection when neither parasite nor ongoing brain inflammation were detectable. This suggests that T. gondii-mediated interruption of mouse innate aversion toward cat urine may occur during early acute infection in a permanent manner, not requiring persistence of parasitecysts or continuing brain inflammation.

💡 Research Summary

Toxoplasma gondii is a ubiquitous neurotropic protozoan that completes its sexual cycle only in felines. When rodents become intermediate hosts, the parasite forms cysts in the brain and has been reported to diminish the animals’ innate fear of cat odors, presumably enhancing transmission to the definitive host. Most previous behavioral studies, however, have focused on Type II strains, which generate high brain cyst burdens and robust immune‑mediated inflammation, making it difficult to separate parasite‑specific effects from nonspecific pathology. To address these limitations, Ingram et al. examined two additional North‑American clonal lineages: a low‑virulence Type III strain and an attenuated Type I strain lacking the ROP5 virulence locus (Δrop5).

Male BALB/c mice (9 weeks old) were infected intraperitoneally with 5 × 10⁵ tachyzoites of either Type I or Type III (or with a low dose of Type II for preliminary work). At 3 weeks, 2 months, and 4 months post‑infection, each mouse was placed in a 15 × 7 inch enclosure equipped with an infrared‑beam tracking system (MotorMonitor SmartFrame). A small dish at one end of the arena contained 400 µL of either bobcat urine (predator cue) or rabbit urine (non‑predator control). The system automatically recorded the time each animal spent in the “Near Target” zone (adjacent to the dish) versus the opposite “Avoidance” zone. Uninfected controls displayed a clear avoidance of bobcat urine, spending significantly more time in the opposite zone, while showing no preference for rabbit urine. In contrast, both Type I‑ and Type III‑infected mice lost this avoidance: they spent comparable time in both zones when exposed to bobcat urine, a phenotype that persisted across all three time points and was observed in every infected animal (no non‑responders). A hidden‑cookie test confirmed that general olfactory function was intact in infected mice.

To determine whether the behavioral change correlated with parasite persistence or ongoing neuroinflammation, the authors performed quantitative PCR for the T. gondii B1 gene on brain‑meningeal homogenates and flow‑cytometric analysis of brain‑derived leukocytes after the final behavioral assay. All Type III‑infected mice harbored detectable parasite DNA and exhibited markedly elevated numbers of CD4⁺ and CD8⁺ T cells in the brain, indicative of chronic inflammation. By contrast, Type I‑infected mice showed no detectable parasite DNA at 4 months and had brain leukocyte counts comparable to uninfected controls. Nevertheless, serum ELISA confirmed that both groups had mounted a robust anti‑Toxoplasma antibody response, proving that infection had been established.

A time‑course experiment further revealed that attenuated Type I parasites transiently entered the brain between days 5 and 20 post‑infection, accompanied by a modest but significant influx of CD4⁺/CD8⁺ T cells. After day 20, both parasite DNA and leukocyte numbers fell to baseline, yet the loss of predator aversion remained. These findings demonstrate that the behavioral alteration does not require persistent cysts or chronic neuroinflammation; instead, it likely originates during the acute phase of infection.

The authors discuss the implications of these results for existing mechanistic models. Prior hypotheses have posited that cysts in specific brain regions (e.g., amygdala) modulate dopamine synthesis or directly disrupt neuronal activity, thereby altering fear responses. The present data challenge the necessity of cysts, as the Type I‑infected mice lost aversion despite complete parasite clearance. Recent work showing that T. gondii can deliver effector proteins into host cells it does not invade suggests an alternative “non‑cyst‑centric” mechanism: secreted effectors during acute infection may rewire neuronal circuits or epigenetically reprogram host gene expression, producing a lasting behavioral phenotype.

In summary, this study establishes that loss of innate cat‑urine aversion is a general trait of infection with any of the three major North‑American T. gondii clonal lineages. Crucially, the effect persists long after the parasite has been cleared from the brain and after neuroinflammation has resolved, indicating that the critical manipulation occurs early in infection and does not depend on chronic cyst presence. These findings call for a revision of current models of parasite‑induced behavioral manipulation, shifting focus toward acute‑phase effector delivery and enduring neural remodeling.