Sensitive, quantitative detection of Besnoitia darlingi and related parasites in intermediate hosts and to assess felids as definitive hosts for known and as-yet undescribed related parasite species

Authors: SCHARES, GEREON - Friedrich-Loeffler-institut; Dubey, Jitender; Rosenthal, Benjamin; TUSCHY, MAREEN - Friedrich-Loeffler-institut; BARWALD, ANDREA - Friedrich-Loeffler-institut; CONRATHS, FRANZ - Friedrich-Loeffler-institut

Submitted to: International Journal for Parasitology: Parasites and Wildlife
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/22/2020
Publication Date: 4/1/2020
Citation: Schares, G., Dubey, J.P., Rosenthal, B.M., Tuschy, M., Barwald, A., Conraths, F.J. 2020. Immunomagnetic separation of Toxoplasma gondii tissue cysts and sporocysts using a single monoclonal antibody. International Journal of Parasitology. 11:114-119. https://doi.org/10.1016/j.ijppaw.2020.01.011.
DOI: https://doi.org/10.1016/j.ijppaw.2020.01.011

Interpretive Summary: Livestock and wild herbivores can contract muscle infections when ingesting water or grazing on vegetation contaminated with the feces of carnivores that have excreted one of several species of parasites in the genus Besnoitia. A fuller understanding of the diversity, distribution, and pathogenesis of such species would benefit from more sensitive and quantitative methods to diagnose infection. Here, USDA researchers working with colleagues in Germany, developed such a test and used it to confirm which tissues (in mice) serve as predilection sites for Besnoitia darlingi and to show that at as few as one parasite could be detected from a naturally-infected fecal sample. The study will assist veterinarians, parasitologists, and wildlife biologists understand the range and habits of such parasites, and may lead to better methods to understand and prevent infections in animals important to agriculture.

Technical Abstract: Background: Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While Besnoitia darlingi uses opossums as intermediate hosts, B. neotomofelis and B. oryctofelisi have been described in Southern Plains woodrats (Neotoma micropus) from the USA and in domestic rabbits from Argentina. In addition, there is another closely related Besnoitia species, B. jellisoni, which was initially described in the USA with the White-footed deer mouse and three species of Kangaroo rats as intermediate hosts. A comparison of the Internal Transcribed Spacer 1 (ITS1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. of new world marsupials, rodents and domestic rabbits showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi DNA in tissues of intermediate hosts and in feces of definitive hosts in order to support studies of these organisms’ epizootiology, geography, and pathogenesis and to provide additional means to differentiate the oocysts of these species from morphologically similar coccidians including congeneric species afflicting livestock hosts. Methods: The established real-time PCR was based on primers located in regions distinct from the ITS1 of ungulate Besnoitia sp. (B. besnoiti, B. bennetti, B. tarandi) and made use of a Besnoitia universal probe earlier established for a B. besnoiti/B. bennettii/B. tarandi real-time PCR. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The analytic specificity of the PCR was assessed using DNA from B. darlingi, B. neotomofelis and B. oyctofelisi as well as DNA form related parasites (B. besnoiti, B. bennetti, B. tarandi, Toxoplasma gondii, Hammondia hammondi, Neospora caninum, H. heydorni, Isospora felis, I. rivolta, I. burrowsi, I. canis, Sarcocystis cruzi, Crytosporidium parvum, Giardia duodenalis, Tritrichomonas fetus). Cell-culture derived tachyzoites of B. darlingi and oocysts-spiked feline faeces were employed to assess the analytic sensitivity and the efficiency of the real-time PCR. The new PCR was used to examine the tissue distribution of B. darlingi in '-interferon knock-out mice infected with oocysts to establish a cell-culture of B. darlingi. Results: The real-time PCR reacted with DNA from B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize DNA of ungulate Besnoitia species (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia lamblia, Tritrichomonas fetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline feces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected '-interferon knock-out mice, the lung was identified the predilection organ. Conclusion: A real-time PCR including an internal control system was established to detect B. darlingi, B. neotomofelis and B. oryctofelisi. It also detects other particularly closely related species; this tool should advance further studies on these parasites and this approach may inspire the development of similar tools efficaceous in studying related parasites, not only in the Americas, but also in other parts of the world.