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Dissection of Besnoitia besnoiti intermediate host life cycle stages: From morphology to gene expression [1]

['Chandra Ramakrishnan', 'Parasite Cell Biology Laboratory', 'Institute Of Parasitology', 'Vetsuisse Faculty', 'University Of Zurich', 'Zurich', 'Aarti Krishnan', 'Department Of Microbiology', 'Molecular Medicine', 'University Of Geneva']

Date: 2022-12 

Cyst-forming Apicomplexa (CFA) of the Sarcocystidae have a ubiquitous presence as pathogens of humans and farm animals transmitted through the food chain between hosts with few notable exceptions. The defining hallmark of this family of obligate intracellular protists consists of their ability to remain for very long periods as infectious tissue cysts in chronically infected intermediate hosts. Nevertheless, each closely related species has evolved unique strategies to maintain distinct reservoirs on global scales and ensuring efficient transmission to definitive hosts as well as between intermediate hosts. Here, we present an in-depth comparative mRNA expression analysis of the tachyzoite and bradyzoite stages of Besnoitia besnoiti strain Lisbon14 isolated from an infected farm animal based on its annotated genome sequence. The B. besnoiti genome is highly syntenic with that of other CFA and also retains the capacity to encode a large majority of known and inferred factors essential for completing a sexual cycle in a yet unknown definitive host. This work introduces Besnoitia besnoiti as a new model for comparative biology of coccidian tissue cysts which can be readily obtained in high purity. This model provides a framework for addressing fundamental questions about the evolution of tissue cysts and the biology of this pharmacologically intractable infectious parasite stage.

The unicellular parasite Besnoitia besnoiti causes besnoitiosis in cattle. This emerging disease is characterized by a chronic phase where the parasite resides in tissue cysts mainly in the skin. Besnoitiosis leads to significant economic losses due to abortions, reduced milk production and/or leather quality and infertility in bulls. Transmission is known to take place by direct contact of infected animals or parasite transfer by blood-sucking insects. However, a definitive host in which sexual development takes place has not yet been identified. Here, we provide a detailed microscopical characterization of the parasite, compare the genome of B. besnoiti to related parasites, reveal the gene expression profiles of acute and chronic stage parasites, and show that the B. besnoiti genome contains genes necessary for sexual development in an as yet unknown definitive host. Our study provides a deeper understanding of B. besnoiti biology, highlights unique features of chronic cyst stages, and discusses its potential as a model organism for other related tissue-cyst forming parasites.

Funding: This work was supported by the Swiss National Science Foundation, SNSF Sinergia grant (CRSII3_160702 to ABH and DS) and the SNSF grant (310030_184662 to AH) https://snf.ch/en/FKhU9kAtfXx7w9AI/page/home . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we performed whole genome sequencing and annotation of B. besnoiti and identified genes that are predicted to be necessary for the development of gametes and oocysts in a putative definitive host. Deep comparative RNA-Seq analysis of bradyzoites extracted from in vivo tissue cysts and tachyzoites cultured in vitro reveal strikingly distinct stage-specific transcriptomes with many similarities to those of the corresponding T. gondii stages.

In contrast to other described CFA, which build walled cysts in muscle and brain, B. besnoiti forms large tissue cysts in cells of the mesenchymal lineage and are prevalent in the dermis, sclera and mucosa. This peripheral niche is conducive to transmission by insects or direct mechanical transfer from chronically infected cattle to a new host. With a diameter of up to 0.5 mm, these cysts are macroscopically detectable. The tissue cysts are enclosed by three distinct layers comprised of host-derived connective tissue surrounding the infected host cell, and a parasite-derived cyst wall on the luminal face of the parasitophorous vacuole membrane (PVM) containing the bradyzoites [ 8 ]. Tissues with high cyst loads, in particular the dermis, show altered architecture, sometimes with significant pericystic leukocyte infiltration and compromised barrier integrity [ 9 , 10 ]. The superficial, and to some extent also the deep dermis, appear expanded by infiltration of immune cells, e.g. macrophages and eosinophilic granulocytes, and shows distinct signs of fibrosis [ 11 , 12 ]. Tissue cyst formation affects animal health and agricultural productivity. Acute phase tachyzoite stages can be cultured in vitro in fibroblast host cells but unlike for T. gondii, there is currently no protocol allowing induction of differentiation to bradyzoites and cyst formation, which precludes systematic molecular genetic characterization of this infectious life cycle stage.

Bovine besnoitiosis caused by Besnoitia besnoiti is an (re-)emerging disease in Southern Europe (reviewed by Álvarez-García et al. [ 1 ]) resulting in considerable economic losses in the cattle industry. Bovine besnoitiosis is responsible for decreased milk production, abortions, bull infertility and reduction of hide quality (reviewed by Álvarez-García et al. [ 1 ] and Cortes et al. [ 2 ]). Thus, besnoitiosis has gained interest in the search for effective therapeutics. Despite this interest, the biology of this parasite remains largely unknown. B. besnoiti–a tissue cyst-forming Apicomplexa (CFA)—is genetically related to Neospora caninum and Toxoplasma gondii but with clear differences in transmission and uncertain definitive host cycle. Transmission is known to take place via close contact (e.g. mating) or horizontally via mechanical transmission by blood-sucking insects (reviewed by Álvarez-García et al. [ 1 ] and Cortes et al. [ 2 ]). Vertical transmission from mother to the foetus–an important route for T. gondii and N. caninum—has never been observed for B. besnoiti [ 3 ]. Thus, as for neosporosis and in contrast to toxoplasmosis, herd management is an important factor in controlling the spread of the besnoitiosis. However, to date, no definitive host in which sexual development can take place has been identified. Studies on wild, domestic and laboratory animals have been conducted to discover the definitive host [ 4 – 7 ], but no conclusive identification could be made.

Results and discussion

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010955

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