Isolation and Propagation of the Ap-Variant 1 Strain of Anaplasma phagocytophilum in a Tick Cell Line

Abstract

The first tissue culture isolates of the unique Anaplasma phagocytophilum strain, Ap-Variant 1, were obtained in the Ixodes scapularis tick-derived cell line ISE6. Two isolates were from goat blood samples: one from a goat infected with I. scapularis ticks from Rhode Island and a second from a goat infected by serial passage of blood from the first infected goat. Eight isolates were made directly from I. scapularis ticks collected from white-tailed deer in Minnesota and represent the first isolations of an Anaplasma species directly from ticks. Each of the 10 isolates had a 16S rRNA gene sequence identical to that previously described for Ap-Variant 1, but differences within the ank gene were found that suggest natural variation. Prevalence of Anaplasma in the Minnesota ticks was 63.9%; 23 of 36 ticks tested by PCR were positive. Six of the tick-derived isolates were obtained from a set of 18 PCR-positive ticks, for a 33.3% isolation success rate. The conservation of host tropism among the Rhode Island and Minnesota isolates of Ap-Variant 1 was examined by use of experimental infections of mice and a goat. A Minnesota tick-derived isolate (MN-61-2) was used to inoculate naïve animals, and this isolate was able to infect a goat but unable to infect each of five mice, confirming that the Minnesota isolates have the same host tropism as Ap-Variant 1 from the northeastern United States. Light and electron microscopy of the Ap-Variant 1 isolate MN-61-2 in ISE6 cells showed cytoplasmic inclusions characteristic of A. phagocytophilum with pleomorphic bacteria in membrane-bound vacuoles and both electron-dense and electron-lucent forms.

Infection of Ixodes spp. tick cells with different Anaplasma phagocytophilum isolates induces the inhibition of apoptotic cell death

“…For comparison, the same sequences were obtained for 9 isolates of which the genomes have been sequenced and are available in Genbank. The isolates CRT35 (NCBI reference sequence JFBI01000001; Massung et al, 2007;Al-Khedery et al, 2012) and CRT38 (NCBI reference sequence NZ_APHI00000000) were isolated from I. scapularis ticks, the isolate Dog2 was isolated from a dog (NCBI reference sequence CP006618; Al-Khedery et al, 2012), the isolate JM was obtained from a rodent (NCBI reference sequence CP006617; Al-Khedery et al, 2012), the MRK isolate was obtained from a horse (NCBI reference sequence JFBH00000000; Al-Khedery et al, 2012), the (Katoh and Standley, 2013). After alignment, regions with gaps were removed and 2354 gap-free sites were used in maximum likelihood (ML) phylogenetic analysis as implemented in PhyML (v3.0 aLRT) (Anisimova and Gascuel, 2006;Guindon and Gascuel, 2003).…”

European Network for Neglected Vectors and Vector-Borne Infections COST Action Guidelines: What Is This About and What Is This For?

“…They can be a good option for the isolation of Anaplasma variants found in vectors, but with low or unknown pathogenicity for vertebrates (Massung et al 2007). Moreover, these cells have already proven their value for bacteria that target mammalian cells that are difficult to continuously propagate in vitro, such as the intraerythrocytic and intrathrombocytic Anaplasma spp.…”

Molecular characterization reveals distinct genospecies of Anaplasma phagocytophilum from diverse North American hosts

“…Nested conventional PCR assays targeting sections of the ank gene and the groESL heat shock operon, as well as the 23S-5S rRNA (rrl-rrs) intergenic spacer region, were then performed on all positive samples. Amplification of the ank gene was performed as described by Massung et al (2007) using external primers ANK-F1 (59-GAAGAAATTACAACTCCTGAAG-39) and ANK-R1 (59-CAGCCAGATGCAGTAACGTG-39), followed by internal primers ANK-F2 (59-TTGACCGCTGAAGCACTAAC-39) and ANK-R2 (59-ACCATTTGCTTCTTGAGGAG-39), yielding an approximately 600 bp product. A segment of the groESL operon was amplified using external primers HS1a (59-AITGGGCTGGTAITGAAAT-39) and HS6a (59-CCICCIGGIACIAIACCTTC-39), followed by internal primers HS43 [59-AT(A/T)GC(A/T)AA(G/A)GAAGCATAGTC-3] and HSVR (59-CTCAACAGCAGCTCTAGTAGC-39), yielding an approximately 1200 bp product as described by Liz et al (2002).…”

“…The North American strain ApVariant 1, which has distinctive 16S rRNA, msp4, msp2 and ank genes, occurs in ticks and deer (Massung et al, 2003); deer, goats and tick-derived cell lines can be infected with Ap-Variant 1, but rodents cannot (Massung et al, 2007). In England, two genetically distinct subpopulations of A. phagocytophilum coexist in separate enzootic cycles, one involving deer and Ixodes ricinus ticks and the other involving field voles (Microtus agrestis) and Ixodes trianguliceps (Bown et al 2009).…”