Besnoitia tarandi in Canadian woodland caribou – Isolation, characterization and suitability for serological tests

Abstract

In the present study, we report the first in vitro isolation of Besnoitia tarandi from North America and the second of B. tarandi at all. The parasite was isolated directly from the skin of a Canadian woodland caribou from the migratory ecotype. The animal belonged to the Leaf River Herd, in Northern Quebec, Canada. The isolate was designated Bt-CA-Quebec1.Sequencing of the 3’-end of the 18S rRNA gene, the complete sequence of the ITS1 and the 5’-end of the 5.8S rRNA gene of Bt-CA-Quebec1 revealed only minor differences to rDNA gene fragments of B. besnoiti. In contrast, the patterns for the microsatellite loci Bt-20 and Bt-21 varied substantially from those reported for B. besnoiti and B. bennetti. Surprisingly, the typing results in the loci Bt-6 and Bt-7 differed between Bt-CA-Quebec1 and results obtained for skin samples from caribou of the Canadian regions of Nunavut and the Northwest Territories reported by other investigators. This indicates that differences might exist among B. tarandi in caribou from different regions in Canada.Mice (γ-interferon knockout) intraperitoneally inoculated with 1.2 × 106 or 1.5 × 106 bradyzoites mechanically released from skin tissue cysts fell ill 8, 9 or 18 days post inoculation. GKO mice inoculated with 3.0 × 104 tachyzoites isolated from the peritoneal cavity of a bradyzoites-inoculated mouse became ill earlier, i.e. 5 days post inoculation. Lung was the predilection site in all mice.Bt-CA-Quebec1 tachyzoites rapidly grew in MARC-145 cells and were used for antigen production. Comparative Western blot analyses revealed only a few differences between B. tarandi Bt-CA-Quebec1 and B. besnoiti Evora antigen when probed with sera collected from chronically infected caribou.Due to its fast growth in vitro, the Bt-CA-Quebec1 isolate may represent an interesting antigen source to establish B. tarandi-specific serological tools and to study the biology of this parasite species further.

First record of besnoitiosis caused by Besnoitia bennetti in donkeys from the UK

“…Unfortunately, due to the retrospective nature of the study, serum samples were only available in this small subset of cases, preventing complete assessment of Besnoitia seropositivity in all 20 donkeys. The Bb1Evora03 strain of B. besnoiti was maintained in MARC-145 cell culture and extracellular tachyzoites were purified as previously described [10]. Purified tachyzoites were used to prepare IFAT slides or were pelleted by centrifugation at 1300×g for 10 min and frozen at − 80 °C until used for immunoblotting.…”

“…These tissues had been frozen at − 20 °C and submitted to the Friedrich-Loeffler-Institut, Germany. Purification was performed as previously described [10]. Eluted parasites were concentrated by centrifugation at 1300×g for 10 min, and frozen at − 80 °C until used for immunoblotting.…”

“…Serial dilutions starting at 1:50 of donkey sera were analyzed for Besnoitia antibodies by IFAT using fluorescein (FITC)-conjugate solution [affinity purified goat antihorse IgG (H + L) (Jackson ImmunoResearch Laboratories, West Grove, USA; diluted 1:50 in PBS)]. The procedure was performed as previously described [10]. Sera from infected or non-infected donkeys were used as positive and negative controls, respectively, sampled during a besnoitiosis outbreak in north-eastern Pennsylvania [7].…”

“…Immunoblots were performed as described previously [10]. Samples containing 2 × 10 6 Besnoitia tachyzoites/ lane were treated for 10 min at 94 °C with non-reducing sample buffer (2% (w/v) SDS, 10% (v/v) glycerol, 62 mM Tris HCl, pH 6.8).…”

“…specific antibodies in the serum using serological assays such as ELISA, immunofluorescent and immunoblotting analysis [4][5][6][7][8][9]. Recently, a more specific genotyping method based on microsatellite markers has been developed to distinguish B. bennetti from the closely related B. besnoiti and B. tarandi, which cause besnoitiosis in cattle and reindeer, respectively [10].…”