Intracellular Parasites Toxoplasma gondii and Besnoitia besnoiti, Unveiled in Single Host Cells Using AP-SMALDI MS Imaging.

The obligate intracellular apicomplexan parasites Toxoplasma gondii and Besnoitia besnoiti are important causes of disease in both humans and cattle. To date, effective specific treatments are lacking for both infections. To counteract severe symptoms leading to, e.g., disabilities and even abortion in the case of human toxoplasmosis and bovine besnoitiosis, novel targets are required for development of drugs and vaccines. A promising emerging technique for molecular characterization of organisms is high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) which enables semiquantitative visualization of metabolite distributions. MSI was here used to trace and characterize lipid metabolites in primary bovine umbilical vein endothelial cells (BUVECs) upon infection with tachyzoites, an early and pathogenic fast-replicating life stage of T. gondii and B. besnoiti. A cell bulk, derived from noninfected controls and parasite-infected cell pellets, was analyzed by AP-SMALDI MSI in technical and biological triplicates. Multivariate statistical analysis including hierarchical clustering and principle component analysis revealed infection-specific metabolites in both positive- and negative-ion mode, identified by combining database search and LC-MS2 experiments. MSI analyses of host cell monolayers were conducted at 5 µm lateral resolution, allowing single apicomplexan-infected cells to be allocated. This is the first mass spectrometry imaging study on intracellular T. gondii and B. besnoiti infections and the first detailed metabolomic characterization of B. besnoiti tachyzoites. MSI was used here as an efficient tool to discriminate infected from noninfected cells at the single-cell level in vitro.

Preparation of (131)I-Pyrimethamine and evaluation for scintigraphy of experimentally Toxoplasma gondii-infected rats.

We aimed to assess the ability of (131)I-Pyrimethamine scintigraphy to detect the lesions of Toxoplasma gondii infection. An experimental model of toxoplasmosis was developed. The presence of toxoplasmosis was confirmed 60 days after implantation. Pyrimethamine was radioiodinated with I-131. The radioligand was validated by the requisite quality control tests to check its radiolabeling efficiency, in vitro stability and radiochemical purity etc. (131)I-Pyrimethamine (specific activity: 7.08 MBq/µmol) was injected intravenously into the tail vein of the control and infected rats. Static whole body images of the rats were acquired under the gamma camera at 5 min, 45 min, 2 h, 6 h, and 24 h following the intravenous administration of the radioactivity (3.7 MBq/rat). Then the scintigraphic data were analyzed both visually and semiquantitatively. Regions of interest (ROIs) were drawn over the organs (thyroid, stomach, liver, bladder, and soft tissues) to calculate the ratios of the radiotracer in infected vs. control rats. The mean ratio of radiotracer in infected/control rats in the liver and diaphragm was over 1 at 45 min which persisted till 24 h. In conclusion, (131)I-Pyrimethamine may be useful agent for diagnosis toxoplasmosis especially involving liver and diaphragm, needs further preclinical validation before being extended for use in clinical applications.

Eosinophilic fibrosing gastritis and toxoplasmosis in a cat.

A 3-year-old, neutered male Tiffany cat was presented to the Animal Health Trust for investigation of pyrexia and a gastric lesion. Radiography and ultrasound showed severe thickening of the gastric wall and regional lymphadenopathy. There was altered gastric wall layering, predominately due to muscularis thickening. Histopathology confirmed eosinophilic fibrosing gastritis. The cat also had evidence of generalised Toxoplasma gondii infection, which may have been responsible for the gastric changes.

Heart failure caused by toxoplasmosis in a fennec fox (Fennecus zerda).

A male fennec fox (Fennecus zerda) kit was examined for lethargy, inappetence, and weight loss. Clinical findings included respiratory distress, a gallop rhythm, and retinochoroiditis. Radiography indicated pleural effusion and cardiomegaly. Echocardiographic findings included left ventricular dilatation, low left ventricular ejection fraction, and atrioventricular valvular regurgitation. Necropsy findings were compatible with a diagnosis of congestive heart failure caused by myocarditis. Histopathology showed a disseminated infection with Toxoplasma gondii causing myocarditis, skeletal polymyositis, gastrointestinal myositis, and panuveitis. Toxoplasma-induced myocarditis should be included in the differential diagnosis of heart failure and retinochoroiditis in the fennec fox.

Toxoplasmosis in Pallas' cats (Otocolobus felis manul) at the Denver Zoological Gardens.

In May 1996 the Denver Zoological Gardens obtained two male and two female Pallas' cats (Otocolobus felis manul) that were wild-caught in the Ukraine. These animals were part of a group of 16 wild-caught adults (eight male and eight female) imported to the United States and Canada between 1995 and 1996. The Denver Zoological Gardens cats were quarantined at the zoo hospital for approximately I mo. During the quarantine period they were immobilized for physical examination, and sera were obtained from them to evaluate for exposure to Toxoplasma gondii. All cats were positive for T. gondii antibodies by latex agglutination (titers from 1:512 to 1:1,024). After being paired for breeding, one pair produced two litters, and another pair produced four litters, a total of 17 kittens between 1997 and 2001. Four kittens and two young adults died from a disseminated granulomatous and necrotizing inflammation consistent with toxoplasmosis. Toxoplasma gondii infection was confirmed in all six deceased cats by polymerase chain reaction performed on formalin-fixed tissues. An additional five kittens disappeared and were not available for necropsy. The fatality rate from toxoplasmosis was 35.3% (6/17) for cats that were available for necropsy and could have been as high as 64.7% (11/17) if it were assumed that the disappeared kittens were also affected. The Pallas' kitten survival rate at the Denver Zoological Gardens was 35.3%. This article describes the clinical and pathologic features of toxoplasmosis in a group of Pallas' cats at the Denver Zoological Gardens.

Diagnosis of naturally occurring toxoplasmosis by bronchoalveolar lavage in a cat.

A 5-year-old cat presented with a history of fever and respiratory distress of 7 days' duration. The cat did not respond to initial symptomatic treatment. Through use of a bronchoalveolar lavage (BAL) technique, the diagnosis of toxoplasmosis was obtained rapidly without evidence of adverse effects to the cat. Antemortem diagnosis of naturally occurring active Toxoplasma gondii infection can be difficult in cats. Identification of organisms obtained by BAL may be beneficial in the diagnosis of feline toxoplasmosis.