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

Abstract

Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While B. 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, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms' epidemiology and pathogenesis. The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate Besnoitia spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize ungulate Besnoitia spp. (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia duodenalis, Tritrichomonas foetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world.

Keywords: Besnoitia darlingi; Besnoitia neotomofelis; Besnoitia oryctofelisi; Besnoitiosis; Lagomorph; Primer; Real-time PCR; Rodent.

Graphical abstract

Fig. 1

Location of the primers and the probe of the Besnoitia darlingi/B.neotomofelis /B. oryctofelisi-specific real-time PCR assay BdanjoRT1 within the ITS-1 region of the rRNA gene. The sequences of the ITS-1 region of B. akodoni (AY545987, bold), B. jellisoni (AF076860, bold), B. neotomofelis (HQ909085, bold), B. darlingi (AF489696, bold) and B. oryctofelisi (AY182000, bold), were aligned relative to sequences of other Besnoitia spp. including B. besnoiti from Portugal, Spain and Germany, B. bennetti reported from the USA and Belgium, B. tarandi from Canada and Finland and those of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and H. hammondi by using Clustal V (DNAStar, Madisin, Wisconsin, USA). Deletions and substitutions in the sequences relative to and within the clade of B. acodoni, B. jellisoni, B. neotomofelis, B. darlingi and B. oryctofelisi are indicated by black background. Sequences of the primer BdanjoRev and the Probe Bb11-12 are displayed in their complementary form. The probe Bb11-12 was established for a real-time PCR to detect B. besnoiti, but it is universal and can be used for the detection of all Besnoitia spp. mentioned here.

Fig. 2

Coccidia-specific PCR to confirm the presence of DNA of various parasite species used to test the analytic specificity of the BdanjoRT1 real-time PCR. (1) Besnoitia darlingi, (2) B. neotomofelis, (3) B. oryctofelisi, (4) B. besnoiti (Evora isolate), (5) B. bennetti (Texas), (6) B. tarandi (Bt-CA-Quebec1), (7) Toxoplasma gondii, (8) Hammondia hammondi, (9) Neospora caninum, (10) H. heydorni, (11) Cystoisospora felis, (12) C. rivolta, (13) C. burrowsi, (14) C. canis, (15) Sarcocystis cruzi and (16) Crytosporidium parvum. Presence of (17) Giardia duodenalis and (18) Tritrichomonas foetus DNA was shown by amplification using species or genus-specific primers, respectively. C, negative control; M, marker.

Fig. 3

Analytic sensitivity (A) and standard curve (B) obtained for the threshold cycle (Cq) values obtained in the BdanjoRT1 real-time PCR using varying amounts of genomic Besnoitia darlingi DNA (approximately equivalent to the DNA content of 10.000 [blue], 1000 [green], 100 [red], 10 [brown] and 1 [black] B. darlingi tachyzoites) diluted in 100 ng/μl mouse DNA. Cq values used for regression are displayed as circles. Results on samples resembling DNA of 0.1 tachyzoite were not included in regression, since only two of four samples had reacted with a Cq value of 37.8 or 38.3 (displayed grey in A and as crosses in B). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)