Animal trypanosomiasis (AT) is a significant livestock disease, affecting millions of animals across Sub-Saharan Africa, Central and South America, and Asia, and is caused by the protozoan parasites Trypanosoma brucei, Trypanosoma vivax, and Trypanosoma congolense, with the largest economic impact in cattle. There is over-reliance on presumptive chemotherapy due to inadequate existing diagnostic tests, highlighting the need for improved AT diagnostics. A small RNA species, the 7SL sRNA, is excreted/secreted by trypanosomes in infected animals, and has been previously shown to reliably diagnose active infection. We sought to explore key properties of 7SL sRNA RT-qPCR assays; namely, assessing the potential for cross-reaction with the widespread and benign Trypanosoma theileri, directly comparing assay performance against currently available diagnostic methods, quantitatively assessing specificity and sensitivity, and assessing the rate of decay of 7SL sRNA post-treatment. Results showed that the 7SL sRNA RT-qPCR assays specific for T. brucei, T. vivax, and T. congolense performed better than microscopy and DNA PCR in detecting infection. The 7SL sRNA signal was undetectable or significantly reduced by 96-h post treatment; at 1 × curative dose there was no detectable signal in 5/5 cattle infected with T. congolense, and in 3/5 cattle infected with T. vivax, with the signal being reduced 14,630-fold in the remaining two T. vivax cattle. Additionally, the assays did not cross-react with T. theileri. Finally, by using a large panel of validated infected and uninfected samples, the species-specific assays are shown to be highly sensitive and specific by receiver operating characteristic (ROC) analysis, with 100% sensitivity (95% CI, 96.44–100%) and 100% specificity (95% CI, 96.53–100%), 96.73% (95% CI, 95.54–99.96%) and 99.19% specificity (95% CI, 92.58–99.60%), and 93.42% (95% CI, 85.51–97.16% %) and 82.43% specificity (95% CI, 72.23–89.44% %) for the T brucei, T. congolense and T. vivax assays, respectively, under the conditions used. These findings indicate that the 7SL sRNA has many attributes that would be required for a potential diagnostic marker of AT: no cross-reaction with T. theileri, high specificity and sensitivity, early infection detection, continued signal even in the absence of detectable parasitaemia in blood, and clear discrimination between infected and treated animals.

In female mammals, reproductive potential is determined during fetal life by the formation of a non-renewable pool of primordial follicles. Initiation of meiosis is one of the defining features of germ cell differentiation, and is well established to commence in response to retinoic acid. WIN 18,446 inhibits the conversion of retinol to retinoic acid, therefore was used to explore the impact of reduced retinoic acid synthesis on meiotic progression and thus germ cell development and subsequent primordial follicle formation. e13.5 mouse fetal ovaries were cultured in vitro and treated with WIN 18,446 for the first 3 days of a total of up to 12 days. Doses as low as 0.01µM reduced transcript levels of the retinoic acid response genes Stra8 and Rarβ without affecting germ cell number. Higher doses resulted in germ cell loss, rescued with the addition of retinoic acid. WIN 18,446 significantly accelerated the progression of prophase I; this was seen as early as 48 hours post treatment using meiotic chromosome spreads, and was still evident after 12 days of culture using Tra98/Msy2 immunostaining. Furthermore, ovaries treated with WIN 18,446 at e13.5 but not at P0 had a higher proportion of growing follicles compared to vehicle controls, thus showing evidence of increased follicle activation. These data therefore indicate that retinoic acid is not necessary for meiotic progression but may have a role in the regulation of its progression and germ cell survival at that time, and provide evidence for a link between meiotic arrest and follicle growth initiation.