A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples

Abstract

The genus Anaplasma (Rickettsiales: Anaplasmataceae), which includes the species Anaplasma capra, Anaplasma bovis, Anaplasma ovis, and Anaplasma phagocytophilum, is responsible for a wide variety of infections in both human and veterinary health worldwide. Multiple infections with these four Anaplasma pathogens have been reported in many cases. We introduce a novel multiplex PCR for the simultaneous detection of A. capra, A. bovis, A. ovis, and A. phagocytophilum, based on species-specific primers against the groEL (A. capra and A. bovis), msp4 (A. ovis), and 16S rRNA (A. phagocytophilum) genes. To verify the specificity of the PCR reactions, we evaluated four sets of primers to analyze samples containing different blood pathogens. The sensitivity of the multiplex PCR was evaluated by amplifying 10-fold dilutions of total genomic DNA extracted from sheep blood infected with A. capra, A. bovis, A. ovis, or A. phagocytophilum. The reproducibility of the assay was evaluated by testing 10-fold dilutions of total genomic DNA extracted from sheep blood infected with these pathogens from 10 0 to 10 −3 ng/µL per reaction in triplicate on three different days. A total of 175 field blood DNA samples were used to evaluate the reproducibility of multiplex PCR compared with the simplex PCRs. PCR primers used in this study were confirmed to be 100% species-specific using blood pathogens previously identified by other methods. The lower limit of detection of the multiplex PCR with good repeatability enabled the detection of A. capra, A. bovis, A. ovis and A. phagocytophilum at concentrations of 3 × 10 −5 , 5 × 10 −7 , 2 × 10 −5 , and 7 × 10 −7 ng/µL, respectively. There was no significant difference between conventional and multiplex PCR protocols used to detect the four Anaplasma species (P > 0.05). The results of the multiplex PCR revealed that the A. capra groEL gene, the A. bovis groEL gene, the A. ovis msp4 gene, and the A. phagocytophilum 16S rRNA gene were reliable target genes for species identification in clinical isolates, being specific for each of the four target Anaplasma species. Our study provides an effective, sensitive, specific, and accurate tool for the rapid differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in sheep and goats.

The first detection of Anaplasma capra, an emerging zoonotic Anaplasma sp., in erythrocytes

“…nov determined in the present study are consistent with those previously reported [1], and different from the other intraerythrocytic Anaplasma spp. [20,23,[33][34][35]. These findings significantly contribute to the research on this novel zoonotic Anaplasma sp.…”

“…For TEM observation, the cells were infiltrated with acetone and Pon 812 epoxy resin (SPI, West Chester, USA) (1/1, 2 h; then, 1/2, 12 h) and cured at 60°C for 48 h. The cured resin blocks were trimmed, thinsectioned, thin sections collected on formvar copper 200 mesh grids, and then post-stained with 2% aqueous uranyl acetate and Reynold's lead citrate. The sections were examined using a HT7700 electron microscope (HITACHI, Japan) [23]. The morulae of A. capra, following lysis of the erythrocytes and centrifuged at 12,000 × g, were observed by TEM.…”

Highly convenient and highly specific-and-sensitive PCR using Se-atom modified dNTPs

“…Encouraged by the high specificity on simplex PCR with dNTPaSe, we explored multiplex PCR (MP-PCR), a powerful tool for simultaneously detecting many target sequences in one tube. [23][24][25] Our results indicated that using canonical dNTPs, the conventional MP-PCRs still formed many by-products even after the time-consuming optimization ( Fig. 5), while in the presence of dCTPaSe, MP-PCRs conveniently offered clean reactions without significant by-products, indicating the suppression of nonspecific amplifications.…”

Refinement of an established large-animal model to understand the tick-pathogen-host interface

“…Three species of Anaplasma infect humans: A. phagocytophilum, Anaplasma platys, Anaplasma ovis and Anaplasma capra (77)(78)(79)(80)(81). The most wellknown of these species is A. phagocytophilum, which was first described in a modern report in Scotland (1932) in which a herd of sheep were reported to have tick-borne fever (TBF) (82).…”

“…The high bacterial load is believed to play a role in induction of a T-cell exhaustion state (142) (80,(148)(149)(150).…”