Toxoplasma gondii

Toxoplasma gondii (Type II) PRU strain was selected for generating a cDNA library because it encodes the TgGRA15, which allows type II strains of T. gondii to be a highly inducer of NF-kB activation, compared to type I strains [28 (link)] and type III strains [11 (link)]. The Type II PRU strain was maintained in our laboratory by the passage of cysts in female BALB/c mice (4 to 5 weeks of age). Mice were purchased from the Center of Experimental Animals, Lanzhou University, Lanzhou, China. Parasite maintenance in the mice experiment was performed in strict compliance with state and institutional animal care guidelines. The tissue cysts of T. gondii PRU strain were harvested from infected mice 30 days post-infection and were used for total RNA extraction.

The previously described RHΔku80Δhxgprt line [40 (link)], referred to as ku80^KO, was used as a parent for the transgenic lines reported here. Toxoplasma gondii lines (S1 Table) were grown in confluent monolayers of human foreskin fibroblasts (HFF), obtained from the Boothroyd laboratory at Stanford University, and harvested as described previously [41 (link)]. Parasite cultures were determined to be mycoplasma negative using the e-Myco plus kit (Intron Biotechnology).

Toxoplasma gondii (RH strain) tachyzoites were maintained by serial passages in MARC-145 (Meat Animal Research Center-145) layers. The infection rate in MARC was 40-50% in all experiments. Therefore, free-released T. gondii tachyzoites were harvested from MARC supernatants, pelleted (400 × g, 12 min), counted in a Neubauer chamber, suspended in HBSS and used for bovine PMN confrontation. All experiments were performed at an MOI of 1:2 (bPMN:parasites).

A retrospective review of the medical records of dogs diagnosed with non-infectious, non-erosive, idiopathic IMPA and SRMA from two referral institutions during the period between 2017 and 2021 was performed. The terms used as identifiers during the search were: “immune-mediated polyarthritis,” “steroid-responsive meningitis arteritis,” “meningitis,” “arthritis,” “polyarthritis,” “SRMA,” “IMPA.” Patients were included if they were diagnosed with non-infectious, non-erosive, idiopathic (primary) IMPA or SRMA and CRP was measured at the time of presentation. Ethical approval was granted by the Research Ethics Committee at the University of Glasgow with a reference number EA39/20.
A diagnosis was made based on consistent medical history, physical, neurological and orthopedic examination, and clinicopathologic findings (results of hematology, serum biochemical analysis for IMPA and SRMA, and thoracic and abdominal imaging for IMPA). These were coupled with the results of routine analysis of CSF collected from the cerebellomedullary or lumbar cistern. For those patients identified for the purposes of the study with SRMA, fluid analysis revealed neutrophilic or mixed neutrophilic and monocytic pleocytosis with no visible organisms, and lack of toxic neutrophils (1 (link), 2 (link)). Patients diagnosed with IMPA were identified for the purposes of the study as those whose results of synovial fluid analysis indicated neutrophilic inflammation in two or more joints.
Other diagnostic tests performed in an attempt to rule out other causes of CSF pleocytosis (imaging of the vertebral column, PCR assays to detect Toxoplasma gondii, Neospora caninum, canine distemper virus in CSF, CSF culture and serum T. gondii and N. caninum antibody titers) or secondary IMPA (echocardiography, serologic and PCR assays to detect vector-borne disease, serologic testing for Bartonella sp. infection, joint radiography, and microbial culture of blood, synovial fluid, or urine samples) were performed at the discretion of the attending clinician, taking into consideration patient demographic and historical factors, physical examination findings, preliminary test results, and client finances. Dogs were excluded if they had incomplete medical history, no definitive diagnosis or had undergone corticosteroid treatment prior to diagnosis. Additionally, dogs with SRMA were excluded if they had neurological deficits.
Data retrieved from the medical records was as follows: breed, age, body weight, gender, neutered status, month of presentation, history, physical and neurologic examination findings, CRP values at the time of diagnosis, CSF routine analysis and joint cytology results, imagining modalities performed and results, infectious disease testing, and final diagnosis.
CRP was measured quantitatively in 142 dogs (84%) and semi-quantitatively in 27 dogs (16%). For the purposes of statistical analysis semi-quantitative results were replaced as follows: 2.5 mg/L instead of <5, 150 mg/L instead of >100 mg/L, and 250 mg/L instead of >200 mg/L. The CRP serum concentration was measured using species-specific immunoturbidimetric assay for canine CRP (Gentian Canine CRP Immunoassay, Gentian AS, Moss, Norway) in one of the hospitals and using IDEXX Catalyst^® CRP Test (a sandwich immunoassay, IDEXX, USA) in the other. The two assays were compared and considered to provide accurate and consistent results (26 ). The reference range for CRP was <10 mg/L.

Toxoplasma gondii tachyzoites (2F1 clone) constitutively expressing the β-galactosidase gene were maintained by serial passages in BeWo cells cultured in medium containing 2% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C and 5% CO₂. 2F1 clone is derived from the highly virulent strain RH and was a gift from Dr. Vern Carruthers, School of Medicine from the University of Michigan (USA).