Milton M. McAllister

Toxoplasma gondii is an intracellular eukaryotic parasite in the phylum Apicomplexa. Reversible, conditional control of gene transcription was achieved previously in this organism using a tet‐off system that was controlled by tetracycline. Although revolutionary for the field at the time, that system in Toxoplasma yielded modest signal‐to‐noise ratios (SNR) and proved to be leaky in the “off” state (i.e., it suffered from high background expression). Here we report the development of a new reversible and robust Conditional Transcription Factor (CTF) that is controlled by rapamycin. Although we originally intended to design a dimerizable drug‐on transcription control system, the CTF unexpectedly functions in a drug‐off fashion. In stably‐transfected tachyzoites, CTF regulates a fluorescent reporter gene in Toxoplasma to achieve almost no background expression in the presence of rapamycin (i.e. near‐zero leakiness), while in the absence of rapamycin 84% of tachyzoites express EYFP in comparison with 87% of constitutively‐expressed positive controls, with 59% mean fluorescence intensity relative to positive controls. Conditional regulation of the median fluorescence intensity of the reporter gene reached an impressive SNR averaging 1489, approximating an “on/off” switch. The modular design of the CTF is expected to facilitate application to varied genes, incremental modifications, and adaptation for use in other apicomplexan organisms.

This study delves into the pathogenesis of virulent genotype VII strains of the Newcastle disease virus (NDV), focusing on experimentally infected birds. Predominant and consistent lesions observed include bursal atrophy and extensive depletion of all lymphoid tissues. Immunohistochemistry (IHC) analysis, targeting apoptosis (Caspase-3), necroptosis (MLKL), and NDV markers, indicates that bursal atrophy is linked to a non-apoptotic programmed cell death pathway known as “necroptosis”. Repair assisted damage detection (RADD) of the bursa reveal oxidative DNA damage patterns consistent with programmed cell death, aligning with MLKL expression. Contrastingly, in the spleen, our findings suggest that necrosis (non-programmed cell death) predominantly contributes to lymphoid depletion. This conclusion is supported by evidence of karyorrhexis, fibrinous inflammation, RADD analyses, and IHC. Moreover, in addition to being pathogenic in its own right, NDV caused extensive and rapid lymphoid depletion that should be expected to contribute to profound immunosuppression. The elucidation of necroptosis in NDV-infected chickens provides a good rationale to investigate this mechanism in other paramyxoviral diseases such as human measles.

Toxoplasma gondiiandNeospora caninum(both Apicomplexa) are closely related cyst-forming coccidian parasites that differ significantly in their host ranges and ability to cause disease. Unlike eutherian mammals, Australian marsupials (metatherian mammals) have long been thought to be highly susceptible to toxoplasmosis and neosporosis because of their historical isolation from the parasites. In this study, the carnivorous fat-tailed dunnart (Sminthopsis crassicaudata) was used as a disease model to investigate the immune response and susceptibility to infection of an Australian marsupial toT. gondiiandN. caninum. The disease outcome was more severe inN. caninum-infected dunnarts than inT. gondii-infected dunnarts, as shown by the severity of clinical and histopathological features of disease and higher tissue parasite burdens in the tissues evaluated. Transcriptome sequencing (RNA-seq) of spleens from infected dunnarts and mitogen-stimulated dunnart splenocytes was used to define the cytokine repertoires. Changes in mRNA expression during the time course of infection were measured using quantitative reverse transcription-PCR (qRT-PCR) for key Th1 (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), Th2 (interleukin 4 [IL-4] and IL-6), and Th17 (IL-17A) cytokines. The results show qualitative differences in cytokine responses by the fat-tailed dunnart to infection withN. caninumandT. gondii. Dunnarts infected withT. gondiiwere capable of mounting a more effective Th1 immune response than those infected withN. caninum, indicating the role of the immune response in the outcome scenarios of parasite infection in this marsupial mammal.

Case report: A 17‐day‐old Bulldog puppy died soon after presentation for weakness and tachypnoea. Gross lesions included diffuse pulmonary oedema and a region of myocardial pallor that resembled an infarct. Inflammation was observed histopathologically in many organs, with numerous clusters of intracellular protozoa that stained positively using Neospora caninum immunohistochemistry. Myocarditis was severe and had associated necrosis of individual myocytes, but the tissue was not infarcted. The bitch had an antibody titre of 1 : 1600 for N. caninum. All six littermates were sold and reported to be healthy at 6 months of age.Conclusion: Unusual aspects of this case include the occurrence of clinical disease in only 1 of 7 neonatal puppies, widespread dissemination of the organism in multiple tissues, and regional pallor associated with myocarditis that gave a false gross appearance of infarction. This report also adds Bulldogs to the list of dog breeds shown to be susceptible to clinical neosporosis.

Toxoplasma gondii is a protozoal parasite with worldwide distribution that is able to infect a wide variety of mammals and birds. Our main goal was to screen for T. gondii antibody titers in a previously untested species, the spotted hyena ( Crocuta crocuta); however, this goal first required us to investigate serological procedures that could be suitable for hyenas. Cats are the closest domestic relations of hyenas, so T. gondii antibody titers were first compared in 26 feral cats with specific or nonspecific fluorophore-labeled secondary reagents, i.e., anti-cat IgG or protein A. Substitution of anti-cat IgG with protein A caused a statistically significant drop in titer measurements in cats (P = 0.01) with a reduction of the geometric mean titer equivalent to 1 doubling-dilution. The same procedures were then applied to captive spotted hyenas. Titers measured in 9 of 10 hyenas were identical whether anti-cat IgG or protein A was used as the secondary reagent: 5 had titers <1:16, 2 had titers of 1:16, and 2 had titers of 1:32. One hyena had maximum titers of 1:64 or 1:32 when anti-cat IgG or protein A was used, respectively. The use of protein A as the secondary reagent in serologic assays can be applied to a range of mammalian species and seems unlikely to affect test specificity; however, the use of protein A may reduce test sensitivity, as suggested in the present study using cats. Despite a control program, some exposure to T. gondii had occurred in the Zoo's spotted hyenas.L.F. Wait, A. Srour, I.G. Smith, P. Cassey, S.K. Sims, and M.M. McAlliste